selenium in dog foods - ghent university · selenium in dog foods mariëlle van zelst dissertation...

Selenium in dog foods

Mariëlle van Zelst

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The studies in this PhD thesis were performed in collaboration with

© 2015 M. van Zelst

Selenium in dog foods

Mariëlle van Zelst

Dissertation submitted in fulfilment of the requirements for the degree of

Doctor of Philosophy (PhD) in Veterinary Sciences

Faculty of Veterinary Medicine

Ghent University

2015

Guidance Committee:

Prof. Dr. Ir. Geert P.J. Janssens (Supervisor)

Ghent University

Prof. Dr. Myriam Hesta (Supervisor)

Ghent University

Dr. Kerry Gray

WALTHAM® Centre for Pet Nutrition

Department of Nutrition, Genetics and Ethology

Faculty of Veterinary Medicine, Ghent University

Heidestraat 19, 9820 Merelbeke, Belgium

WALTHAM® Centre for Pet Nutrition

Freeby lane, Waltham-on-the-Wolds,

Leicestershire, England

"If you can look at a dog and not feel vicarious excitement and affection,

you must be a cat"

- Carrie Latet -

TTTTable of contentsable of contentsable of contentsable of contents

Table of contents

List of figures

List of tables

List of acronyms, abbreviations and symbols

1. General introduction____________________________________________________________________________ 23

1.1 History of selenium _______________________________________________________________________________ 25

1.2 Geological distribution of selenium ___________________________________________________________ 25

1.3 Selenium metabolism in monogastric animals ________________________________________________ 27

1.3.1 Absorption __________________________________________________________________________________ 27

1.3.2 Distribution __________________________________________________________________________________ 28

1.3.3 Excretion _____________________________________________________________________________________ 31

1.4 Selenoproteins ____________________________________________________________________________________ 33

1.4.1 Glutathione peroxidase ___________________________________________________________________ 37

1.4.2 Thioredoxin reductase ____________________________________________________________________ 39

1.4.3 Iodothyronine deiodinase ________________________________________________________________ 40

1.4.4 Selenoprotein P _____________________________________________________________________________ 41

1.5 Selenium related diseases ______________________________________________________________________ 41

1.5.1 Cancer _______________________________________________________________________________________ 42

1.5.2 Cardiovascular disease ___________________________________________________________________ 43

1.5.3 Diabetes mellitus type II __________________________________________________________________ 44

1.5.4 Joint conditions _____________________________________________________________________________ 45

1.5.5 Urolithiasis ___________________________________________________________________________________ 46

1.6 Selenium in pet foods____________________________________________________________________________ 46

1.7 Bioaccessibility, -availability, and -activity of selenium ____________________________________ 49

1.8 Current recommendations on selenium intake for adult dogs ____________________________ 54

1.9 Biomarkers of selenium status __________________________________________________________________ 56

1.10 References _______________________________________________________________________________________ 59

2. Scientific aims ____________________________________________________________________________________ 77

3a. In vitro selenium accessibility in dog foods ____________________________________________ 81

3a.1 Abstract___________________________________________________________________________________________ 83

3a.2 Introduction ______________________________________________________________________________________ 83

3a.3 Experimental methods __________________________________________________________________________ 84

Table of contentsTable of contentsTable of contentsTable of contents

3a.3.1 Diet selection ______________________________________________________________________________ 84

3a.3.2 Sample preparation ______________________________________________________________________ 85

3a.3.3 In vitro digestion__________________________________________________________________________ 85

3a.3.4 Chemical analyses ________________________________________________________________________ 86

3a.3.5 Calculations ________________________________________________________________________________ 87

3a.3.6 Statistical analyses _______________________________________________________________________ 88

3a.4 Results ____________________________________________________________________________________________ 88

3a.5 Discussion ________________________________________________________________________________________ 91

3a.6 Conclusions ______________________________________________________________________________________ 93

3a.7 Acknowledgements _____________________________________________________________________________ 93

3a.8 References _______________________________________________________________________________________ 93

3b. Predictive equations of in vitro selenium accessibility in dry pet foods ________ 97

3b.1 Abstract __________________________________________________________________________________________ 99

3b.2 Introduction ______________________________________________________________________________________ 99

3b.3 Experimental methods __________________________________________________________________________ 99

3b.3.1 Diets and chemical analyses ___________________________________________________________ 99

3b.3.2 Calculations ________________________________________________________________________________ 100

3b3.3 Statistical analyses ________________________________________________________________________ 100

3b.4 Results ____________________________________________________________________________________________ 101

3b.5 Discussion ________________________________________________________________________________________ 104

3b.6 References _______________________________________________________________________________________ 105

4. Association between diet type and selenium bioavailability and -activity

in dogs _________________________________________________________________________________________________ 107

4.1 Abstract ____________________________________________________________________________________________ 109

4.2 Introduction _______________________________________________________________________________________ 109

4.3 Experimental methods ____________________________________________________________________________ 110

4.3.1 Study design ________________________________________________________________________________ 110

4.3.2 Dogs __________________________________________________________________________________________ 111

4.3.3 Diets __________________________________________________________________________________________ 111

4.3.4 Blood samples_______________________________________________________________________________ 113

4.3.5 Urine samples _______________________________________________________________________________ 114

4.3.6 Faeces samples _____________________________________________________________________________ 114

4.3.7 Chemical analyses _________________________________________________________________________ 114

4.3.8 Statistical analyses _________________________________________________________________________ 115

TTTTable of contentsable of contentsable of contentsable of contents

4.4 Results ______________________________________________________________________________________________ 117

4.5 Discussion __________________________________________________________________________________________ 117

4.6 Acknowledgements ______________________________________________________________________________ 121

4.7 References _________________________________________________________________________________________ 121

5. Biomarkers of selenium status in dogs _____________________________________________________ 125

5.1 Abstract ____________________________________________________________________________________________ 127

5.2 Introduction _______________________________________________________________________________________ 127

5.3 Experimental methods ____________________________________________________________________________ 129

5.3.1 Study design ________________________________________________________________________________ 129

5.3.2 Dogs __________________________________________________________________________________________ 129

5.3.3 Diets __________________________________________________________________________________________ 131

5.3.4 Blood samples_______________________________________________________________________________ 131

5.3.5 Urine samples _______________________________________________________________________________ 132

5.3.6 Hair growth measurements _______________________________________________________________ 132

5.3.7 Chemical analyses _________________________________________________________________________ 132

5.3.8 Statistical analyses _________________________________________________________________________ 137

5.4 Results ______________________________________________________________________________________________ 137

5.5 Discussion __________________________________________________________________________________________ 139

5.6 Conclusion _________________________________________________________________________________________ 145

5.7 Acknowledgements ______________________________________________________________________________ 145

5.8 References _________________________________________________________________________________________ 145

6. General discussion ______________________________________________________________________________ 149

6.1 Methods to measure selenium availability ___________________________________________________ 151

6.2 Factors affecting selenium availability ________________________________________________________ 152

6.2.1 Dietary selenium concentration _________________________________________________________ 152

6.2.2 Selenium speciation ________________________________________________________________________ 154

6.2.3 Processing conditions _____________________________________________________________________ 157

6.3 Bioavailability versus bioactivity _______________________________________________________________ 158

6.4 Factors affecting measurement of glutathione peroxidase activity _______________________ 159

6.4.1 Expressing glutathione peroxidase activity ___________________________________________ 160

6.4.2 Serum versus whole blood glutathione peroxidase activity _______________________ 161

6.5 Other factors to consider when assessing selenium requirements of dogs _____________ 163

6.5.1 Gender _______________________________________________________________________________________ 163

6.5.2 Age ___________________________________________________________________________________________ 164

Table of contentsTable of contentsTable of contentsTable of contents

6.5.3 Breed _________________________________________________________________________________________ 165

6.5.4 Metabolic changes associated with selenium status ________________________________ 166

6.6 Future perspectives ______________________________________________________________________________ 167

6.7 Conclusions ________________________________________________________________________________________ 168

6.8 References _________________________________________________________________________________________ 169

Supplement. Storage of whole blood for glutathione peroxidase activity

analysis ________________________________________________________________________________________________ 173

S.1 Abstract ____________________________________________________________________________________________ 175

S.2 Introduction ________________________________________________________________________________________ 175

S.3 Experimental methods ____________________________________________________________________________ 175

S.4 Results and discussion ___________________________________________________________________________ 176

S.5 References _________________________________________________________________________________________ 179

Summary ______________________________________________________________________________________________ 181

Samenvatting ________________________________________________________________________________________ 187

Curriculum vitae ____________________________________________________________________________________ 193

Bibliography _________________________________________________________________________________________ 197

Doctoral training programme ___________________________________________________________________ 201

Acknowledgements ________________________________________________________________________________ 205

List of figuresList of figuresList of figuresList of figures

List of figures

Figure 1.1 World map indicating areas with low, adequate and high

selenium concentrations in vegetation ____________________________________________________________ 26

Figure 1.2 Metabolic pathway of selenium ____________________________________________________ 29

Figure 1.3 Selenocysteine incorporation into selenoproteins _______________________________ 33

Figure 1.4 Activity of glutathione peroxidase _________________________________________________ 38

Figure 1.5 Activity of thioredoxin reductase ___________________________________________________ 38

Figure 1.6 The involvement of iodothyronine deiodinases in the deiodination

of thyroid hormones __________________________________________________________________________________ 40

Figure 1.7 Influence of selenium on components of the insulin

signalling cascade ____________________________________________________________________________________ 44

Figure 1.8 Fractionation of ingested selenium into bioaccessible,

-available and -active fractions_____________________________________________________________________ 50

Figure 3a.1 Correlations (r) between in vitro selenium accessibility (%) and

crude protein digestibility (%) of pet foods _______________________________________________________ 89


parameters of canned (black bars, n=16) and kibble (grey bars, n=19) diets in

g/100g DM, except where specified _______________________________________________________________ 90

Figure 3b.1 Predicited versus observed in vitro selenium accessibility in kibble

and pelleted diets (n=27) _____________________________________________________________________________ 103

Figure 3b.2 Predicited versus observed in vitro selenium accessibility in kibble

diets (n=19) _____________________________________________________________________________________________ 103


Figure 4.1 Selenium digestibility (A), serum selenium (B), serum isoprostanes (C)

and serum T3:T4 ratio (D) in dogs in relation to crude protein intake of four

canned and four kibble diets ______________________________________________________________________ 116

Figure 4.2 Whole blood glutathione peroxidase responses in dogs in relation

to crude protein intake of four canned and four kibble diets ________________________________ 118

Figure 4.3 Urinary selenium to creatinine ratio relative to selenium intake in

dogs in relation to crude protein intake of four canned and four kibble diets ___________ 119

Figure 5.1 Urinary selenium to creatinine ratio of dogs fed a low or

adequate selenium diet _____________________________________________________________________________ 138

Figure 5.2 Glutathione peroxidase activity (U/L) in whole blood of dogs

fed a low or adequate selenium diet _____________________________________________________________ 138

Figure 5.3 Cumulative hair growth (mm) of dogs fed a low or adequate

selenium diet __________________________________________________________________________________________ 139

Figure 6.1 In vitro and in vivo selenium availability and apparent dry matter

and crude protein digestibility coefficients ______________________________________________________ 152

Figure 6.2 Dietary selenium and apparent in vivo and corrected† in vitro selenium

availability coefficients of canned and kibble pet foods ______________________________________ 154

Figure 6.3 Supplemented selenite and selenium from ingredients in

kibble diets (n=18) _____________________________________________________________________________________ 156

Figure 6.4 Effect of selenite supplementation on apparent in vivo and

corrected† in vitro selenium availability coefficients in kibble diets (n=18) _________________ 156

Figure 6.5 Effect of digestible crude protein on in vitro selenium accessibility

and apparent in vivo selenium bioavailability ___________________________________________________ 158


Figure 6.6 Effect of dog age on serum selenium (A), whole blood glutathione

peroxidase (B), serum T3:T4 ratio (C) and urinary selenium:creatinine ratio (D) _____________ 165

Figure S.1 Mean differences in glutathione peroxidase activity (U/L) between

fresh and stored canine heparinised whole blood _____________________________________________ 178

List of tablesList of tablesList of tablesList of tables

List of tables

Table 1.1 Selenoproteins currently identified in humans and their validation

number in dogs _______________________________________________________________________________________ 34

Table 1.2 Typical ingredients of dog foods and their selenium concentration

in mg/kg dry matter* _________________________________________________________________________________ 48

Table 1.3 Dietary factors affecting selenium bioavailability and/or bioactivity __________ 51

Table 1.4 Current recommended selenium intakes for adult dogs by FEDIAF

and the NRC __________________________________________________________________________________________ 55

Table 3a.1 Chemical composition (g/100g DM, except where specified),

gross energy content (MJ/kg DM) and in vitro digestibility (%) of

pet foods (n=60) per diet type ______________________________________________________________________ 86

Table 3a.2 In vitro selenium accessibility of pet foods (%) per diet type _________________ 88

Table 3a.3 In vitro selenium accessibility (%) in processed versus

unprocessed canned and kibble diets ____________________________________________________________ 91

Table 3b.1 Chemical composition (g/100g DM, except where specified),

gross energy content (MJ/kg DM), in vitro digestibility and selenium accessibility

(%) of dry pet foods (n=27) ___________________________________________________________________________ 102

Table 4.1 Gender, age, body weight and energy intake of the study dogs

per dog group ________________________________________________________________________________________ 111

Table 4.2 Analysed chemical composition (g/MJ ME, except where specified),

dry matter (DM) and metabolisable energy concentration (ME) of four canned

and four kibble single batch dietsa with differing protein concentrations __________________ 112

Table 4.3 Energy, selenium and crude protein intakes per diet type of four

canned and four kibble diets ______________________________________________________________________ 115

List of List of List of List of tablestablestablestables

Table 5.1 Gender*, age, body weight and energy intake of the study

dogs per dog group _________________________________________________________________________________ 129

Table 5.2 Analysed* chemical composition of two semi-purified diets with an

adequate or low selenium concentration _________________________________________________________ 130

Table 5.3 Primer and probe sequences used for positive control and

qPCR assays ___________________________________________________________________________________________ 134

Table 5.4 Serum biomarker concentrations in dogs fed a diet with a low or

adequate selenium concentration _________________________________________________________________ 136

Table 5.5 Relative† mRNA expression in dogs fed a diet with a low or

adequate selenium concentration _________________________________________________________________ 140

Table 6.1 Effect of diet type on selenium intake and biomarkers __________________________ 162

Table 6.2 Effect of gender* on selenium parameters __________________________________________ 164

Table S.1 Mean glutathione peroxidase activity and coefficient of variation

of fresh and stored (-80°C) canine blood _________________________________________________________ 177

List of acronyms, abbreviations and symbolsList of acronyms, abbreviations and symbolsList of acronyms, abbreviations and symbolsList of acronyms, abbreviations and symbols

List of acronyms, abbreviations and symbols

6-PGDL 6-phospho-glucono-δ-lactone

AAFCO association of American feed control officials

AI adequate intake

Akt protein kinase B

Ala alanine

Arg arginine

B2M beta-2-microglobulin

BCS body condition score

BW body weight

BW0.75 metabolic body weight

C Celsius

Ca2+ calcium

cDNA complementary deoxyribonucleic acid

CK creatine kinase

CP crude protein

Ct threshold value

CT creatinine

Cu copper

CV coefficient of variation

Cys cysteine

DIO iodothyronine deiodinase

DM dry matter

DMSe dimethylselenide

DNA deoxyribonucleic acid

EDTA ethylenediaminetetraacetic acid

EFSec selenocysteine-specific translation elongation factor

e.g. exempli gratia, for example

eIF4a3 eukaryotic translation initiation factor 4a3

ELISA enzyme-linked immunosorbent assay

et al. et alia, and others

FEDIAF European pet food industry federation

FoxO1a forkhead box class O 1a


g gram

G6-P glucose 6-phosphate

G6PD glucose 6-phosphate dehydrogenase

GAPDH glyceraldehyde 3-phophate dehydrogenase

Glut4 glucose transporter 4

GPx glutathione peroxidase

GR glutathione reductase

GS glutathione-sulphur conjugation

GSH glutathione

H hydrogen

H2O water

H2O2 hydrogen peroxide

HPRT1 hypoxanthine phosphoribosyltransferase 1

ID identification

i.e. id est, in other words

IR insulin receptor

IRS insulin receptor substrate

IsoPs isoprostanes

ISO international organization for standardization

IU international unit

kcal kilo calorie

kDa kilodalton

kg kilogram

L30 ribosomal protein for selenocysteine incorporation

LC-HR-MS liquid chromatography-high resolution mass spectrometry

Leu leucine

Lys lysine

ME metabolisable energy

Met methionine

mg milligram

min minutes

MJ mega joule

ml millilitre

mRNA messenger ribonucleic acid

MSe methylselenol


N nitrogen

n number

n.a. not applicable

NADP+ nicotinamide adenine dinucleotide phosphate

NADPH reduced nicotinamide adenine dinucleotide phosphate

NEFA non-esterified fatty acids

NFκB nuclear factor kappa-light-chain-enhancer of activated B cells

ng nanogram

nm nanometer

Nox4 NADPH oxidase 4

nr number

NRC national research council

O2 dioxygen

O2·- superoxide

OM organic matter

oxLDLs oxidized low-density lipoproteins

P phosphorus

PGC-1a proliferator-activated receptor gamma coactivator 1a

Phe phenylalanine

PI3K phosphoinositide-3-kinase

PIP2 phosphatidylinositol diphosphate

PIP3 phosphatidylinositol triphosphate

ppm parts per million

pred. predicted

PTEN phosphatase and tensin homolog

PTP-1B protein tyrosine phosphatase 1B

qPCR quantitative polymerase chain reaction

RA recommended allowance

ROH alcohol

ROOH hydroperoxide

RT reverse transcription

rT3 reversed triiodothyronine

SAH S-adenosyl homocysteine

SAM S-adenosyl methionine

SBP2 selenocysteine insertion sequence (SECIS)-binding protein


SD standard deviation

SE standard error

Se selenium

SECIS selenocysteine insertion sequence

SeCys selenocysteine

Sel selenoprotein (H, I, K, M, N, O, R, S, T, V)

SeMet selenomethionine

SEM standard error of the mean

Sep15 15 kilodalton selenoprotein

SepP selenoprotein P

SepW1 selenoprotein W

Ser serine

SHAPE size, health and physical evaluation

SPS2 selenophosphate synthetase 2

T2 diiodithyronine

T3 triiodothyronine

T4 thyroxine

TDF total dietary fibre

TiO2 titanium dioxide

TMSe trimethylselenonium

TNF-α tumor necrosis factor alpha

tRNA transfer ribonucleic acid

TrxR thioredoxin reductase

U unit

UGA uracil, guanine, adenine, genetic (stop) codon, coding for selenocysteine

UK United Kingdom

USA United States of America

< less than

> more than

% percent

° degrees

g gravitational force

µg microgram

µl microlitre


α alpha

β beta

γ gamma

δ delta

κ kappa

ᴅ dexter, spatial configuration of an isomer

ᴸ laevus, spatial configuration of an isomer

® registered

™ trademark

_____________________________________________________________________________________________________________

General introduction_____________________________________________________________________________________________________________

Chapter 1

General introduction _____________________________________________________________________________________________________________

Chapter 1

General introductionGeneral introductionGeneral introductionGeneral introduction

25

Selenium (Se) is a non-metal, or less commonly considered a metalloid with both metallic and

nonmetallic properties. It has atomic number 34, which lies between sulphur (S) and tellurium

(Te) in group VI of the periodic table. The atomic weight of Se is 78.96 and it has six

naturally occurring stable isotopes; 74Se (natural abundance 0.82 %), 76Se (natural

abundance 8.66 %), 77Se (natural abundance 7.31 %), 78Se (natural abundance 23.21 %), 80Se

(natural abundance 50.65 %), and 82Se (natural abundance 8.35 %)(1).

1.1 History of selenium

Selenium was discovered in 1817 in Uppsala by the Swedish chemist Jöns Jacob Berzelius

(1779 - 1848), in collaboration with the chemists J.G. Gahn and M.H. Klaproth. They were

examining a reddish deposit that remained in the lead chambers after roasting copper

pyrites. This was first thought to be tellurium, which was recently discovered by Klaproth as

a residue of copper ore roasting. When Berzelius examined the material, he found no trace

of tellurium, but instead, he found an unknown substance with properties very much like

tellurium. Since tellurium has been named after the Latin word tellus (earth), Berzelius

decided to name the new chemical after the Greek goddess of the moon, selênê(2-3). In hind

sight, Marco Polo may already have come across the less desirable qualities of selenium on

his travels in western China around 1295. He documented the hoofs of animals to drop off

after eating poisonous plants. This was later called "alkali disease" and even much later

found to be caused by Se accumulation in plants due to high Se levels in the soil. Until the

late 1950's Se was merely known as a toxicant, causing diseases, like alkali disease, in farm

animals(4). It was not until 1957 that Se was first recognised as an essential nutrient, when it

was found to spare the anti-oxidant action of vitamin E in the diets of rats and chicks(5-7).

Since then, more research was conducted on this trace mineral and more beneficial

properties were discovered.

1.2 Geological distribution of selenium

Quantitatively, Se is the 70th of the 88 elements that naturally occur in the earth's crust(8).

However, geologically it is very unevenly distributed. Research on soil Se content focuses

on specific parts of the world where either Se deficiencies or toxicities in farm animals have

been reported, and therefore, is rather limited.

Selenium in soils is mainly present as the water-soluble selenate in well-drained alkaline

soils, which is a highly available chemical form for plant roots, and thus can lead to toxic

amounts of Se in vegetation(9). Several plants can accumulate Se, such as plants from the

Brassica (e.g. rapeseed), Astragalus (e.g. milk-vetch root) and Allium (e.g. onion) genera(10-12),

26 Chapter 1


but Brazil nuts are the richests known food source of Se(13). In some parts of the world, the

soil contains high levels of Se, but the water-soluble Se, and thus the risk of toxic quantities

in vegetation, is low(9). These soils are often acidic and poorly drained and contain Se in the

form of selenides and elemental Se, which are unavailable to plant roots(9). This indicates

that absolute soil Se levels do not necessarily reflect a level of toxicity to animals.

Vegetation containing more than 0.1 mg Se/kg prevents livestock from Se deficiency

disorders(14), while dietary levels above 1 mg Se/kg may lead to toxicity(15-16). Figure 1.1

shows an overview of seleniferous and Se deficient areas in the world based on

documented Se deficiencies and toxicities and on Se levels in the vegetation. Low Se areas

(yellow) are defined as areas where the vegetation has a concentration of 0.1 mg Se/kg

vegetation or less, or where deficiency diseases were reported. Seleniferous areas (red) are

those where vegetation contains 1 mg Se/kg vegetation or more, or where signs of Se

toxicity have been reported. Serum Se concentrations of inhabitants were not taken into

consideration, as it was not clear if their diet was solely from their own region.

Figure 1.1 World map indicating areas with low, adequate and high selenium

concentrations in vegetation

Yellow represents low selenium areas, ≤ 0.1 mg Se/kg vegetation or reported deficiencies; green

adequate, 0.1-1 mg Se/kg vegetation; red seleniferous, ≥ 1 mg Se/kg vegetation or reported toxicities.

For white areas, no information was available. Based on Fleming(17), Rosenfeld & Beath(4), NRC(9), Combs

& Combs(18-19), Gissel-Nielsen(20), Fordyce et al.(21), Murphy & Cashman(22), Oldfield(23), Dhillon & Dhillon(24),

Surai & Taylor-Pickard(25), Garcia Moreno et al.(26), and De Temmerman et al.(27).

Chapter 1


27

In general, plants with a deficient Se concentration for animals are produced in large parts

of Canada(28), Scandinavia(23; 29-31), United Kingdom(23; 32-33), Italy(34), Greece(35-36), Serbia(37),

Russia(25), China(19; 38-39), Sri Lanka(21), Australia(40) and New Zealand(23; 41). The Se concentration

in plants in large areas of Africa are not mapped, but are likely to be Se-deficient as well(42-

45). American plant derived products are generally considered to contain adequate or even

toxic amounts of Se(23; 46). There are only a few seleniferous areas in the world, but this can

have severe implications to the health of livestock, but also on humans and pets consuming

their meat. Beside parts of America, other seleniferous areas are the Limerick, Tipperary and

Meath counties in Ireland(4; 17), Enshi county in China(47), Punjab area in India(48) and the

northern part of Queensland(23). This shows that it is important to know from which geological

area pet food ingredients originate to get an understanding of its Se concentration.

1.3 Selenium metabolism in monogastric animals

Unlike plants, Se is an essential trace mineral for all mammals(49). There are quite some

discrepancies in literature about the metabolism of Se. The majority of studies on Se

metabolism have been performed in humans and rats. In this paragraph an overview is

given of the current knowledge, with the assumption that Se metabolism is similar for all

monogastric animals. Although there are many different Se species present in foods, focus is

placed on the organic Se species selenomethionine and selenocysteine and the inorganic

species selenite and selenate. The metabolic pathway is schematically displayed in Figure

1.2.

1.3.1 Absorption

Selenoaminoacids are released from the protein during protein digestion. The main

absorption site of Selenomethionine (SeMet) and selenite is the duodenum(50), while that of

selenate is the ileum, as investigated in rats(51-52). SeMet is absorbed transcellularly through

active transport by a sodium-dependent transporter, as depicted in Figure 1.2. This energy

requiring transport is the common absorption mechanism for neutral amino acids, and

therefore, methionine (Met) and SeMet compete for absorption(53). Wolffram et al.(53) also

showed an important inhibition of SeMet uptake in pig brush border membranes by

addition of cysteine (Cys), leucine (Leu), phenylalanine (Phe), alanine (Ala) and serine (Ser).

Selenocysteine (SeCys) is similarly absorbed (Figure 1.2), but then by the sodium-dependent

transport mechanism of basic amino acids and it competes for absorption with Cys, lysine

(Lys) and arginine (Arg)(53).

28 Chapter 1


Selenate is also actively absorbed by a transcellular co-transport pathway with sodium-ions

in pig brush border membranes(54-55) and competes for absorption with sulphur compounds(55).

It has been proven to compete with sulphate and thiosulphate in pig brush border

membranes(54). The same research group found that selenate can also be absorbed by

active anion exchange(55), as shown in Figure 1.2.

Selenite is absorbed passively, by paracellular diffusion, as studied in rats and humans(51; 56).

However, there are indications that a large part of selenite is bound to the brush border

membranes instead of being absorbed into the circulation(54). Selenite can also form a

covalent adduct with glutathione (GSH) in the intestine (Figure 1.2) to form

selenodiglutathione, which seems to be more readily absorbed in rats and human caco-2

cells than selenite(51; 57).

Absorption of SeMet in the human intestinal tract is also better than of selenite(58). SeMet is

reported to be the most efficiently absorbed Se species at 97% in humans and 83-97% in

rats(59). SeCys has an apparent absorption of 81% in rats(60). The level of absorption of

selenate is similar to that of SeMet. It has been determined that the absorption rate of

selenate in humans is approximately 91% and that of selenite only 50%(61) of the total

intake. However, selenite seems to be better absorbed in rats(59) and dogs(62), with absorption

levels of 75-93% and 90%, respectively. It is unlikely that this difference is caused by

differences in Se status between the subjects used in the different studies, as it is known

that Se absorption is not homeostatically mediated(51; 63). However, there are several other

factors that may underlie this difference, one of them being the amount of GSH present in

the gut to be able to transform selenite to selenodigluthatione. Another possibility is that the

bioavailability of the dietary Se differed between the studies. Factors affecting Se

bioavailability, such as diet composition, will be discussed in paragraph 1.7.

1.3.2 Distribution

After absorption, SeMet can potentially be non-specifically incorporated into body proteins

in the place of Met, as shown in Figure 1.2. The genetic code and tRNA do not discriminate

between the two(64). Non-specific incorporation of SeCys also occurs, as found in rats, but

this is believed to be an unimportant metabolic pathway(65). Skeletal muscle is the major site

of SeMet storage in the human body (28-46% of the total Se pool)(66-67). However, kidneys

contain the highest amount of Se on a per-weight basis; the average human kidney Se

concentration is 470 ng/g as is, compared to 51 ng/g as is in skeletal muscle(66). This finding

was verified in dogs(68-70). Kidney Se concentrations may be high because this is the primary

organ of urinary excretion.

Figure 1.2 Metabolic pathway of selenium

Na+, sodium; A-, anion; GSH, glutathione;

tRNA, transfer ribonucleic acid; GS, gluta

1. Glutathione reductase, 2. Thioredoxin reductase, 3

Methyltransferase, 7. Selenophosphate synthetase,

kinase, 10. Selenocysteine synthase. After passing the intestinal barrier, the majority of the processes

take place in the liver, except for transformation from sel

selenomethionine incorporation into body prote

and all other cells, respectively. The g

brain and testes. Based on Levander & Bauman

& Pannier(73), Suzuki(74), Lu et al.(75)


Metabolic pathway of selenium

GSH, glutathione; SAM, S-adenosylmethionine; SAH, S-adenosylhomocysteine;

ic acid; GS, glutathione-sulphur conjugation. Numbers represent the enzymes;

. Thioredoxin reductase, 3. β-lyase, 4. γ-lyase,

nophosphate synthetase, 8. Seryl-tRNA synthetase, 9

. Selenocysteine synthase. After passing the intestinal barrier, the majority of the processes

take place in the liver, except for transformation from selenite to selenodiglutathione and

selenomethionine incorporation into body proteins, which can also take place in the erythrocytes

s, respectively. The grey area represents selenoprotein formation

Based on Levander & Bauman(71), Wolffram et al.(53; 55), Gyurasics

(75), Hoefig et al.(76), Thiry et al.(77), Roman et al.(78), and

Chapter 1


29

adenosylhomocysteine;

represent the enzymes;

, 5. Demethylase, 6.

9. Phosphoseryl-tRNA

. Selenocysteine synthase. After passing the intestinal barrier, the majority of the processes

enite to selenodiglutathione and

ich can also take place in the erythrocytes

resents selenoprotein formation in liver, kidney,

, Gyurasics et al.(72), Francesconi

and Jäger et al.(79).

30 Chapter 1


The average residence time of Se in the tissue pool is 115-285 days as measured by the

stable isotope 74Se in humans(80). However, results may be taken with caution, as the Se

status of the subject and the Se species ingested may have had an influence on retention.

Selenium turn-over is also organ specific, with a much longer retention in muscle than in

kidney, as investigated in rats(81). In addition, there may be a gender difference in the tissue

retention of Se. Se deprived female rats showed a higher tissue Se retention than males(82).

In humans this gender difference is also recognized and is believed to be mainly caused

by the increased demand of Se from the male reproductive tract(83). Studies in dogs,

although numerically higher in males than females (neuter status not reported), did not show

significant differences in tissue Se concentrations based on gender(68; 70). Age did seem to

have an impact on liver and kidney Se concentrations. They were reported to be higher in

middle aged dogs (4 - 10 years) compared to young (<4 years) and older dogs (>10

years)(68). However, dietary nutrient intake of the dogs was unknown.

After degradation of regenerating proteins (so not those of hair and nails), SeMet can again

be used in Se metabolism and be transselenated to SeCys (in a similar manner to the

transsulphuration pathway)(64), which in turn is transformed into hydrogen selenide via

reductive cleavage of the C-Se bond by β-lyase, as studied in rats(84). This has been

depicted in Figure 1.2. Hydrogen selenide is used for the incorporation of Se into

selenoproteins (discussed in paragraph 1.4), but can also be transformed to methylselenol by

a S-adenosylmethionine (SAM)-mediated methylation(85) or be converted to selenosugars(74).

Another possible route for SeMet is to be directly metabolised into methylselenol by γ-

lyase(74; 86). However, this is demonstrated in mice and rats to only occur with high Se

intakes(84; 86). Methylselenol can subsequently be demethylated into hydrogen selenide by

demethylase(84; 87-88) (Figure 1.2).

Uptake of selenite by erythrocytes is very rapid. An in vitro study using human erythrocytes

showed an uptake of 50-70% of the administered selenite within 1 minute(89). Selenite is

taken up via a specific anion exchange carrier, band 3 protein(90-91). Thereafter, it is reduced

to hydrogen selenide(74; 92), either via the thioredoxin system by thioredoxin reductase(93) or

after the formation of a covalent adduct with GSH to selenodiglutathione, which can be

reduced to hydrogen selenide by glutathione reductase(78; 94). Studies in humans show that

the hydrogen selenide is effluxed into the blood stream and immediately bound to

albumin(95-96) or hemoglobin(91) to be transported to the liver.

Selenate, on the other hand, is believed to be directly taken up by hepatocytes from the

blood stream, as studied in rats(97). In the liver, it can be reduced to selenite(98) and

subsequently metabolised to hydrogen selenide, or, as demonstrated in rat and human

Chapter 1


31

studies and shown in Figure 1.2, it is excreted in the urine(79; 97). Studies in mice have

demonstrated that the liver synthesizes most of the selenoproteins(99-100). Selenoprotein P

(SepP) is one of those proteins. SepP is an important transporter of Se to the testes, brain and

kidneys, where other selenoproteins can be synthesized(100-101). The liver also regulates Se

excretion(99-100).

1.3.3 Excretion

The main route of Se excretion is via the urine(73; 102). Urinary Se excretion is dependent on

the amount of dietary Se intake in rats(103-104). The majority of human urinary Se exists of

selenosugars(73-74), which are formed from hydrogen selenide, via an intermediate

glutathione-sulphur conjugated selenosugar(105), as depicted in Figure 1.2. Three different

selenosugars have been identified in human and rat urine; methyl 2-acetamido-2-deoxy-1-

seleno-β-ᴅ-glucopyranoside (selenosugar 1), methyl 2-acetamido-2-deoxy-1-seleno- β-ᴅ-

galactopyranoside (selenosugar 2), and methyl 2-amino-2-deoxy-1-seleno- β-ᴅ-

galactopyranoside (selenosugar 3)(73). Selenosugars are detected in both, urine from rats fed

with selenite(105-106) and SeMet(107), and in humans supplemented with selenized yeast(108-110),

which suggests that urinary selenosugar excretion is not specific to a certain dietary Se

species.

Selenosugar 2 has been reported to provide the highest Se concentration in human urine(109;

111). However, the studies reporting this have used supra-nutritional (3.6 to 36.4x the

European recommended intake for humans, which is 55 µg/day) levels of Se. It is known

from a study in humans that selenosugar 2 can be deacylated to selenosugar 3(112) and that

urinary excretion of selenosugar 3 is not increased by Se supplementation(108). This suggest

that selenosugar 3 is the main selenosugar in Se adequacy and that excess Se is excreted

as selenosugar 2. Gammelgaard & Bendahl(109) reported that selenosugar 1 only consisted of

2% of the concentration of selenosugar 2 in the urine of human subjects supplemented with

selenized yeast (1000 or 2000 µg).

Selenium can also be excreted in the urine as the monomethylated methylselenol (MSe)(104)

and trimethylselenonium (TMSe) (Figure 1.2), as found in rats(74; 104). In case of excessive Se

intake, the excretion of TMSe in the rat urine increases(113). However, in some cases urinary

TMSe is even excreted with adequate Se intake. It has recently been reported in humans

that TMSe excretion seems to be affected by genetic variation(111) and that, in the German

human population, 18% eliminates TMSe in Se adequacy(114).

A small fraction of selenite(73) and selenate(79; 97; 115) from the diet may not even be

metabolised and is directly excreted in the urine (Figure 1.2). A study in which 40 µg of

32 Chapter 1


labeled 75Se was administered to humans per day, showed that selenate is more quickly

excreted in the urine than selenite (90% of total after 40 and 121 hours, respectively)(61).

In rats administered with labeled 75Se as SeCys or SeMet (max. 5 µg), the urinary excretion

after one week was more than twice as high in SeCys (13.9% of administered Se) than

SeMet administered rats (5.8% of administered Se), while the intestinal absorption was similar

for both Se species (81 and 86%, respectively)(60). This is in accordance with findings of

Nahapetian et al.(116), who reported that urinary Se excretion after 48 hours was similar in

rats administered with a single dose of 16 µg labeled 75Se/kg BW as selenite or SeCys

(approximately 45%), but was significantly lower when SeMet was administered in the same

amount (approximately 30%). Interestingly, the percentage of urinary Se excretion at high

intakes of selenite (1500 µg Se/kg BW) decreased to the percentage excreted with SeMet

administration(116). This is probably caused by an alternative route of excretion, possibly

respiratory.

Studies in rats and humans showed that, in subtoxic Se status, a small amount of Se may be

eliminated via exhaled breath(117-119). The primary form of respiratory excretion is

dimethylselenide (DMSe)(117-118), as shown in Figure 1.2. Se expiration is higher in subjects fed

selenite than SeMet(119). However, the exhalation via breath is generally negligible(58; 103) and

was found not to be dose-dependent in adequate levels in rats(103). With excess dietary Se

intakes, on the other hand, DMSe excretion increases in humans(117). DMSe is responsible for

the characteristic garlic-like smell in Se intoxication(84; 119).

A few percent of the dietary Se is also excreted via the bile, as demonstrated in rats(71) and

dogs(120). Enterohepatic recycling occurs, where the Se that is excreted via the bile can in

turn be absorbed in the intestine (Figure 1.2). Selenodiglutathione seems to be the major Se

metabolite in bile of rats when selenite is the source of dietary Se(72). It is not known in what

form Se is excreted when other Se species are in the diet. There are indications that it may

be bound to the erythrocyte catabolism product, bilirubin(120-121). Organically-bound Se may

also be converted to selenotaurine, in the same manner as sulphur-containing Cys would be

converted(122), and excreted in the bile as taurine conjugate. Over 99% of the bile acids of

dogs are taurine conjugated, like taurocholic acid, taurodeoxycholic acid, and

taurochenodeoxycholic acid(123). No information is available on the amount of biliary Se

excretion from the organically-bound Se species. However, selenite and selenate are

excreted in similar amounts in the bile of steers when administered in the same quantity(124).

Selenite is proven in rats and steers to be excreted in the bile in a dose-dependent

manner(72; 124), but again, for the other Se species this is unknown.

Chapter 1


33

1.4 Selenoproteins

The existence of between 30 and 50 selenoproteins have been estimated(125) based on

electrophoretic separation studies like that of Behne et al.(126). So far, 25 have been

identified in humans(3; 78; 127-128), and 21 of them have been validated in dogs(129) (Table 1.1).

However, studies into the selenoproteome identification of dogs are limited. In this

paragraph, an overview will be given of the selenoproteins and some will be briefly

described.

All selenoproteins contain SeCys as their active centre(130). Interestingly, SeCys from the diet

cannot immediately be incorporated, but similar to other dietary Se species, are first

converted to hydrogen selenide(94; 131), as indicated in figure 1.2. Consequently, hydrogen

selenide is transformed into selenophosphate by selenophosphate synthetase 2 (SPS2) and

combined with phosphoseryl-tRNA to form selenocysteine-tRNA. The latter reaction is

mediated by selenocysteine synthase(132). Figure 1.3 shows an overview of the factors

involved in selenoprotein formation. When the selenoproteins are catabolised, the released

SeCys is recycled and again transformed to hydrogen selenide(78).

Figure 1.3 Selenocysteine incorporation into selenoproteins

Factors that are currently known to be required for selenocysteine

incorporation are; eukaryotic initiation factor eIF4a3, nucleolin,

selenocysteine insertion sequence (SECIS), SECIS binding protein 2

(SBP2), SeCys-specific translation elongation factor (EFsec), and

ribosomal protein L30. From Labunskyy et al.(133).

Figure 1.3 shows an overview of the factors involved in selenoprotein formation. SeCys is

inserted into the peptide chain encoded by a specific UGA codon, which is genetically

encoded in the ribosome-mediated system and is recognized as the 21st amino acid(78). UGA

is generally a stop codon, but a conserved stem-loop structure, called the SeCys insertion

34 Chapter 1


Ta

ble

1.1

Se

lenopro

tein

s cu

rrently identified in h

um

ans

and their v

alid

ation n

um

ber in

dogs

Ge

nB

an

k I

D

nr

for

do

gs

NM

_001115119

NM

_001115135

NM

_001164454

- NM

_001256320

NM

_001007126

NM

_001122645

NM

_001164188

NM

_001122673

Pre

d. transc

ript

variants:

XM

_005636584,

XM

_005636585,

XM

_005636586,

XM

_845088

NM

_001122778

Tis

sue

dis

trib

uti

on

Ubiq

uitous, c

yto

sol

Live

r, e

pithelium

, gastro

inte

stin

al

tract

Pla

sma

Cell

mem

bra

ne

Olfacto

ry e

pithelium

, em

bry

o's

Live

r, k

idney, th

yro

id,

pituitary

gla

nd, ova

ry

Thyro

id, heart, bra

in, sp

inal cord

,

skele

tal m

usc

le, pla

centa

, kid

ney,

pancre

as, b

row

n a

dip

ose

tissu

e

Pla

centa

, fe

tus, b

rain

, sk

in

Ubiq

uitous

Ubiq

uitous

Testes

Fu

ncti

on

Antioxi

dant pro

tection

Antioxi

dant pro

tection

Main

tenance o

f cellu

lar re

dox

statu

s

Lipid

hydro

pero

xide d

eto

xification

Antioxi

dant pro

tection

Conve

rsio

n o

f T4 to T

3

Conve

rsio

n o

f T4 to T

3

Conve

rsio

n o

f T4 to reve

rse T

3

NA

DPH-d

ependent re

duction o

f

oxi

dized thio

redoxi

n

NA

DPH-d

ependent re

duction o

f

oxi

dized thio

redoxi

n

NA

DPH-d

ependent re

duction o

f

oxi

dized thio

redoxi

n

Ab

bre

via

tio

n

GPx1

GPx2

GPx3

GPx4

GPx6

DIO

1

DIO

2

DIO

3

Trx

R1

Trx

R2

Trx

R3

Se

len

op

rote

in

Glu

tath

ione p

ero

xidase

1

Glu

tath

ione p

ero

xidase

2

Glu

tath

ione p

ero

xidase

3

Glu

tath

ione p

ero

xidase

4

Glu

tath

ione p

ero

xidase

6

Iodoth

yro

nin

e d

eio

din

ase

1

Iodoth

yro

nin

e d

eio

din

ase

2

Iodoth

yro

nin

e d

eio

din

ase

3

Thio

redoxi

n reducta

se 1

Thio

redoxi

n reducta

se 2

Thio

redoxi

n reducta

se 3

Chapter 1


35

Ge

nB

an

k I

D

nr

for

do

gs

NM

_001114736

NM

_001114760

NM

_001164506

NM

_001164486

NM

_001114878

NM

_001115014

- NM

_001137603

NM

_001115118

NM

_001114749

(SelX

)

NM

_001114757

Tis

sue

dis

trib

uti

on

Live

r

Pro

state

, th

yro

id, lu

ng, bra

in,

kid

ney, H9 T

cells

Various, m

ain

ly in e

mbry

o's a

nd

tum

or cells

Various, m

ain

ly in b

rain

Various, m

ain

ly in h

eart

Various, m

ain

ly in b

rain

, kid

ney

and lung

Ubiq

uitous, e

ndopla

smic

reticulu

m

Ubiq

uitous

Pla

sma, live

r, h

eart, bra

in, kid

ney

Heart, live

r, s

kele

tal m

usc

le, kid

ney

Ubiq

uitous, p

lasm

a, endopla

smic

reticulu

m

Fu

ncti

on

Form

ation o

f se

lenophosfate

fro

m

hydro

gen s

ele

nid

e

Thio

redoxi

n-lik

e, ro

le in u

nfo

lded

pro

tein

resp

onse

DN

A b

indin

g p

rote

in, re

gula

tion o

f

glu

tath

ione s

ynth

esis

genes

Unknow

n

Poss

ible

antioxi

dant pro

tection in

card

iom

yocyte

s

Pro

tein

fold

ing in the e

ndopla

smic

reticulu

m, antioxi

dant pro

tection

Unknow

n, m

ay b

e im

portant in

musc

le

and d

eve

lopm

ent

Unknow

n

Se h

om

eostasis

and tra

nsp

ort o

f

Se to tissu

es, a

ntioxi

dant pro

tection

Reduction o

f oxi

dized m

eth

ionin

e

residues

in d

am

aged p

rote

ins,

antioxi

dant pro

tection

Regula

tion o

f in

flam

ato

ry c

yto

kin

es

(inte

rleukin

1β a

nd 6

, and tum

or

necro

sis

facto

r α),

rem

ova

l of

misfo

lded p

rote

ins

from

the

endopla

smatic reticulu

m

Ab

bre

via

tio

n

SPS2

Sep15

SelH

SelI

SelK

SelM

SelN

SelO

SepP

SelR

SelS

Ta

ble

1.1

(c

ontinued)

Se

len

op

rote

in

Sele

nophosp

hate

synth

eta

se 2

Sele

nopro

tein

15

Sele

nopro

tein

H

Sele

nopro

tein

I

Sele

nopro

tein

K

Sele

nopro

tein

M

Sele

nopro

tein

N

Sele

nopro

tein

O

Sele

nopro

tein

P

Sele

nopro

tein

R

Sele

nopro

tein

S

36 Chapter 1


Ge

nB

an

k I

D

nr

for

do

gs

NM

_001164487

- NM

_001115012

ID, id

entification; nr., num

ber; T

3, triiodoth

yro

nin

e; T4, th

yro

xine; N

ADPH, re

duced n

icotinam

ide a

denin

e d

inucle

otide p

hosp

hate

; pre

d.,

pre

dic

ted; DN

A, deoxy

ribonucle

ic a

cid

; Se, se

leniu

m, Ca

2+ ,

calc

ium

. A

dapte

d fro

m P

appas

et al.(1

27) , Fairw

eath

er-Tait e

t al.(1

28) , Tanguy e

t

al.(1

34) , Rom

an e

t al.(7

8) , El-Ram

ady e

t al.(3

) , G

enBank

(129) .

Tis

sue

dis

trib

uti

on

Ubiq

uitous, m

ain

ly e

ndopla

smic

reticulu

m

Testes

Various, m

ain

ly m

usc

le

Fu

ncti

on

Unknow

n, poss

ibly

intracellu

lar

Ca

2+ h

om

eostasis

and a

ntioxi

dant

pro

tection

Unknow

n, poss

ible

role

in redox

regula

tion

Invo

lved in s

kele

tal and c

ard

iac

musc

le m

eta

bolism

, antioxi

dant

pro

tection

Ab

bre

via

tio

n

SelT

SelV

SepW

1

Table

1.1

(continued)

Se

len

op

rote

in

Sele

nopro

tein

T

Sele

nopro

tein

V

Sele

nopro

tein

W

Chapter 1


37

sequence (SECIS), can distinguish between the UGA for amino acid incorporation and the

UGA for termination(135). In addition to SECIS, there are five other factors required for

efficient recoding of UGA as SeCys. SECIS binding protein 2 (SBP2) binds the SECIS element

with high affinity and specificity and interacts with the SeCys-specific translation elongation

factor (EFSec), which recruits SeCys-tRNA and facilitates its incorporation into proteins(133).

Ribosomal protein L30 is also involved in SeCys incorporation, while eukaryotic translation

initiation factor 4a3 (eIF4a3) and nucleolin regulate selenoprotein synthesis and are

involved in deciding the hierarchy of selenoprotein expression(133). However, the underlying

mechanism for selenoprotein hierarchy is not yet fully understood(136). When the

selenoproteins are catabolised, the released SeCys is recycled and again transformed to

hydrogen selenide(78).

1.4.1 Glutathione peroxidase

The main function of GPx is to reduce hydrogen peroxide (H2O2) and non-esterified fatty

acid hydroperoxides(137), thereby preventing the formation of free radicals(138). Free radicals

may cause oxidative damage, and destruction of cell membranes and deoxyribonucleic acid

(DNA)(139). GPx uses GSH as a reducing agent to form two water molecules and glutathione

disulfide (GSSG)(140). The nicotinamide adenine dinucleotide phosphate (NADPH) and

hydrogen (H+) generated by the pentose phosphate cycle is consequently used to reduce

the glutathione disulfide back to two GSH molecules, catalysed by glutathione reductase

(GR)(140) (Figure 1.4). Vitamin E (α-tocopherol) and Se can partially spare each other(138). Vitamin

E is located in cell membranes and scavenges free radicals that may attack the

membrane(138). Thus, both Se and vitamin E serve the same purpose, namely the prevention of

lipid peroxidation, but act at different points in the sequence.

There are three homologs of the GPx family which are not selenoproteins (GPx5, 7 and 8).

They contain Cys in their active site instead of SeCys(133). Also, GPx6 in mice and rats

contains Cys instead of SeCys in the active centre(141). In dogs, GPx6 does contain SeCys(129)

(Table 1.1). The non-Se-dependent GPx homologs also act as anti-oxidants, but show very

little affinity for H2O2(142) and thus function slightly different compared to the Se-dependent

GPx's. The various Se-dependent isoforms of GPx vary in their protein structure, but have

overlapping substrate specificity and tissue distibution(133).

GPx1 is the most abundant isoform in mammals(133; 143). It is an intracellular enzyme and its

only known function is the reduction of H2O2(144). GPx1 knockout mice are characterized by

a higher susceptibility to oxidative stress(145). Overexpression of GPx1, on the other hand,

38 Chapter 1


Figure 1.4 Activity of glutathione peroxidase

ROOH, hydroperoxide; H2O2, hydrogen peroxide; ROH, alcohol;

H2O, water; GPx, glutathione peroxidase; GSH, glutathione; GSSG,

glutathione disufide; GR, glutathione reductase; NADP+,

nicotinamide adenine dinucleotide phosphate; NADPH, reduced

nicotinamide adenine dinucleotide phosphate; H+, hydrogen; G6-P,

glucose 6-phosphate; G6PD, glucose 6-phosphate dehydrogenase;

6-PGDL, 6-phospho-glucono-δ-lactone. Adapted from Lehninger et

al.(140).

Figure 1.5 Activity of thioredoxin reductase

TrxR, thioredoxin reductase; NADP+, nicotinamide adenine

dinucleotide phosphate; NADPH, reduced nicotinamide adenine

dinucleotide phosphate; H+, hydrogen; G6-P, glucose 6-phosphate;

G6PD, glucose 6-phosphate dehydrogenase; 6-PGDL, 6-phospho-

glucono-δ-lactone. Adapted from Roman et al.(78).

Chapter 1


39

may disrupt H2O2 signalling(133). It has a low position in the hierarchy of selenoprotein

expression(133), and thus is highly regulated by Se availability(146).

GPx1 and 4 are expressed in all cells(143), while GPx2 is primarily found in the epithelium of

the gastrointestinal tract(133) and GPx3 is primarily synthesized in the kidneys(147) and

secreted into the blood stream(143). As GPx3 is the major isoform of GPx in plasma, it is often

referred to as plasma GPx. Plasma GPx is well correlated with GPx tissue levels(148) and with

Se intake at low dietary concentrations(148). GPx4 reduces complex phospholipid

hydroperoxides. In studies with mice, GPx4 is proven to be essential for embryonic

development(149) and is associated with disulfide bond formation during spermiogenesis(150).

GPx6 is closely related to GPx3(78), but can merely be found during embryonic development

and in the olfactory epithelium(141). It is assumed to also act as antioxidant, but its specific

function is unknown(78).

1.4.2 Thioredoxin reductase

Together with the GSH system, the thioredoxins are the major systems of cellular redox

homeostasis(143). In mammals, three isoforms of TrxR are identified and all contain SeCys(75), as

listed in Table 1.1. However, not much is known about the individual functions of these

isoforms. TrxR1 and 2 are found to be essential for mammalian embryonic development(143).

TrxR3 contains an additional glutaredoxin domain, and is therefore also called thioredoxin

glutathione reductase(151). It has been suggested to be involved in sperm maturation(152).

Like GR for GSH, thioredoxin reductase (TrxR) uses NADPH and H+ for the reduction of

thioredoxin(78), as shown in Figure 1.5. Next to thioredoxin, TrxR's reduce selenite,

selenodiglutathione, lipoic acid, lipid hydroperoxides, Ca-binding proteins and many other

endogenous substrates(152-155). TrxR can also reduce ascorbyl free radicals(156), which suggests

the involvement of TrxR in the recycling of ascorbic acid (vitamin C). However, this function

will be of less importance to dogs who can synthesize ascorbic acid in the liver(157),

compared to for example humans, who evolutionary lost that ability(158). The action of

riboflavin (vitamin B2) is also associated with TrxR, as TrxR includes flavin adenine

dinucleotide (FAD) as a cofactor, of which riboflavin is the central component(159).

Since TrxR's are the only enzymes known to reduce oxidised thioredoxin(78), it is likely that

changes in TrxR activity have an impact on the functioning of thioredoxin. Thioredoxin is

involved in cell growth, inhibition of apoptosis and it facilitates the reduction of several

enzymes by cysteine thiol-disulfide exchange(78). Ribonucleotide reductase, thioredoxin

peroxidase and some transcription factors are examples of such enzymes, making

40 Chapter 1


thioredoxin an important factor in DNA synthesis, anti-oxidant potential and gene

transcription, respectively(78).

1.4.3 Iodothyronine deiodinase

There are three isoforms of the selenium-dependent iodothyronine deiodinase (DIO) family,

as indicated in Table 1.1, which are all involved in the thyroid hormone metabolism by

reductive deiodination(133). Thyroid hormones are involved in the regulation of various

important metabolic processes such as growth, thermogenesis, and lipid metabolism(78). DIO1

and -3 can be found on plasma membranes and DIO2 in the endoplasmic reticulum(160). Both

DIO1 and -2 can be mainly found in the thyroid and pituitary gland. Aditionally, DIO1 is

expressed in liver and kidney, and DIO2 in the central nervous system and skeletal

muscle(161). DIO3, on the other hand, is considered a foetal enzyme and is mainly present in

embryonic and neonatal tissues(161).

Figure 1.6 The involvement of iodothyronine deiodinases in the

deiodination of thyroid hormones

Selenoprotein iodothyronine deiodinase 1 (DIO1) unspecifically catalyzes the

deiodination of thyroid hormones. Iodothyronine deiodinase 2 (DIO2)

deiodinates thyroxine (T4) into triiodothyronine (T3) and reversed

triiodothyronine (rT3) into diiodothyronine (T2). Iodothyronine deiodinase 3

(DIO3) catalyzes the deiodination of T4 into rT3 and T3 into T2. Adapted from

Roman et al.(78).

Chapter 1


41

DIO1 mainly controls circulating triiodothyronine (T3) concentrations by unspecifically

catalysing monodeiodination of thyroid hormones, whereas DIO2 and 3 locally regulate

deiodination(162). DIO2 catalyses monodeiodination of thyroxine (T4) to form T3 and of

reversed T3 (rT3) to form diiodothyronine (T2), while DIO3 catalyses deiodination of T4 and

T3 to form rT3 and T2, respectively(78), as shown in Figure 1.6. The DIO family is highly

ranked in the selenoprotein hierarchy(161) and thus is not quickly affected by Se deficiency.

1.4.4 Selenoprotein P

Selenoprotein P (SepP) is the most abundant form of Se in plasma, it makes up more than

60% of plasma Se in rats with an adequate Se status(163). In humans this is approximately

50%(164) and the exact concentration of SepP compared to total plasma Se in dogs is

unknown. SepP is mainly synthesized in the liver(101), but the brain may also synthesize it(165).

In humans, SepP contains up to 10 SeCys' per SepP molecule(166), while in dogs this seems to

be 15(129). The half life of labelled 75Se in plasma SepP is only 3-4 hours(137), suggesting a

rapid turn-over.

SepP primarily functons as a Se transporter(167). In SepP knockout mice, transport of Se from

the liver to peripheral tissues was found to be interrupted(168). SepP is particularly important

for Se supply to brain and testes and in Se deficiency Se transport to these organs is

prioritised(169). At the brain and testes, SepP is taken up via the apolipoprotein E receptor-

2(170). Uptake in the kidney is performed via the lipoprotein receptor megalin(171). When SepP

has entered the tissue, it is catabolised by β-lyase to Ala and hydrogen selenide(172) and the

latter can be reused in Se metabolism to form other selenoproteins.

In addition to its function as Se transporter, there are indications that SepP is involved in

anti-oxidant defence. In an in vitro study, SepP has been demonstrated to protect low

density lipoproteins against oxidation(164). The binding of SepP to endothelial cells, as found

in rats(173), is also an indication of its function as anti-oxidant. Endothelial cells are assumed

to be sites of oxidative stress, as they release free radicals such as nitric oxide and super

oxide(78).

1.5 Selenium related diseases

Because too much Se in the diet can be toxic, and too little can cause chronic and

sometimes fatal deficiency, and the range between the two is very narrow, Se has been

called "the essential poison"(18). Selenium deficiency signs in dogs include muscular

weakness, anorexia, subcutaneous oedema, dyspnea, coma, muscular degeneration and

kidney mineralisation(174). Toxicity in dogs can be recognized by refusal of food, anorexia,

42 Chapter 1


nausea, vomiting, diarrhoea, apprehension, respiratory stimulation, cardiovascular changes,

nervous disorders, pathological lesions, especially in liver and spleen(175), and severe toxicity

may lead to death(176).

No clinical cases of either Se deficiency or toxicity are reported in dogs(177). However, the

lack of short term clinical signs does not imply that a suboptimal Se status may not be

involved in the onset of long-term diseases. There are indications that Se, due to its large

scala of functions, is involved in a broad spectrum of diseases, such as cancer(178-180),

neurological/brain diseases(181-182) and an impaired immune function(139; 183-185). In this

paragraph, some of the Se-related conditions which can also occur in dogs are briefly

described.

1.5.1 Cancer

Cancer is reported to be one of the major causes of death in UK and Swedish insured

dogs(186). The incidence rate for neoplasia in UK insured dogs was 2671 per 100,000 dogs

per year(186). In Italy, the number of malignant neoplasm in dogs was investigated between

1985 and 2002 and the incidence was 272 per 100,000 dogs per year in female dogs and

in 99 males(187). The incidence increased with age(187). The most common forms in dogs were

mammary cancer, non-Hodgkin's lymphoma and skin cancer(187).

In the last decades, many studies on the relationship between Se and cancer have been

described(188-193), however, only a limited amount of research has been in dogs(178-180; 194). The

results on the success of Se supplementation on the reduction of cancer incidence are

inconsistent(195). There are even indications that SeMet may promote cellular proliferation in

human cancer cells, by which it induces cancer growth(196). The effect of Se supplementation

seems to be, amongst other things, dependent on the Se status and amount of

supplementation. A quadratic (U-shaped) realtionship was found between toenail Se

concentration (i.e. Se status) and DNA damage in canine prostate cells (i.e. prostate cancer

risk)(193). Another study reported Se supplementation to reduce the risk of lung cancer in

human subjects with a baseline serum Se concentration of <106 ng/ml, while people with a

baseline serum Se concentration of >122 ng/ml had an increased risk(197). The reducing

effect of Se on the formation of cancer is mainly attributed to its anti-oxidant function(78), and

thus the prevention of DNA damage. In addition, the capacity of Se to bind and excrete

carcinogenic compounds, such as mercury(198-200) and arsenic(71; 175), may reduce cancer

formation.

Se does not only prevent cancer formation, but there are also indications that it acts as a

cancer specific cytotoxic agent(201). Selenite and, to a lesser extent, selenate are more potent

Chapter 1


43

than organic Se species to act as a prooxidant and cause apoptosis in cancer cells. Selenite

can oxidize thiol-containing cellular substrates to form superoxide (O2-) and other reactive

oxygen species (ROS), where SeMet and SeCys lack oxidation capacity(191). Cancer cells are

particularly sensitive to this prooxidative apoptosis, because of their activated oncogenes,

leaving the healthy cells unharmed(75). It should be noted, however, that a supranutritional

concentration of selenite (in humans 200 µg/day, which is approximately four times the daily

recommended allowance) is required for this process(191).

1.5.2 Cardiovascular disease

Cardiovascular disease and congestive heart failure are common conditions in dogs. A

study in the USA showed that 9 to 11% of dogs had reliable signs of heart disease and also

in Italy in 11% of the dogs suffered from hart disease(177). Studies into chronic heart failure in

humans have revealed that dietary Se intake and serum Se levels are negatively correlated

with the severity of chronic heart failure(202). Also, the recovery after ischaemia was improved

at adequate (0.16 mg Se/kg diet as sodium selenite) compared to deficient Se intake (0.01

mg Se/kg diet), as studied in rat hearts(203). This protection of Se against heart failure and its

consequences is, amongst other selenoproteins, related to the action of GPx(204). GPx1 knock-

out mice were more sensitive to reperfusion injury after myocardial ischaemia than the

control mice(205), while mice which overexpressed the GPx1 gene were more resistant than

the controls(206).

TrxR also has an important function in the prevention of cardiovascular diseases via its

involvement in cellular redox control(207). TrxR1 was upregulated in human artherosclerotic

plaques and dose-dependently by oxidized low-density lipoproteins in the mRNA of human

macrophages(208). Knock-out of the mitochondrial TrxR2 caused fatal cardiomyopathy directly

after birth in mice(209). The symptoms reported in this study were similar to that of an

endemic cardiomyopathy that occurs in some parts of China, which is called Keshan

disease. This disease is named after the region in North-East China, where the disease was

first described. Myocardial necrosis and eventually congestive heart failure are typical

symptoms of this disease. So it may be that underexpression of TrxR is a factor in the

incidence of Keshan disease. The underlying mechanisms for the preventive effect and the

enhanced recovery after cardiovascular damage are only partly understood(210). Based on a

study in rats, it is thought to be related to the anti-oxidant properties of TrxR and GPx(211).

Additionally, the reduction of thioredoxin by TrxR enables thioredoxin to perform its

regulatory function in ventricular remodelling(212). DIO1 is also associated with cardiovascular

disease, through its action in thyroid metabolism (converting T4 into T3) and thereby

44 Chapter 1


playing a key role in lipid metabolism. Hypothyroidism leads to changes in lipoproteins,

which increases their artherogenicity(78).

1.5.3 Diabetes mellitus type II

Diabetes mellitus type II is hallmarked by insulin resistance, which causes dyslipidemia,

hyperglycemia and an increased insulin secretion by pancreatic beta cells(213). The

incidence of diabetes in American dogs was 58 per 10,000 dogs in 1999 and seemed to

have increased since the 1970's, when it was only 19 in 10,000 dogs(214). The effect of Se on

Figure 1.7 Influence of selenium on components of the insulin

signalling cascade

O2, oxygen; O2·-, superoxide; H2O2, hydrogen peroxide; Nox4, NADPH oxidase

4; IR, insulin receptor; p, phosphorus; IRS, insulin receptor substrate; PIP2,

phosphatidylinositol diphosphate; PI3K, phosphoinositide-3-kinase; PIP3,

phosphatidylinositol triphosphate; Akt, protein kinase B; Glut4, glucose

transporter 4; PTP-1B, protein tyrosine phosphatase 1B; PTEN, phosphatase and

tensin homolog; GPx1, glutathione peroxidase 1; SeP, selenoprotein P; FoxO1a,

forkhead box class O 1a; PGC-1a, proliferator-activated receptor gamma

coactivator 1a. From Steinbrenner et al.(215).

Chapter 1


45

the onset of type II diabetes mellitus is two-fold. It has long been thought of as an anti-

diabetic agent(215), due to its anti-oxidant function which inhibits reactive oxygen species

that result from hyperglycemia and is associated with the progression of diabetes mellitus(216).

Interest in the diabetogenic effect of Se is relatively recent. During a large human cancer

prevention trial by the Nutritional Prevention of Cancer study group, an unexpected

increase in the incidence of type II diabetes was found in subjects supplemented with 200

µg Se/day compared to subjects assigned to a placebo(217). Diabetes incidence increased

with increasing baseline plasma Se concentrations(217). These findings may be attributed to

the insulin mimetic properties of Se(218-219), which may accelerate insuline resistance

development. Also, overexpression of GPx1 has been associated with an impaired contol of

insulin release by beta cells in mice(220) and a decreased phosphorylation (activation) of

protein kinase B(221), which enables glucose to enter the cell via the glucose transporter 4. A

study in rats showed a higher liver GPx1 activity and an increased activity of protein

tyrosine phosphatase 1B (PTP-1B) in Se supplemented animals compared to rats on a Se

deficient diet(222). PTP-1B dephosphorylates the insulin receptor and its substrate, and

therefore has an antagonistic effect on the insulin signaling pathway(223).

An overview of the current knowledge on Se involvement in the onset of diabetes mellitus

type II is given in Figure 1.7. However, the mechanisms are not exactly understood yet. A

more detailed explanation of these mechanisms can be found in the review of Steinbrenner

et al.(215).

1.5.4 Joint conditions

In humans, several positive associations were reported between serum and toenail Se

concentrations and osteo- and rheumatoid arthritis(224). Symptoms of the Se and iodine

deficiency disease "Kashin-Beck disease", which are for example joint deformation,

decreased joint mobility and necrosis of joint cartilage(225), also indicate involvement of Se in

joint conditions.

A study in rats indicated that a combination of Se and iodine is necessary for normal

growth and repair of bone and cartilage(226). This study revealed that type X collagen was

decreased in the hypertrophic zones in growth plate cartilage in Se deficient rats. Collagen

type X is only expressed in hypertrophic chondrocytes, which is the terminal differentiation

step during endochondral ossification(227). The study also found that a Se or iodine

deficiency or a deficiency in both resulted in an increase of the parathyroid hormone-

related protein(226). Overexpression of this protein delays the appearance of hypertrophic

chondrocytes(228) and it can regress hypertrophic chondrocytes to a prehyperthrophic

46 Chapter 1


proliferating stage(229), which indicates an impaired cell maturation during bone development

in Se deficiency.

Also, a large range of selenoproteins were found in human mesenchymal stem cells(230),

which can differentiate to osteoblasts, chondrocytes and adipocytes, strengthening the idea

that Se is involved in bone and cartilage formation. However, the exact action of the

selenoproteins in these cells is unknown(230). To our knowledge, there are no studies on the

association between Se and joint conditions performed in dogs.

1.5.5 Urolithiasis

A few recent studies in rats and dogs have reported a reducing effect of Se

supplementation on the formation of calcium oxalate urolithiasis(231-232), which is a common

urological disease in dogs(233). One of the possible mechanisms behind these findings is that

antioxidants, such as the Se-dependent GPx, inhibit renal tubular damage caused by free

radicals, which is a key factor in the formation of calcium oxalate calculi(234). Oxalic acid is a

constituent of calcium oxalate calculi and may act as a free radical to damage the renal

tubular epithelial cells(234). Se supplementation in rats have been reported to decrease the

urinary oxalic acid concentration(231), but this was not confirmed in dogs(232).

Se has also been reported to inhibit osteopontin, an acidic glycoprotein involved in the

formation of calcium oxalate calculi. Liu et al.(232) showed that osteopontin was significantly

lower in Se supplemented dogs (0.3 mg/kg diet, as sodium selenite or Se yeast) than in dogs

without Se supplementation (Se concentration in basal diet not mentioned), reducing the

formation of calium oxalate calculi. Dogs supplemented with Se yeast showed a larger

decrease in osteopontin than sodium selenite supplemented dogs(232).

1.6 Selenium in dog foods

Safety, nutritional adequacy and health promoting effects of dog foods are an important

issue for dog owners(235). Although clinical cases of dietary-induced selenium deficiency and

toxicity have not been reported in dogs(177), it is important for the dogs' health to estimate

the requirements as reliably as possible. Also, long-term effects of dietary Se provision

should be investigated, because no such information has ever been published.

Diet types differ in the way they are processed and in the type of ingredients that are

used. Kibble and canned dog foods are the most popular diet types(235). Canned diets are

produced by retorting, which is a time/temperature dependent heat and pressure-cooking

and sterilisation process(236). Fresh and or frozen meats are ground, and potentially mixed

with pre-ground or blanched grains, starches, gums, vitamin and mineral premixes and

Chapter 1


47

water. This mixture is heated to 25 to 85°C to gelatinize starches and start protein

denaturation(177). The heated mixture is transferred into cans by a filler/seamer machine that

also places the lids on the cans. Before the can is closed, steam is injected over the product

to displace air and create a vacuum when the can is cooled. The closed cans are sterilised

in a retort to preserve the food and get rid of harmful pathogens. The retorting process

includes three steps; in the first step hot water heats the contents of the cans to

approximately 80 to 100°C. The second step uses pressurised steam of between 116 and

129°C in order to raize the core temperarture of the product to above 116°C for 60 to 90

minutes. In the final step, the product is cooled to a temperature between 38 and 49°C

using water of 18 to 25°C(177).

Kibble diets are produced by extrusion. Grains, meat meals, vitamin and minerals are often

ground together in a hammer mill, to achieve a uniform particle size, and are then

thoroughly mixed. The dry mixture is transferred into a pre-conditioner where water and

steam is added and potentially fat or meat slurries can be added. In the pre-conditioner the

ingredients are further mixed and starches are started to gelatinise(177). The pre-conditioned

mixture is transferred into an extruder barrel where a combination of friction, shear, and

indirect heat gelatinise the starch. The residence time in the extruder (cooking time) can be

influenced by screw speed or type (single or double screw), insertion rate from the pre-

conditioner and the composition of the mixture (e.g. fat lubricates the extruder, resulting in

less friction and a shorter residence time). Generally, residence times vary between 10 to

270 seconds, temperatures between 80 and 200°C, moisture contents between 25 and 27%

and pressure between 34 and 37 atmospheres(177). Kibbles are formed at the end of the

extruder barrel when they expand by reaching atmospheric pressure after passing a die

and been cut off by a knife(236). After extrusion, the kibbles are dried to reach a moisture

content between 8 and 10% and coated with fat and/or palatants(177).

The retorting and extrusion processes also influence the ingredients that can be used for

the different diet types. For extrusion, the mixture should contain a certain amount of starch.

Therefore, kibble diets mainly contain plant derived ingredients, like wheat(236). Animal

protein in kibble diets is mainly present in the form of meat meal(237-238), due to their lower

moisture content as compared to fresh or frozen meat. Canned diets, on the other hand,

have a much higher moisture content than kibble diets. They mainly contain fresh or frozen

animal derived ingredients(236).

Selenium is not essential for plants, so the Se content of plants reflects the Se content of the

soil(64). As a consequence of the Se availability in soils (see paragraph 1.2), Se levels of plant-

derived dog food ingredients may vary greatly. A plant-derived ingredient may have more

48 Chapter 1


Table 1.2 Typical ingredients of dog foods and their selenium concentration in mg/kg dry

matter*

Plant derived Animal derived

Ingredient Average Ingredient Average

Rice bran 0.20 Chicken meat and skin 0.37

Rice brewers, broken 0.30 Chicken gizzard 2.35

Rice flour, white 0.17 Chicken liver 2.42

Corn bran 0.18 Turkey, mechanically deboned 0.87

Corn distillers grain, dried 0.37 Egg, dried 1.24

Corn gluten feed, dried 0.19 Fish meal, menhaden 2.26

Corn gluten meal 0.40 Fish meal, tuna 4.30

Corn grits 0.19 Fish meal, white 1.71

Corn grain 0.15 Shrimp meat 1.58

Corn meal, degermed 0.09 Beef meat 0.52

Corn meal, whole kernel 0.18 Beef heart 0.90

Corn starch 0.03 Beef kidney 6.48

Wheat flour, whole grain 0.79 Beef liver 1.32

Wheat flour, white 0.39 Beef tripe 2.47

Wheat germ 0.89 Pork, cured 0.37

Wheat germ meal 0.67 Lamb meat 0.47

Wheat middlings 0.50 Lamb liver 2.87

Sorghum grain 0.23 Poultry by-product meal 0.83

Oats grain 0.48 Meat meal 0.45

Barley 0.17 Meat and bone meal 0.27

Cereal by-product 0.45 Whey, dried 0.07

Flaxseed 0.07

Soybean flour, roasted 0.08

Soybean hulls 0.21

Soybean meal 0.21

Soybean meal without hulls 0.13

Brewers' yeast, Torula, dried 1.08

Chicory root 0.05

Beet pulp, dried 0.14

Sugar, granulated 0.01

Patato, flesh and skin 0.00

Carrots 0.08

Peas 0.09

Average 0.28 Average 1.63 * For all ingredients applies that Se concentration is largely dependent on the origin. These data are

assumed to be based on products with American origin and may overestimate the selenium

concentration in European produced ingredients. From NRC(236).

Chapter 1


49

than a 10-fold difference in Se concentration, depending on where it was produced(239). In

contrast, Se is essential for animals, so meat will always contain a minimum amount of Se, but

depending on the part of the animal (muscle, kidney, liver) it contains more or less Se; the

kidney cortex contains the highest Se concentration and skeletal muscle contains

considerably less Se(240-241). The amount of Se in the feed (the area in which the animal is

raised) can also have an impact on the Se concentration of the meat(242). Fish is also known

to contain high amounts of Se(243) and pet foods with seafood-based flavours are found to

contain more Se than differently flavoured pet foods(244).

Table 1.2 gives an overview of the Se concentration in typical plant and animal derived

dog food ingredients. Considering the fact that the major part of Se in food ingredients is

incorporated into proteins(10) and that canned dog foods have higher protein concentrations

than kibble diets(236), it is generally assumed that canned diets contain more Se than kibble

diets. This was, however, not confirmed in a study by Simcock et al.(244). Although canned

dog foods available in New Zealand had a numerically higher Se concentration than kibble

dog foods (approximately 0.45 and 0.35 mg/kg DM, respectively, estimated from a figure),

this was not significantly different. Canned cat foods, on the other hand, did contain

significantly more Se than kibble cat foods and canned and kibble dog foods(244).

It is known that SeMet, and possibly to some extent also SeCys, are incorporated into

proteins in both animals(64-65) and plants(245). Selenium in meat products is for over 90%

organically bound(246), while plants contain both, organic and inorganic forms(48; 247-250). This

potentially results in a higher amount of organically bound Se in canned diets compared to

kibble diets. Due to the variable Se concentrations in dog food ingredients, diets may also

be supplemented with sodium selenite(236). This is more likely in kibble compared to canned

diets, due to their generally lower Se concentration in the ingredients.

1.7 Bioaccessibility, -availability, and -activity of selenium

Bioavailability and -activity are often used interchangeably. In this thesis, bioavailability will

be defined as the fraction of the dietary Se that reaches the systemic circulation(251)

(including digestibility, absorption, blood Se levels). Apparent bioavailability will be defined

as the amount of dietary Se that is apparently absorbed (intake minus faecal excretion). A

part of the dietary Se may be physiologically inert, such as SeMet that is non-specifically

incorporated into body proteins or Se that directly after absorption is methylated and

excreted. Bioactive Se is defined as the Se that is incorporated into selenoproteins (e.g. GPx).

Dietary Se that is potentially available for gastrointestinal absorption will be called

bioaccessible Se(251). These definitions are briefly summarised in Figure 1.8.

50 Chapter 1


It should be noted that the bioavailable fraction may also have an impact on metabolism of

Se, even if it does not increase the bioactivity. It may, for example, be involved in the

detoxification process of heavy metals; binding the metals for excretion. Also important to

emphasise is that Se may also be active in the intestine without being absorbed. For

example, when protecting epithelial cells from DNA oxidative damage in the protection

against intestinal cancer(251). Therefore, it should be kept in mind that the bioactivity and

bioavailability may slightly underestimate the actual effect of Se in the body.

Figure 1.8 Fractionation of ingested selenium into

bioaccessible, -available and -active fractions

Adapted from Thiry et al.(77).

The bioavailability and -activity of Se from pet foods is generally considered to be low.

Wedekind et al.(252) reported Se bioactivities (in the article called bioavailability) of 30% and

53% in canned and kibble petfoods, respectively, using GPx measurements in a chicken

bioassay. Todd et al. reported Se bioavailabilities of the same canned cat food in two

different studies of 25.3%(253) and 21.2%(69) in cats. Interestingly, this cat food had a higher

bioavailability in dogs (37.4%). The exact bioavailability and -activity from complex matrices

like pet food are, however, difficult to determine, as these may be influenced by various

factors.

The type of Se species (chemical form) is often reported to have an effect on the

bioavailability and -activity of Se(77; 254). The organically-bound Se species SeMet and SeCys

are generally accepted to have a higher bioavailability than inorganic Se(58; 255-256), but

selenite has been reported to have a higher bioactivity than organically-bound Se(257-258).

However, there are large differences in Se bioavailability and -activity within Se species(77).

This variation indicates that other factors also play an important role in the bioavailability

and -activity of Se. Some of these possible factors are summarised in Table 1.3.

Chapter 1


51

Table 1.3 Dietary factors affecting selenium bioavailability and/or bioactivity

Factor Animal model Effect ref.

Type of ingredient

Chicken Plant-derived ingredients had a higher Se bioactivity (60-90%) than animal-derived ingredients (15-25%).

(259)

Chicken Plant-derived ingredients had a higher Se bioactivity (47%) than animal-derived ingredients (28%).

(252; 260)

Chicken Fish-derived ingredients had a Se bioactivity of 9-28%. (260-261) Rat Se bioavailability and -activity were higher in Se from wheat

and selenite than from tuna, at dietary Se concentrations of 0.05, 0.10, and 0.15 mg/kg diet.

(262)

Selenium species Human SeMet had a higher bioavailability than selenite when supplemented at 100 µg/day.

(255)

Human SeMet (2 µg radiolabeled Se) had a higher bioavailability

than selenite (10 µg radiolabeled Se).

(58; 256)

Rat Selenite had a higher bioactivity than SeMet at dietary Se concentrations <0.5 mg/kg diet.

(257)

Rat Selenite had a higher bioactivity than SeCys at dietary Se concentrations of 0.1 and 0.2 mg/kg diet.

(258)

Heat processing Cat Bioavailability of inorganic Se and retention of organic Se in a canned diet decreased after heat processing.

(263)

Sheep Heat processed soy beans at 130°C for 45 min. had a higher

Se bioavailability than unprocessed soy beans or soy beans processed for 30 min at 150°C.

(264)

Dietary protein Rat Se bioactivity decreased with increasing dietary protein (range 5.3-13.4%) with a Se concentration of 0.03 mg/kg diet.

(265)

Chicken Se from a low protein diet (16.9%) had a higher bioactivity than from an adequate protein diet (22.5%).

(266)

Dietary methionine

Rat Se bioavailability decreased with increasing Met supplementation (0, 4, or 9 g/kg), but bioactivity maintained or increased.

(267)

Rat Bioactivity of SeMet was higher on a diet with 0.64% Met compared to a diet containing 0.24% Met.

(257)

Dietary sulphur Sheep Se bioavailability decreased with increasing dietary sulphur (25 vs. 37%).

(268)

Dietary fat Chicken Bioactivity, but not bioavailability, of selenite increased with increasing concentration of butter or olive oil (from 4 to 20%).

(269)

Dietary fibre Human Guar gum supplementation (2.5g/day) decreased Se bioavailability and bioactivity regardles of dietary Se concentration (range: 124-155 µg/day).

(270)

α-Tocopherol

(vit. E)

Chicken Increased Se bioavailability when an α-tocopherol-free diet

containing 0.05-0.07 mg Se/kg was supplemented with 100 IU α-tocopherol/kg diet.

(271)

Chicken Increased Se bioactivity when an α-tocopherol-free diet

containing <0.02 mg Se/kg diet was supplemented with 100

IU α-tocopherol/kg diet.

(272)

Retinol (vit. A) Chicken Increased Se bioactivity when an α-tocopherol-free diet

containing 0.04-0.08 mg Se/kg diet was supplemented with 1,000,000 IU retinyl palmitate/kg diet.

(273)

Chicken Increased Se bioavailability when an α-tocopherol-free diet

containing <0.02 mg Se/kg diet was supplemented with

1,500,000 IU retinyl palmitate/kg diet.

(272)

52 Chapter 1


Table 1.3 (continued)

Factor Animal model Effect ref.

Pyridoxine (vit. B6)

Rat The bioavailability of SeMet was less than for selenite when pyridoxine-deficient, but equal when receiving an adequate intake of pyridoxine (2.5 µg/g diet).

(274)

Riboflavin (vit. B2) Pig Bioactivity was decreased when feeding a riboflavin-free diet compared to a diet containing 25 mg/kg diet.

(275)

Ascorbic acid (vit. C)

Human Bioavailability of selenate was increased by the supplementation of ascorbic acid (600 mg/day). Bioactivity also increased, but not significantly.

(276)

Chicken Increased Se bioavailability and -activity with ascorbic acid supplementation (from 10 mg/kg) to a Se-deficient and α-

tocopherol-free diet.

(271)

Mercury LNCaP cells

In vitro bioactivity of selenite, but not SeMet and methylated SeCys, decreased after administration of mercury (0-100 µM

HgCl2).

(198)

Copper Rat A copper deficient diet (7.9 nmol Cu/g diet) decreased Se bioactivity compared to a diet containing 125.9 nmol Cu/g diet.

(277)

Sheep Se bioavailability decreased with increasing dietary copper (8 vs. 25 mg/kg DM).

(268)

ref., reference; Se, selenium; SeMet, selenomethionine; SeCys, selenocysteine; min, minutes; Met, methionine; vit, vitamin; LNCaP cells, androgen-sensitive human prostate adenocarcinoma cells.

Fish generally contains high amounts of Se (see paragraph 1.6) and the bioavailability of Se

from fish is high(278). However, the bioactivity is sometimes reported to be low(261; 279). This may

be due to the potential of fish to accumulate heavy metals in their muscle tissue(280). In the

presence of heavy metals, Se will likely be used for detoxification (binding and excretion)

instead of being used for the incorporation into selenoproteins.

Processing can reduce the Se concentration of diets and ingredients. The Se content of

milled wheat and corn products may decrease to 70% of the original crop(281). Dry heating

of cereals appears to result in a reduction to 77-93% of the original selenium content(282).

Not only the Se concentration is affected by processing, but also the bioavailability. In cats,

Todd et al.(263) found that the bioavailability of selenite was reduced from 83.3% to 53.7%

when Se was added before retorting a canned cat food compared to addition after

processing. Also, the retention of Se from Se-yeast in the cats was lower when the Se was

added before the diet was processed. Hendriks et al.(283) showed that heat processing of

canned diets reduces the digestibility of amino acids, also of the sulphur- or Se-containing

amino acid Met, suggesting that heat processing may reduce the bioavailability of Se. The

protein digestibility after extrusion processing pet foods, on the other hand, is not

affected(284-285) or can even be increased(286).

Chapter 1


53

Protein concentration of a diet may have a negative effect on Se bioactivity(265-266). This

seems to have to do with the Met and Cys levels in the diet, because when human subjects

were given a diet low in protein supplemented with Met and Cys, Se retention was lower

than when given the same diet without supplementation(287). Also in rats, the negative effect

of Met on Se bioavailability was reported(267). This is possibly due to the competition of Met

with SeMet for absorption. However, in the same study Met had a positive effect on Se

bioactivity. If the percentage SeMet of the whole dietary Met pool reduces (by Met

supplementation), the chance of non-specific SeMet incorporation is lower and Se bound to

Met can be used for selenoprotein formation.

Sulphur may also play a role in the bioavailability and -activity of Se. Although this is an

apparently logical statement, as Se and sulphur compete for absorption and organically-

bound Se may replace the S-amino acids in body proteins, by which it becomes unavailable

for incorporation into biologically active selenoproteins, there are only limited amount of

reports specifically studying the effect of sulphur on Se bioavailability and -activity. Ganther

& Bauman(288) reported in rats that sulphate in the diet (1%) or an injection with sodium

sulfate (2.5 mg) significantly increased urinary selenate excretion, indicating a potential

reduction in bioactivity.

Shen et al.(289) reported that the removal of milk fat increased the in vitro Se accessibility of

the milk. This suggests a negative impact of fat on Se bioavailability. This was however, not

found in a study with chickens(269), in which 4 and 20% of the diets consisted of butter, olive

oil, rape oil, corn oil or sunflower oil. On the contrary, the 20% butter and olive oil diets

increased the Se bioactivity compared to the 4% butter and olive oil diets, respectively.

Fibre depresses overall nutrient digestibility(290-291), by making the absorption surface smaller,

which also may lead to a decrease in Se bioavailability. This was verified by a study of

Choe & Kies(270) who reported a decrease in Se bioavailability and -activity in humans after

supplementation with the soluble non-starch polysaccharide guar gum. In dogs, guar gum

(3.4% and 7% DM) was reported to decrease apparent protein digestibility(292-293), which may

cause a decrease in the bioavailability of protein-bound Se.

Van Vleet(174) showed that Se-deficiency signs were more severe when beagle puppies were

fed a Se- and α-tocopherol-deficient diet compared to a diet only deficient in Se, which

indicates that α-tocopherol is an important factor in the biological action of Se or may spare

Se partly. A deficiency in pyridoxine and riboflavin were also reported to reduce the

bioavailability in rats and the bioactivity in pigs, respectively. In chickens, high

concentrations of retinyl palmitate (1,000,000 and 1,500,000 IU/kg diet) are shown to have a

positive effect on Se bioavailability and -activity. The Se-vitamin E deficiency disease

54 Chapter 1


exudative diathesis in chicks, was effectively prevented with 100 mg ascorbic acid/kg diet,

with a basal diet containing <0.02 mg Se/kg and was free of α-tocopherol(271). This was

possibly due to the increased plasma GPx concentration which was also reported. Both,

ascorbic acid (200 mg/kg) and α-tocopherol (100 IU/kg) supplementation decreased Se

accumulation in the liver(271), indicating that the Se is excreted or used for selenoprotein

formation. The anti-oxidants GPx, TrxR, α-tocopherol and ascorbic acid all work together in

the anti-oxidant cascade and thereby can partially spare each other. When α-tocopherol

reduces lipid peroxides it is converted into an α-tocopheroxyl radical, which can be

recycled by ascorbic acid resulting in dehydroascorbate. Dehydroascorbate can, in turn, be

recycled into ascorbate by the thiol cycle, which includes the selenoproteins GPx and

TrxR(294-295).

Selenium is involved in the detoxification of heavy metals(77). Heavy metals like arsenic(71; 175),

cadmium(92), mercury(198-200), and copper(268) form Se-metal bonds and, therefore, are likely to

decrease Se bioactivity. As Se will be occupied to bind and excrete these metals, it cannot

be used for the formation of selenoproteins. However, in an in vitro model with human

LNCaP cells (an androgen-sensitive human prostate adenocarcinoma cell line), mercury only

seemed to decrease Se bioactivity when selenite was the Se species used and not when

organically-bound Se was used(198). Copper was reported to also have a beneficial effect on

the presence of GPx in rat plasma(277). The mechanism behind this finding is not understood,

but may be due to a reduction in copper-zinc-superoxide dismutase (CuZn-SOD) activity

leading to a decrease in substrate for GPx, by which there is a reduced need for GPx. As

some of these metals are essential to dogs, more research is required to determine the exact

effects of heavy metals on Se bioavailability and -activity. In general, the current knowledge

on dietary factors that affect Se bioavailability and -activity and their interactions are not

enough understood to be able to estimate the bioactive Se fraction from pet foods.

1.8 Current recommendations on selenium intake for adult dogs

The guidelines for Se inclusion in pet foods for European pet food producing companies are

created by the European Pet Food Industry Federation (FEDIAF), and by the National

Research Council (NRC) and the Association of American Feed Control Officials (AAFCO) for

American companies. Generally, nutritional recommendations by FEDIAF and AAFCO are

based on state-of-the-art scientific research. However, research on the optimal intake of Se

in adult dogs is lacking. Current recommendations of Se intake for adult dogs by FEDIAF

and the NRC are shown in Table 1.4. These are extrapolated from data of growing cats and

dogs(261), respectively. In rats, it has been demonstrated that the Se requirement of adults is

Chapter 1


55

at least 50% lower than that of growing rats(296-297). In addition, liver Se concentrations in

dogs were highest in adult dogs compared to young and geriatric dogs(68), which is an

indication for lower Se requirements of adult dogs.

The current recommendations do not take into account the highly variable Se bioavailability

and -activity of pet food ingredients. One fixed "bioavailability" (=bioactivity) correction

factor is used, which is adapted from a study in which purified pet foods were fed to

chickens(252). In this study the bioactivity, calculated with breakpoint analysis, was 30% for

canned diets and 53% for kibble diets. These values are not species-specific and their

relevance for dogs may be questioned. In the minimum recommendation of FEDIAF, not only

the correction factor is not species-specific, but also their requirement, which was based on

data from cats. Studies by Todd et al.(69) showed that there are significant differences in Se

availability and retention between dogs and cats, indicating that an extrapolation of these

data to adult dogs may not be correct.

Table 1.4 Current recommended selenium intakes for adult dogs by FEDIAF and the NRC

unit FEDIAF(298)a NRC(236)b

Recommended allowance µg/MJ n.a. 20.9

Min. recommended allowance µg/MJ 17.9 n.a.

Based on

kitten study(261) puppy study(261)

Assumed ME concentrationc MJ/kg diet 16.7 16.7

Minimum adequate concentration in study

µg/kg diet 120 210

µg/MJd 7.2 12.6

Assumed bioavailabilitye % 40 60 FEDIAF, European Pet Food Industry Federation; NRC, national research council; n.a., not applicable; MJ, megajoule.

a based on a maintenance energy requirement of 110 kcal/kg BW0.75 b based on a maintenance energy requirement of 130 kcal/kg BW0.75 c Energy concentration of the diets used in the studies is not mentioned and therefore

an assumption is made. d Dietary selenium concentration is calculated by deviding the selenium concentration

per kg by the assumed ME concentration. e Assumed bioavailability is calculated by deviding the recommendation by the

dietary selenium concentration of the study diet (µg/MJ) multiplied by 100.

Wedekind et al.(299) have attempted to define the minimum and maximum requirements of

adult dogs. This study was performed in Se depleted beagles using SeMet supplements in a

Se deficient synthetic basal canned diet. Results indicated a minimum requirement of 110

µg/kg diet. A maximum was not specified. This study may have overestimated the

requirements because SeMet may be non-specifically incorporated into proteins. Also, it is

56 Chapter 1


not likely that dogs will be Se depleted, as commercially available diets always contain

some Se. It is plausible that Se is more efficiently used in depleted dogs than in dogs with

an adequate Se status. Therefore, the use of Se depleted dogs may give an inaccurately

higher requirement of adult dogs. As SeMet was supplemented after the diet had been

processed (retorted), the recommended minimum was corrected for the chicken bioassay

bioactivity for canned diets (30%)(252) and resulted in a recommendation of 0.33 mg/kg

diet(299).

High dietary Se concentrations may increase its health-beneficial properties, but it may also

have a negative effect on health, like diabetes mellitus type II (see paragraph 1.4.3) or other

unknown consequences. A legal upper limit of 0.5 mg Se/kg diet for pet foods is set by the

European Commision (70/524/EEC, 2004)(300). This accounts for 12% moisture in the diet and

thus is 0.568 mg Se/kg on a DM basis. This upper limit is only applicible to the total dietary

Se concentration in diets to which Se is supplemented. Sodium selenite is the main Se

species used to supplement diets(236) of which the calculated amount of Se from the

ingredients is not sufficient to reach the recommended minimum. In addition to sodium

selenite, sodium selenate, selenium yeast and selenomethionine are also approved Se

supplements by the European Union(301). It is not specified on which research the legal upper

limit is based. However, Wedekind et al.(299) have reported decreased hair growth in dogs as

a sign of Se toxicity at a SeMet intake of 5 mg Se/kg, while at intakes of 1 mg Se/kg no

such signs were reported.

Due to the extrapolations of data from animals with presumable different requirements and

the lack of accounting for variations in bioavailability and -activity, it can be concluded that

the current recommendations on Se intake for adult dogs are premature and need further

research.

1.9 Biomarkers of selenium status

Dietary recommendations for dogs are currently based on the optimisation of serum Se(302)

and GPx activity(252; 261). These are frequently used biomarkers in Se studies in dogs(69; 303-307).

However, it is not known whether these are the most sensitive markers for Se status. A

biomarker is defined as a characteristic that can be objectively measured and evaluated as

an indicator of normal biological processes, pathogenic processes or as a pharmacological

response to a therapeutic intervention(308). Se status refers to the amount of bioactive Se or

potentially bioactive Se in the body and may be used to assess the risk of Se deficiency,

potential health beneficial properties of Se, or Se toxicity(309).

Chapter 1


57

Se retention can be calculated by the difference between Se intake and the sum of urinary

and faecal excretion(309) and is useful as an indicator of the degree to which requirements

have been met and the presence of potential Se reserves. Other mechanisms of Se excretion

from the body, such as expired breath(117), and hair and toenail Se may be negligible when

estimating Se retention(309). Urinary Se excretion can be conveniently measured in a single

void sample by correcting the Se concentration by the urinary creatinine concentration, to

reduce the error associated with variation in urinary output. This method has been

validated against 24-hour urinary Se excretion by Hojo(310) in humans. No validation has

been done in dogs, but urinary concentrations corrected for urinary creatinine

concentration are widely accepted for other urinary excretion products, such as protein(311-

312) and aldosterone(313).

It was previously believed that the excretion of TMSe in urine was a valid urinary marker

for excess Se intake(116). However, since the finding of Kuehnelt et al.(111), the excretion of

TMSe should be interpreted with caution. These authors found that TMSe can also be

excreted in Se adequacy, possibly depending on genetic variation(111). There is no support

for the findings of Kuehnelt et al. in dogs. From an exploratory study in preparation to the

study reported in chapter 4 of this thesis, a small number of urine samples (n=5) from dogs

that have been fed a commercially available kibble diet (approximately 20 µg Se/MJ) have

been analysed and TMSe was not detected in any of the samples (detection limit of 50

ng/ml, unpublished data). Also, because it is known that the excretion of high dietary Se

levels shifts partly from urinary to respiratory excretion(117), the measurement of urinary Se

should be interpreted with care in dietary Se concentrations that exceed the minimum

recommendation.

Hair and toenail Se concentrations may also be measured as excretory products, as the Se

is no longer accessible to the body. This is a measure of long-term Se status and correlates

well with blood Se concentrations(47; 314). However, they should be interpreted with caution as

Se intake should be stable for an extended period before hair and toenail Se

concentrations could be accurately measured, and also, contamination with Se from outside

the body may occur, for example by anti-dandruff shampoos that contain Se(309).

Retention of Se can occur in many organs, with the highest concentrations in kidney(68) and

liver(59). Tissue Se concentrations contain only non-specifically incorporated Se and thus may

be used as a measure of Se reserves. Tissue Se concentrations have been reported to

correlate well with whole blood Se concentrations(314). However, whole blood contains Se

that could be used for selenoproteins as well as non-specific incorporation(309). Plasma/serum

Se also consist of both specific and non-specifically incorporated Se(309) and ar highly

58 Chapter 1


correlated with Se intake(315). Plasma and serum deviate in that serum does not contain

clotting proteins such as fibrinogen, and plasma does. Forrer et al.(307) indicated a reference

range for serum Se in healthy dogs between 1.90 and 4.31 µmol/L.

More specific biomarkers of Se bioactivity may also be useful in the assessment of Se status.

Selenoprotein activity or concentration may be measured in whole blood, plasma, serum or

tissues like the liver. In humans, erythrocyte GPx activity is 25 to 100 times higher than

plasma GPx activity(316). It may be most applicable in determining Se deficiency rather than

high Se concentrations, as selenoprotein activity level plateaus at adequate Se intake(317), as

the enzyme protein becomes the limiting factor instead of selenium(318).

Selenoproteins can also be measured by means of their mRNA expression. However,

responses of mRNA concentrations do not necessarily reflect the response of the

corresponding selenoprotein, as a complex mechanism at post-transcriptional and -

translational level regulates protein expression(319). Yet, these markers can offer an early

prediction of individuals at risk for disease and disease progression(320). mRNA transcripts of

selenoproteins can be determined in tissue biopsies, but also in blood, as this shares more

than 80% of the selenoprotein transcriptome with major tissues(320-321). Not all mRNA

transcripts respond to changes in Se intake. Sunde et al.(322) found that SelH, SepW1, and

GPx1 in mice liver and kidney are most decreased compared to other selenoproteins by a

decreasing dietary Se intake (0 or 0.05 vs. 0.2 µg/g diet). While also in rats most

selenoprotein mRNA concentrations were down-regulated in Se deficiency, SPS2 was up-

regulated(323). This is understandable, because this enzyme is involved in the first step of

selenoprotein formation, namely by transforming hydrogen selenide into

selenophosphate(132). So the up-regulation of SPS2 mRNA may facilitate enhanced recycling

of limiting Se. Unfortunately, no dog-specific information on the effect of dietary Se intake

on selenoprotein mRNA expression is yet available.

Non-specific biomarkers of Se status are markers that may indirectly be altered by Se.

These may be useful as secondary marker to get an idea of the adequacy of the Se status.

Examples of non-specific biomarkers for Se status are thyroid hormone ratio (T3:T4),

isoprostanes (IsoP) as marker for lipid peroxidation, and creatine kinase (CK) as a marker of

muscle damage. Van Vleet(174) reported increased plasma CK activities in combination with

muscular degeneration in puppies fed a vitamin E and Se deficient diet.

In conclusion, more research is needed into specific biomarkers for Se status in dogs to

accurately assess their Se requirements. Also, the effect of these biomarkers with different

dietary Se concentrations and their relation to the incidence of disease in dogs should be

studied.

Chapter 1


59

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selenium (Se) in adult dogs. FASEB Journal 15151515, A992-A993. 300. European Commission (2004) List of the authorised additives in feedingstuffs (1)

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measure urine protein:creatinine ratios. Veterinary Clinical Pathology 39393939, 53-56. 313. Gardner SY, Atkins CE, Rausch WP et al. (2007) Estimation of 24-h aldosterone secretion in

the dog using the urine aldosterone:Creatinine ratio. Journal of Veterinary Cardiology 9999, 1-7.

314. Longnecker MP, Stram DO, Taylor PR et al. (1996) Use of selenium concentration in whole blood, serum, toenails, or urine as a surrogate measure of selenium intake. Epidemiology 7777, 384-390.

315. Combs GF, Jr., Watts JC, Jackson MI et al. (2011) Determinants of Selenium Status in Healthy Adults. Nutrition journal 10101010, 75.

316. Rucker RB, Fascetti AJ, Keen CL (2008) Trace Minerals. In Clinical Biochemistry of Domestic

Animals (Sixth Edition), pp. 663-693 [JJKWHL Bruss, editor]. San Diego: Academic Press. 317. Brown KM, Pickard K, Nicol F et al. (2000) Effects of organic and inorganic selenium

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318. Horvath PM, Ip C (1983) Synergistic Effect of Vitamin E and Selenium in the Chemoprevention of Mammary Carcinogenesis in Rats. Cancer Research 43434343, 5335-5341.

319. Glanemann C, Loos A, Gorret N et al. (2003) Disparity between changes in mRNA abundance and enzyme activity in Corynebacterium glutamicum:implications for DNA microarray analysis. Applied Microbiology and Biotechnology 61616161, 61-68.

320. Sunde RA (2010) mRNA transcripts as molecular biomarkers in medicine and nutrition. The

Journal of nutritional biochemistry 21212121, 665-670. 321. Evenson JK, Wheeler AD, Blake SM et al. (2004) Selenoprotein mRNA Is Expressed in Blood

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76 Chapter 1


322. Sunde RA, Raines AM, Barnes KM et al. (2009) Selenium status highly-regulates selenoprotein mRNA levels for only a subset of the selenoproteins in the selenoproteome. Bioscience Reports 29292929, 329-338.

323. Barnes KM, Evenson JK, Raines AM et al. (2009) Transcript analysis of the selenoproteome indicates that dietary selenium requirements of rats based on selenium-regulated selenoprotein mRNA levels are uniformly less than those based on glutathione peroxidase activity. Journal of Nutrition 139139139139, 199-206.

________

Scientific aims_____________________________________________________________________________________________________________

Chapter 2

Scientific aims _____________________________________________________________________________________________________

Chapter 2

Scientific aimsScientific aimsScientific aimsScientific aims

79

Selenium is an essential trace mineral in dogs and due to the many biological functions in

which it is involved via its selenoproteins, it is important to optimize Se intake in dogs. Acute

deficiencies and toxicities are rare in dogs. However, there is growing concern for the long-

term health effects, such as cancer, of Se over- and underfeeding in dogs. Bioavailability

and -activity are important indicators for the amount of Se that is available for the many

biological processes in which Se plays a role. The bioavailability and -activity of dietary Se

may be subject to many dietary factors, such as Se concentration and diet type. To

accurately assess the (potential) bioactivity of a diet and its effect on Se status, valid

biomarkers need to be determined. The current lack of a "gold-standard" assay method for

Se status in dogs makes it difficult to assess effects of Se status on long-term disease

development. In order to define accurate minimum recommendations on Se for dogs,

biomarkers that are sensitive to a change in Se status need to be determined.

The overarching objective of this PhD was to identify selected dietary factors that contribute

to the biological utilisation of dietary Se in dogs, by studying the following:

1. As a first step, the impact of a broad range of dietary factors will be assessed on

in vitro selenium accessibility.

2. The predominant factors resulting from this study will be used to determine their

effect on in vivo Se bioavailability and -activity.

3. To monitor selenium status, a large range of potential selenium biomarkers are

evaluated for their quickness of response.

The studies in this thesis will provide information on which factors contribute to the

bioavailability and -activity of selenium and how this can be measured. This is important to

provide a tool to determine long-term health effects in dogs.

In vitro seleniu _____________________________________________________________________________________________________________

Chapter 3

selenium accessibility in dog foo_____________________________________________________________________________________________________________

Chapter 3a

in dog foods _____________________________________________________________________________________________________________

"In vitro selenium accessibility in dog foods is affected by diet composition and type"

Mariëlle van Zelst1, Myriam Hesta1, Lucille G. Alexander2, Kerry Gray2, Guido Bosch3, Wouter

H. Hendriks3,4, Gijs Du Laing5, Bruno De Meulenaer6, Klara Goethals7, Geert P.J. Janssens1

1 Department of Animal Nutrition, Genetics and Ethology, Faculty of Veterinary Medicine, Ghent

University, Merelbeke, Belgium

2 WALTHAM® Centre for Pet Nutrition, Waltham-on-the-Wolds, Leicestershire, United Kingdom

3 Animal Nutrition Group, Department of Animal Sciences, Wageningen University, Wageningen,

the Netherlands

4 Department of Animal Nutrition, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The

Netherlands

5 Department of Applied Analytical & Physical Chemistry, Faculty of Bioscience Engineering, Ghent

University, Ghent, Belgium

6 Department of Food Safety & Food Quality, Faculty of Bioscience Engineering, Ghent University,

Ghent, Belgium

7 Department of Comparative Physiology & Biometrics, Faculty of Veterinary Medicine, Ghent


Adapted from British Journal of Nutrition (2015) 113: 1888-1894.

Chapter 3a

In vitroIn vitroIn vitroIn vitro selenium accessibilityselenium accessibilityselenium accessibilityselenium accessibility

83

3a.1 Abstract

Selenium (Se) bioavailability in commercial pet foods has been shown to be highly variable.

The aim of this study was to identify dietary factors associated with in vitro accessibility of

Se (Se Aiv) in pet foods. Selenium Aiv is defined as the percentage of Se from the diet that is

potentially available for absorption after in vitro digestion. Sixty-two diets (dog, n=52; cat,

n=10) were in vitro enzymatically digested: 54 were commercially available (kibble, n=20;

pellet, n=8; canned, n=17; raw meat, n=6; steamed meat, n=3) and eight were unprocessed

(kibble, n=4; canned, n=4) from the same batch as the corresponding processed diets. The

effect of diet type, dietary protein, methionine, cysteine, lysine and Se content, DM, organic

matter (OM), and crude protein (CP) digestibility on Se Aiv was tested. Se Aiv differed

significantly among diet types (p<0.001). Canned and steamed meat diets had a lower Se

Aiv than pelleted and raw meat diets. Se Aiv correlated positively with CP digestibility in

extruded diets (kibbles, n=19; r= 0.540, p=0.017) and negatively in canned diets (n=16; r= -

0.611, p=0.012). Moreover, the canning process (n=4) decreased Se Aiv (p=0.001), whereas

extrusion (n=4) revealed no effect on Se Aiv (p=0.297). These differences in Se Aiv between

diet types warrant quantification of diet type effects on in vivo Se bioavailability.

3a.2 Introduction

Selenium (Se) is an essential micronutrient that is required by dogs and cats to sustain the

basic functions of life, such as antioxidant, immune, and thyroid functions(1). There is a large

variability in Se content within and between raw materials used in pet foods(2). For example

beef muscle contains on average 11.9 µg Se/MJ with a range of 6.6 - 22.0 µg Se/MJ and

whole grain wheat contains on average 1.7 µg Se/MJ, ranging between 0.8 and 4.6 µg

Se/MJ(2). Moreover, the bioavailable Se fraction, i.e. the Se fraction that reaches the systemic

circulation(3), can also vary considerably between raw ingredients and processed pet foods,

although information is limited. Wedekind et al.(4) used a chicken bioassay and found Se

bioavailability (BA) relative to the Na2SeO3 content of pet food ingredients ranged from 9%

in mackerel to 38% in beef spleen. These authors found Se BA in a canned dog and cat

food to be 25 and 17%, respectively. In subsequent studies, using the same methodology,

greater relative Se BAs of canned (30%) and dry pet foods (53%) were reported(5). Todd et

al.(6-7) reported the Se BA of two canned cat foods, as measured in Se balance studies in

adult cats, of 25.3 and 21.2%, respectively. The reason for these variable and often low

values is unknown.

There are several factors that might underlie the variation in Se BA. An important factor is

the chemical form of Se, with organic Se being absorbed in the intestine through active

84 Chapter 3a

In vitroIn vitroIn vitroIn vitro seleniumseleniumseleniumselenium accessibilityaccessibilityaccessibilityaccessibility

transport, whereas sodium selenite appears to be absorbed through diffusion(8). Due to

competition for absorption sites, sulphur and methionine can also influence Se absorption(3; 9).

Moreover, Se BA might also be affected by factors such as fibre content(10-14) and food

processing(15-16) due to their effect on overall nutrient digestibility. Dietary fat is another

potential factor since it correlates negatively with the concentration of Se containing

enzymes (Glutathione Peroxidase, GPx) in plasma of chickens(17), suggesting that dietary fat

reduces the intestinal transport of Se. In vitro, dietary fat is also negatively associated with

Se accessibility (Se Aiv) , i.e. the dietary Se fraction in the filtrate after in vitro digestion in

milk(18).

The aim of this study was to identify dietary factors that affect the Se Aiv in commercial pet

foods. Selenium Aiv was used as an estimate for Se BA. For this study, 62 pet foods were

selected based on their variability in nutrient content and diet type.

3a.3 Experimental Methods

3a.3.1 Diet selection

A set of 54 commercial (dog, n=44; cat, n=10) and 8 unprocessed pet foods were sourced to

meet a broad range of diet types (kibble [extruded], n=24; pellet [pressed], n=8; can [retorted],

n=21; meat, n=9, [6 raw/frozen and 3 steamed]) and nutrient composition (protein content,

mean 34.4% of DM, range 7.9-93.8%; fat content, mean 20.7% of DM, range 3.1-52.4%; and

fibre content, mean 4.0% of DM, range 0.7-13.2%). A broad range in nutrient composition

was achieved based on listing label information of the parameters mentioned above. A

small number of cat foods were included to broaden the range of dietary protein content.

The number of diets per diet type was chosen to reflect the market availability of these diet

types. Diet types differed by virtue of both their processing (heat and pressure treatment)

and raw ingredients (cereals, fresh meat products, meat and/or bone meal). As the majority

of pet food producers did not include Se content or speciation on the package or website,

this information was not available for diet selection. It is possible that diet types differ in

their typical amount and speciation of Se, but this was considered as an inherent trait of the

diet types.

In addition to the commercially available diets, eight standard recipes of unprocessed diets

(kibble, n=4; can, n=4) were included in the diet selection to assess the effect of processing.

The unprocessed diets came from the same batch as the corresponding commercially

available diet that was selected. Each of the 62 diets was analysed for DM, crude ash,

crude protein (CP, 6.25× nitrogen), crude fat (Cfat), sulphur (S), amino acid profile, total dietary

Chapter 3a


85

fibre (TDF), gross energy (GE), and total selenium (Se) (methodologies as described in

"chemical analyses").

3a.3.2 Sample preparation

Dry diets were ground over a 1 mm sieve in a centrifugal mill (Retsch ZM200, Retsch GmbH,

Haan, Germany). The wet diets (canned and meat) were homogenised using a hand mixer

(Philips HR1561) and then frozen at -20°C until in vitro digestion. A portion of each wet diet

was also freeze dried and ground over a 1 mm sieve for chemical analyses.

3a.3.3 In vitro digestion

Stomach and small intestinal digestion were simulated using a modified procedure

described by Hervera et al.(19). The method consisted of a 2 h pepsin (2000 FIP U/g,

International Pharmaceutical Federation, expressed as micromoles of tyrosine equivalents

liberated per minute at 25°C, Merck art no. 7190) incubation step at a pH of 2.0 and a

second pancreatin (Porcine pancreas grade VI, Sigma no. p-1750) incubation step for 4 h at

a pH of 6.8. The method was scaled up tenfold to increase the amount of residue required

for chemical analyses. The amount of fresh matter required that equated to 10 g of DM (±

0.75) was calculated, to account for the different moisture contents of each diet. De-

mineralised water was added to all diets to achieve a moisture content of 85%. A hypoxic

environment was used by addition of CO2 for 30 seconds before every incubation step, to

prevent Se oxidation, which might have an influence on the Se Aiv(20-22). Glass covers were

placed over the beakers during incubation and pH was measured after every incubation

step. As dietary fat content may influence Se Aiv(18), samples were not defatted before

incubation, as was done in the original method, but 1.5 g bile extract (Sigma Porcine Bile

Extract B8631, Sigma Aldrich, St.Louis, MO, USA) was added to the small intestine incubation

step to mimic fat digestion. Type and amount of bile extract is based on publications of

Hedrén et al.(23), Clegg et al.(24), and Intawongse and Dean(25). Due to the larger amount of

sample, the filtration step as described by Hervera et al.(19) was not feasible. Filtration was

performed with a Büchner funnel and a nylon cloth, based on a method of Jha et al.(26),

resulting in a digested (filtrate) and undigested (residue) fraction.

The filtrates were stored at -20°C for total Se analysis. Residues were scraped from the cloth

and dried overnight in an oven at 70°C and stored at room temperature. Residues were

pooled per diet and ground to a powder before analyses for DM, crude ash, and CP. In

order to obtain at least 3 g of residue for analyses, in vitro digestion was repeated two to

nine times according to the digestibility of the diets. Diets with an in vitro DM digestibility

86 Chapter 3a


higher than 97% were eliminated from the study, because more than ten repeats would

have been necessary. With every new batch of buffers, quality controls (blanks and one of

the pelleted study diets) were incubated for assessment of repeatability between runs. The

CV for DM digestibility over the incubation runs was 0.5%. Diet filtrates were corrected for

total Se in the blank filtrates (2.31 µg/l, only containing de-mineralised water, buffers, pepsin,

pancreatin and bile solutions).

Table 3a.1 Chemical composition (g/100g DM, except where specified), gross energy content

(MJ/kg DM) and in vitro digestibility (%) of pet foods (n=60) per diet type

Kibble

(n=23)†

Pellet

(n=8)

Canned

(n=20)†

Raw Meat

(n=6)

Steamed

Meat (n=3)

Component Mean SD Mean SD Mean SD Mean SD Mean SD

DM (g/100 g as is) 92.3 1.4 90.7 0.7 24.6 7.6 35.4 3.1 27.3 4.1

Crude ash 7.5 1.8 7.6 1.4 8.9 3.0 6.9 2.5 11.2 1.1

Crude protein (N×6.25) 30.9 11.4 24.1 5.0 42.6 17.8 38.0 4.2 45.6 5.0

Lysine 1.5 0.7 1.2 0.5 2.4 1.4 2.3 0.3 3.3 0.5

Methionine 0.5 0.2 0.3 0.2 0.9 0.5 0.8 0.1 1.1 0.2

Cysteine 2.9 1.0 2.3 0.6 4.3 1.8 4.4 0.4 5.6 1.2

Crude fat 14.6 4.9 13.1 2.8 20.9 7.7 39.1 3.5 36.6 3.9

Total dietary fibre 12.6 4.8 13.1 5.7 10.5 3.6 13.6 4.5 11.3 0.8

Sulphur (mg/100 g DM) 8.0 3.0 6.1 1.9 2.6 1.2 2.8 0.7 2.7 0.4

Selenium (µg/100 g DM) 47.6 24.7 39.0 15.3 81.1 66.0 461.4 562.9 36.9 4.6

Gross energy 21.2 1.2 20.4 0.8 23.3 1.7 27.6 1.1 26.2 0.8

DM digestibility 87.8 5.7 87.0 5.8 91.4 2.6 82.8 5.3 87.1 2.1

OM digestibility 87.8 5.8 87.3 5.6 91.7 2.7 83.1 5.5 87.8 2.3

CP digestibility 93.0 3.9 93.3 2.1 95.9 2.1 92.1 3.1 96.4 0.4

DM, dry matter; MJ, megajoule; SD, standard deviation; N, nitrogen; OM, organic matter; CP, crude

protein

† Including 4 unprocessed diets

3a.3.4 Chemical analyses

Diet sample preparation for total Se analysis was adapted from Lavu et al.(27). Diets were

prepared Filtrates resulting from the in vitro digestion were pooled per sample and

transferred into a 20 ml centrifuge tube. Samples were centrifuged for 10 min at 10,000 ×g

(Sorvall RC-5B Refrigerated Superspeed Centrifuge, SA-600 rotor). Supernatants were

Chapter 3a


87

pipetted into 1.5 ml Eppendorf tubes and stored at -20°C until analysis. Samples were

analysed for total Se using inductively coupled plasma-MS (ICP-MS, Elan DRC-e, PerkinElmer,

Waltham, MA, USA), as described by Lavu et al.(27). Spikes with Certipur® selenium standard

solution (SeO2 in HNO3, 1000 mg Se/l, Merck art no. 119796) at a concentration of 200 or

400 µg/l were added as a quality control to 4 samples before and 3 samples after

microwave destruction. Selenium recoveries of 87.7% (standard deviation, SD 1.1%) and

82.6% (SD 4.5%) were obtained when spikes were added after or before microwave

destruction, respectively.

Diets and residues were analysed in duplicate for DM and crude ash by drying to a

constant weight at 103°C and combusting at 550°C, respectively. The Kjeldahl method (ISO

5983-1, 2005)(28) was used to determine CP (6.25× nitrogen). Crude fat in the diets was

assayed according to the Berntrop-method (ISO 6492, 1999)(29) and GE was analysed by

bomb calorimetry. The microwave digests prepared for Se analyses in diets, were also

analysed for S using inductively coupled plasma-optical emission spectrometry (ICP-OES, Iris

intrepid II XSP, Thermo Fisher Scientific Inc., Waltham, MA, USA) according to ISO 11885

(2007)(30). Diets were defatted by fat extraction with petroleum ether and extracted in line

with the procedure of the Commission Directive (98/64/EC)(31) for amino acid analyses; an

HPLC method was used (Agilent 1100; Fluorescence Detector; ZORBAX eclipse AAA Rapid

Resolution 4.6 × 150 mm, 3.5 micron column, PN 963400-902, Agilent Technologies, Santa

Clara, USA) according to the method of Henderson et al.(32). TDF analyses were performed

using the enzymatic-gravimetric method described by Prosky et al. (AOAC 985.29)(33).

3a.3.5 Calculations

Se Aiv was calculated with the following formula:

SeAiv�%

=Seinthe�iltrate�μ g L⁄ × [dilutionduring�� digestion �ml 1000]⁄

sampleweighedinfor�� digestion�gDM × 100Seinthediet�μg/gDM,

Digestibility coefficients were calculated with the formulae:

DMdigestibility�% = 100 −residue�gDM × 100

sampleweighedinfor�� digestion�gDM

Organicmatter�OMdigestibility�%

= 100 −residue�gDM × �100 − ashinresidue �%DM 100⁄ × 100

sampleweighedinfor�� digestion�gDM × [100 − ashinthediet �%DM 100⁄ ]

88 Chapter 3a


CPdigestibility�% = 100 − CPintheresidue�gDM × 100CPinsampleweighedinfor�� digestion�gDM

3a.3.6 Statistical analyses

Data were analysed using the Statistical Analysis System (SAS) version 9.3 for Windows (SAS

Institute Inc., Cary, NC). Data were initially screened for linearity, normality, outliers and

homogeneity of variance. The effect of diet type on Se Aiv was analysed using analysis of

variance (proc glm). Pairwise comparisons between diet types were tested at a total

significance level of 0.05 using the Tukey-Kramer adjustment for multiple comparisons. The

effect of the variables GE, CP, Fat, TDF, S, Se, lysine (Lys), cysteine (Cys), and methionine

(Met) of the diets, and calculated variables CP (g/MJ), Met/CP, Met (g/MJ), Cys/CP, Cys (g/MJ),

Lys/CP, Lys (g/MJ), Se (µg/MJ), DM digestibility, OM digestibility, and CP digestibility on Se

Aiv was analysed, per diet type, using regression (proc reg). The effect of processing was

analysed with a paired Student's t-test. In all cases statistical significance was evaluated at

p≤0.05.

Table 3a.2 In vitro selenium accessibility of pet foods (%) per diet type

Range

Diet type n mean min max SD

Kibble† 19 72a,b 37 97 17

Pellet 8 79a 62 101 13

Canned† 16 58b 29 91 16

Raw Meat 6 91a 79 98 23

Steamed Meat 3 47b 39 54 8

n, number of animals; min, minimum; max, maximum; SD, standard deviation

a,b Groups with a common letter in superscript do not significantly differ

† Unprocessed diets are not included in the data

3a.4 Results

One commercial canned diet and one commercial extruded diet were eliminated for further

analyses based on an in vitro DM digestibility >97%. The inter-assay CV for DM

digestibility over the incubation runs was 0.5%. pH remained constant within each

incubation step. Total Se in the blank samples was on average 2.31 µg/l, which was used to

correct Se in filtrates. Selenium analyses had a recovery of 87.7% as measured using spiked

Chapter 3a


89


crude protein digestibility (%) of pet foods

Solid trend line, all diets (n=52); dotted trend line and ♦, kibble (n=19); ƒ, pellet (n=8);

striped trend line and ∆, canned (n=16); o, raw meat (n=6); X, steamed meat (n=3).

Values with an asterisk show a significant correlation (p<0.05). Unprocessed diets are

not included in the data.

samples after microwave destruction and 82.6% when samples were spiked before

microwave destruction.

Table 3a.1 provides an overview of the chemical composition and digestibility results of the

diets per diet type. The high variation in CP, Cfat and TDF reflect the diet selection criteria.

The large range in Se content of the diets was mainly due to two high values from raw

meat diets (1173.2 and 1195.5 µg/100 g DM), which also increased the mean value over all

diets (98.4 µg/100 g DM, results not shown). The median for dietary Se over all diets was

44.8 µg/100 g DM (results not shown).

Selenium Aiv differed among diet types (p< 0.001) (Table 3a.2). Canned and steamed meat

diets had lower Se Aiv than pelleted and raw meat diets. Within all diet types, a large range

of Se Aiv was found, but the range was largest for kibble and canned diets. There was no

significant correlation between Se Aiv and CP digestibility when all diet types were pooled

(Figure 3a.1: r= -0.095, p= 0.504). However, when diet type was considered, there was a

positive correlation between Se Aiv and in vitro CP digestibility within the kibble diets (r=

0.540, p= 0.017) versus a negative correlation within the canned diets (r= -0.611, p= 0.012).

r = - 0.095all diets

r = 0.540*kibble

r = - 0.611*canned

0

20

40

60

80

100

120

80 85 90 95 100 105

In v

itro

sele

niu

m a

ccess

ibili

ty (%

)

Crude protein digestibility (%)

90 Chapter 3a


Figure 3a.2 Correlations (r) between in vitro selenium accessibility (%) and parameters of

canned (black bars, n=16) and kibble (grey bars, n=19) diets in g/100g DM, except where

specified

dig, digestibility; MJ, megajoule. Asterisks indicate the level of significance: * p<0.05, **p<0.001.

Unprocessed diets are not included in the data.

Selenium Aiv was not significantly correlated to any of the measured parameters in the

pelleted diets. Raw meat diets showed a negative correlation between the amount of Se in

the diet and the Se Aiv (in µg/g DM: r = -0.823, p= 0.044; in µg/MJ: r = -0.843, p= 0.035). For the

steamed meat diets a positive correlation was found between Se Aiv and the amount of

cysteine per MJ (r = 0.999, p= 0.012). The correlations for the canned and kibble diets are

displayed in Figure 3a.2. Among the kibble diets a negative correlation was found between

Se Aiv and dietary CP, Lys, Cys, Met, S, ash, CP/MJ, Cys/MJ, and Lys/MJ. In canned diets Se

Aiv was negatively correlated with dietary TDF.

The extrusion process did not affect Se Aiv (p=0.297, Table 3a.3). In contrast, retorting almost

halved Se Aiv (p=0.001). Dietary Se concentrations did not differ before and after processing

(canned p=0.863, kibble p=0.335; results not shown). In the canned diets, the S content was

higher before processing (p=0.028) and the TDF content tended to be higher after

processing (p=0.085).

-0.061

0.187

-0.258

-0.548*

0.038

-0.063

-0.054

-0.071

0.245

0.230

-0.382

0.102

0.086

-0.272

0.174

0.161

0.389

0.363

-0.169

-0.205

-0.422

-0.545*

-0.052

-0.016

-0.479*

-0.056

-0.100

0.237

-0.443

-0.480*

0.261

-0.749**

-0.765**

-0.077

-0.808**

-0.802**

-0.752**

-0.769**

0.317

0.288

-1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6

Gross energy (MJ/kg DM)

Crude ash

Crude fat

Total dietary fibre

Sulphur (mg/100g DM)

Se (µg/MJ)

Se (µg/g DM)

Met/Crude protein

Met (g/MJ)

Met

Cys/Crude protein

Cys (g/MJ)

Cys

Lys/Crude protein

Lys (g/MJ)

Lys

Crude protein (g/MJ)

Crude protein

Organic matter dig (%)

Dry matter dig (%)

Correlation (r) with in vitro selenium accessibility

Chapter 3a


91

Table 3a.3 In vitro selenium accessibility (%) in processed versus

unprocessed canned and kibble diets

Range

Diet category n mean min max SD p*

Unprocessed kibble 4 60 53 68 8 0.297

Processed kibble 4 67 52 77 11

Unprocessed canned 4 102 83 112 13 0.001

Processed canned 4 53 29 67 17

n, number of animals; min, minimum; max, maximum; SD, standard deviation * p-value from paired Student's t-test

3a.5 Discussion

To verify the factors that may contribute to the Se BA of pet foods, this study examined a

large range of commercially available pet foods differing in diet type and protein, fat, and

fibre content. There was a clear difference in Se Aiv between diet types (canned and

steamed meat Se Aiv < pellet and raw meat Se Aiv) which may have been due to the way

the diets were processed. A number of factors that influence the processing of pet foods

may have contributed to the observed variation such as time, temperature, pressure and

shear. Typically, raw meat diets and pelleted diets, which undergo no or relatively low heat

treatment, showed a higher Se Aiv (91 and 79%, respectively) compared to steamed meat

and canned diets (47 and 58%, respectively). These findings suggest a negative effect of

heat processing on Se accessibility. This is in accordance with findings of Todd(16), who

found that non-processed inorganic Se in canned diets had a higher apparent Se

absorption in cats (83.3%) than processed inorganic Se (53.7%)(16). Canned diets had a

higher Se Aiv before, compared to after processing, which also indicates an effect of

processing on Se Aiv, which was not demonstrated in the kibble diets. The differences in the

effect of processing on Se Aiv between canned and kibble diets may be due to variations

in Maillard reactions caused by the different processing types. In the baking process, some

Se may be lost because the Maillard reaction of SeMet and glucose yields volatile

seleniferous compounds(34). This did not appear to have a major influence on total Se in this

study because total dietary Se did not differ before and after processing. Processing did

decrease the S content in canned diets, which might be due to the conversion of dietary S

to volatile compounds and volatilisation after opening the processed cans. Another possible

effect in heat processed diets is that cross-linkages between amino acids, within and

92 Chapter 3a


between proteins occur. Cross-linking reduces the rate of protein digestion by preventing

enzyme penetration or by masking the sites of enzyme attack(35). Cysteine seems to be one

of the most susceptible amino acids for cross-linking(36), by which Se linked to Cys might be

less available for digestion.

Hendriks et al.(15) showed that heat processing a cat food at 121°C for 80-120 minutes did

not destroy amino acids, but that it did decrease ileal apparent digestibility of the diet. In

this study, there was no impact on CP digestibility when comparing the same diets pre- and

post-processing. However, the effect of CP digestibility on Se Aiv differed between canned

and kibble diets. In kibble diets, there was a positive relationship between CP digestibility

and Se Aiv. This may indicate that when more protein is digested, more protein bound-Se

becomes available. Similar findings were reported by Shen et al. in milk products(18).

Interestingly, the opposite effect was found for canned diets. The lack of correlation

between Se Aiv and Cys, Met and Lys suggests that they are not an explanation for the

negative correlation between CP digestibility and Se Aiv in canned diets. Therefore, this

correlation is likely due to a factor that was not accounted for in this study.

The source of TDF in the canned diets could play a role in Se Aiv, because fibre is known

to reduce nutrient digestibility(10-14). Total dietary fibre in canned diets in the present study

was negatively correlated with Se Aiv and tended to be increased in canned diets after

processing compared to TDF in unprocessed diets (p=0.085). Azizah and Zainon(37) also found

an increase in TDF after roasting wheat, rice, mung beans and soya beans at 80°C for 5

min. During heat treatment, fibre-protein complexes can be formed(38) which might be the

cause for the negative impact on Se Aiv and the tended increase in TDF. A particular TDF

component that is commonly used in canned diets is the soluble non-starch polysaccharide

guar gum. Guar gum increases viscosity and has been shown to decrease the digestibility

of protein in diets in cats(39) and dogs(40), which can be an additional explanation for the

negative relationship between TDF and Se Aiv in canned diets, because protein bound-Se

may then not become available. Choe and Kies(41) reported an increase in faecal Se

excretion by 14% and a decreased Se balance (53%) and whole blood GPx activity (9%) in

humans when guar gum was supplemented to a standardised diet, although, in their study

both Se and guar gum where not processed.

The difference in Se Aiv between the diet types might also be explained by a difference in

the raw materials that are used in their manufacture, and consequently, the Se species in

the diets. Supplemental Se in the form of sodium selenite or sodium selenate is commonly

employed in dry pet foods (pellet and kibble), whereas, in canned diets Se is mainly present

in the form of SeMet from raw materials. Furthermore, reactions of sodium selenite with other

Chapter 3a


93

components during storage may change its speciation, possibly to elemental Se(42). Due to

detection limit issues, Se speciation was not analysed in this study, however it may have an

effect on the Se BA(6; 20-22; 43).

Finally, the very high Se content in two of the raw meat diets could be due to the type of

raw materials used. Tissues with a high rate of protein synthesis such as erythrocytes,

skeletal muscle, pancreas, liver and kidney generally contain high amounts of Se(44).

This study aimed to identify factors that influence Se Aiv. It is possible that in vitro Se Aiv

from the pet foods in this study may differ quantitatively from what may be found in vivo.

Hervera et al.(45) confirmed that apparent in vivo CP digestibility is lower than when

measured in vitro. The current European recommended allowance (RA) set by the European

Pet Food Industry Federation (FEDIAF)(46) and the adequate intake of the National Research

Council (NRC)(1) for Se in dogs and cats only take into account a fixed Se BA percentage,

despite the large number of factors that influence the BA. The Association of American

Feed Control Officials (AAFCO)(47) do not give any information on which bioavailability

factor they have used for the RA of Se in pet foods. The results of this study can be used to

help design in vivo studies to confirm and quantify the impact of diet composition and type

on Se Aiv found in this study. This may enable the pet food industry to formulate diets that

meet canine Se requirements by taking into account the Se BA for each specific diet type.

3a.6 Conclusions

This study reported that diet type and processing affect Se Aiv. Among other factors, crude

protein digestibility is positively correlated with Se Aiv in kibble diets, but negatively in

canned diets. Retorting strongly decreased Se Aiv. In vivo work is warranted to confirm

these in vitro data and to verify if recommendations of Se inclusion levels in complete and

balanced diets need to take such factors into account.

3a.7 Acknowledgements

Special thanks to Joachim Neri of the Department of Applied Analytical and Physical

Chemistry at the Faculty of Bioscience Engineering of Ghent University for total Se analyses

and Sofie Coelus of the Department of Food Safety and Food Quality at the Faculty of

Bioscience Engineering of Ghent University for assistance during amino acid analyses.

3a.8 References


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Predictive equations ofselenium

___________________________________________________

Chapter 3b

Predictive equations ofselenium accessibility in dry pet foods

_____________________________________________________________________________________________________________

Chapter 3b

Predictive equations of in vitro dry pet foods

__________________________________________________________

"Predictive equations of selenium accessibility in dry pet foods"

Mariëlle van Zelst1, Myriam Hesta1, Kerry Gray2, Klara Goethals3, Geert P.J. Janssens1




3 Department of Comparative Physiology & Biometrics, Faculty of Veterinary Medicine, Ghent


Submitted as short communication to Journal of Animal Physiology and Animal

Nutrition

Chapter 3b

PredictiPredictiPredictiPredictiveveveve equationsequationsequationsequations

99

3b.1 Abstract

The trace element selenium is essential to both dogs and cats. Dry diets are formulated with

a large range of ingredients, which may vary in selenium concentration and accessibility.

This paper reports equations to predict the average in vitro selenium accessibility from dry

pet foods based on essential dietary nutrient concentrations, including crude protein, amino

acids and crude fat. Predictive equations were made using stepwise linear regression for

extruded and pelleted diets. The equations can be used to aid diet formulation to optimize

selenium accessibility within the diet and to prevent selenium deficiency or toxicity.

3b.2 Introduction

Selenium (Se) is an essential micro-mineral involved in anti-oxidant protection, thyroid

metabolism and other biological processes(1). Acute Se deficiency in puppies has been

reported to cause muscular weakness, depression, subcutanous oedema, dyspnoea and

eventual coma(2). High Se intake may be associated with reduced food intake, hypochromic

microcytic anemia and severe liver damage in dogs(3). No deficiency and toxicity signs have

been reported for cats(4).

Dry pet foods are the most popular commercial diet type selected for pet dogs(5). Dry pet

foods can be processed by either extruding to kibbles or pelleting. Pellets and kibbles are

formulated with a large range of both plant and animal derived ingredients. Depending on

the region of origin and type of ingredient, the Se concentration in pet food ingredients

can differ(6-7). In addition, the bio-availability of Se from pet foods has been reported to be

low(8) and dependent on various factors such as protein, fat, sulphur, and fibre content of the

diet(9). Therefore, the total dietary Se concentration may not reflect the amount of Se that is

available to the animal.

This study aimed to develop equations to predict the average availability of Se from dry

pet foods, expressed as in vitro accessibility (Aiv). This formed an estimation for in vivo

availability. Accessibility here is defined as the percentage of dietary Se that is potentially

available for absorption after in vitro digestion.

3b.3 Experimental methods

3b.3.1 Diets and chemical analyses

Commercially available dry pet foods (23 dog, 4 cat); 19 extruded (15 dog, 4 cat) and 8

pelleted (all dog), were in vitro digested according to a method adapted from Hervera et

al.(10). Full sample preparation and analysis methods for Se are described in van Zelst et al.(7).

Residues (undigested fractions) were analysed for DM, ash, and N, and filtrates (digested

100 Chapter 3b

PredictiPredictiPredictiPredictive equationsve equationsve equationsve equations

fractions) for total Se. Diets were analysed for dry matter (DM, % as is), ash (% DM), nitrogen

(N, ×6.25 = crude protein (CP) % DM), crude fat (Cfat, % DM), sulphur (S, mg/100g DM), amino

acids (% DM), total dietary fibre (TDF, % DM), gross energy (GE, MJ/kg), and total Se (mg/kg

DM). Total Se concentration was determined using inductively coupled plasma-mass

spectrometry (ICP-MS, Elan DRC-e, PerkinElmer, Waltham, MA, USA). Diets and residues were

analysed in duplicate for DM and crude ash by drying to a constant weight at 103°C and

combusting at 550°C, respectively. The Kjeldahl method (ISO 5983-1)(11) was used to

determine CP (6.25× nitrogen). Cfat in the diets was assayed according to the Berntrop-

method (ISO 6492)(12) and GE was analysed by bomb calorimetry. Sulphur was determined

using inductively coupled plasma-optical emission spectrometry (ICP-OES, Iris intrepid II XSP,

Thermo Fisher Scientific Inc., Waltham, MA, USA) according to ISO 11885(13). Diets were

defatted by fat extraction with petroleum ether and extracted in line with the procedure of

the Commission Directive (98/64/EC)(14) for amino acid analyses; an HPLC method was used

(Agilent 1100; Fluorescence Detector; ZORBAX eclipse AAA Rapid Resolution 4.6 × 150 mm,

3.5 micron column, PN 963400-902, Agilent Technologies, Santa Clara, USA) according to the

method of Henderson et al.(15). TDF analyses were performed using the enzymatic-gravimetric

method described by Prosky et al.(16).

3b.3.2 Calculations

Se Aiv was calculated with the following formula:

SeAiv�% = 4567895�6:8;<85�=> ?⁄ ×[@6:A86B7@A;67>CDECFGH@6>5I86B7�J: KLLL]⁄I<JM:5N56>95@67OB;CDECFGH@6>5I86B7�>PQ×KLL Seinthediet�μg/gDM,

Digestibility coefficients were calculated with the formulae:

DMdigestibility�% = 100 − ;5I6@A5�>PQ×KLLI<JM:5N56>95@67OB;CDECFGH@6>5I86B7�>PQ

Organicmatter�OMdigestibility�% =100 − ;5I6@A5�>PQ×�KLLR<I967;5I6@A5�%PQ KLL⁄ ×KLL

I<JM:5N56>95@67OB;CDECFGH@6>5I86B7�>PQ×[KLLR<I967895@658�%PQ KLL⁄ ]

CPdigestibility�% = 100 − ST67895;5I6@A5�>PQ×KLLST67I<JM:5N56>95@67OB;CDECFGH@6>5I86B7�>PQ

4.3.3 Statistical analyses

Data were initially screened for normality, outliers and homogeneity of variance. All data

were analysed using stepwise linear regression in Statistical Analysis System (SAS) software

Chapter 3b


101

version 9.3 for Windows (SAS Institute Inc., Cary, NC) to construct predictive equations. A

simple equation, excluding in vitro digestibility coefficients and amino acids, and a complex

equation, including all measured parameters, were constructed for all dry diets and

separately for the extruded diets. Differences in nutrient composition between the diet types

was analysed by an analysis of variance in SPSS version 21.0, with diet type as factor and

dietary and digestibility parameters as dependent variables. In all cases statistical

significance was evaluated at p≤0.05. Results are reported as mean ± standard error.

3b.4 Results

The chemical composition, in vitro digestibility coefficients and Se Aiv of the diets are shown

in Table 3b.1. Neither dietary Se concentration nor Se Aiv differed between the diet types

(p=0.289 and 0.303, respectively). Se Aiv was on average 73.8% (Kibble: 71.7%; pellet: 78.9%).

Dry matter, methionine, Cfat and GE content were higher in the kibble diets compared to

the pellets (p=0.005, 0.037, 0.046, and 0.017, respectively).

The variables in the complex predictive equation for dry diets were shown to be good

predictors of Se Aiv (n=27; R2=0.86; p<0.001):

Se Aiv (%) = -151 (±37) - 19 (±2) lysine (% DM) + 2.1 (±0.5) CP digestibility (%) + 0.6 (±0.3) DM

digestibility (%).

When only extruded diets were considered, a different predictive equation with a higher

coefficient of determination was obtained (n=19; R2=0.92; p<0.001):

Se Aiv (%) = -90 (±34) - 1.6 (±0.7) ash (% DM) - 18.0 (±1.9) lysine (% DM) + 2.1 (±0.3) CP

digestibility (%).

Coefficients of determination were lower for the simple predictive equations. For all dry

diets (n=27; R2=0.59; p<0.001):

Se Aiv (%) = 104 (±6) + 2.6 (±1.3) S (mg/100g DM) - 1.8 (±0.4) CP (% DM).

For extruded diets a similar predictive equation was found (n=19; R2=0.71; p<0.001):

Se Aiv (%) = 104 (±7) + 3.5 (±1.4) S (mg/100g DM) - 2.0 (±0.4) CP (% DM).

102 Chapter 3b


Table 3b.1 Chemical composition (g/100g DM, except where specified), gross energy

content (MJ/kg DM), in vitro digestibility and selenium accessibility (%) of dry pet foods

(n=27)

Kibble (n=19) Pellet (n=8)

Component Mean SE min max Mean SE min max p

Dry matter (g/100 g as is) 92.4 0.3 89.9 95.1 90.7 0.3 89.9 92.3 0.005

Crude ash 7.3 0.4 3.1 9.3 7.6 0.5 6.2 10.0 0.664

Crude protein (N×6.25) 29.7 2.6 11.2 49.2 24.1 1.8 14.4 30.7 0.193

Methionine 0.5 0.1 0.1 0.9 0.3 0.1 0.1 0.7 0.037

Cysteine 2.9 0.2 1.3 4.8 2.3 0.2 1.2 3.1 0.187

Lysine 1.5 0.2 0.6 3.3 1.2 0.2 0.5 1.8 0.234

Crude fat 16.2 0.9 8.5 22.6 13.1 1.0 8.5 16.4 0.046

Total dietary fibre 12.3 1.0 6.0 22.3 13.1 2.0 6.5 26.1 0.681

Sulphur (mg/100 g DM) 7.6 0.7 3.0 14.5 6.1 0.7 3.2 8.6 0.208

Selenium (µg/100 g DM) 49.9 6.1 20.9 129.6 39.0 5.4 22.6 56.2 0.289

Gross energy 21.4 0.2 19.0 23.4 20.4 0.3 19.2 21.4 0.017

Dry matter digestibility 89.0 1.1 79.5 96.6 87.0 2.0 73.8 91.7 0.351

Organic matter digestibility 89.0 1.1 79.5 96.6 87.3 2.0 74.5 92.0 0.444

Crude protein digestibility 93.6 0.8 84.7 99.7 93.3 0.7 90.2 96.5 0.842

Selenium Aiv 71.7 4.0 36.9 97.4 78.9 4.8 62.2 101.0 0.303

p-values indicate whether dietary components differ between the kibble and pelleted diets, true at

p≤0.05. DM, dry matter; MJ, megajoule; n, number of animals; SE, standard error; min, minimum

value; max, maximum value; N, nitrogen; Aiv, in vitro accessibility

The predicted versus observed data has been plotted in Figures 3b.1 and 3b.2.

Chapter 3b


103

Figure 3b.1 Predicited versus observed in vitro selenium

accessibility in kibble and pelleted diets (n=27)

Solid trend line and solid bullets, complex predictive equation; dotted

trend line and open bullets, simple predictive equation.

Figure 3b.2 Predicited versus observed in vitro selenium

accessibility in kibble diets (n=19)

Solid trend line and solid bullets, complex predictive equation; dotted

trend line and open bullets, simple predictive equation.

Complex: R² = 0.86

Simple: R² = 0.59

0

20

40

60

80

100

120

0 20 40 60 80 100 120

Pre

dic

ted in v

itro

sele

niu

m

acc

ess

ibili

ty (%

)

Observed in vitro selenium accessibility (%)

Complex: R² = 0.92

Simple: R² = 0.71

0

20

40

60

80

100

120

0 20 40 60 80 100 120

Pre

dic

ted in v

itro

sele

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acc

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ibili

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)

Observed in vitro selenium accessibility (%)

104 Chapter 3b


3b.5 Discussion

This study reported average predictive equations of Se Aiv of dry pet foods. The large

range of Se Aiv in this study is evidence for the highly variable Se provision in dry pet

foods. The complex equations constructed in this study can be used in nutrition research for

the formulation of new diets to optimize Se accessibility. For cases where in vitro

digestibility and/or amino acids cannot be determined, such as in routine food formulation

in practice, the simple predictive equations were formulated. These can be used to obtain

an indication of the dietary Se availability to prevent Se deficiency or toxicity caused by a

diet.

The negative intercepts of the predictive equations suggest a negative Se Aiv when all

parameters are zero. This is, however, very unlikely. Although this study has been performed

with a wide range in all dietary parameters measured, it should be kept in mind that the

predictive equations only apply for parameters within this range.

The equations based only on the extruded diets provided a prediction with a higher

confidence than the model for all dry diets. Therefore, these can best be used for extruded

diets, which is the majority of commercially available dry pet foods(17). The lower coefficients

of determination of the equations for pelleted plus extruded dry diets compared to

extruded diets only, may be due to a difference in ingredients used or the different

conditions under which the diets are processed. Pellets are processed using lower

temperatures and less shear compared to kibbles(18), which may have a beneficial effect on

the protein quality(19). However, no difference in crude protein digestibility was found in this

study. Tran et al.(18) reported a lower dietary Cfat and starch concentration in pellets than

kibbles, but only four diets per diet type were included in their study. The current study

found the opposite for Cfat. Starch concentration was not analysed in this study. The

difference in Cfat, DM, methionine and GE content between the diet types may have

contributed to the lower coefficients of determination when both diet types were included

for the formation of the equation, although this study does not allow attribution of causality.

Although the parameters in the equations do not necessarily have a causal relationship

with Se Aiv, some of the parameters have previously been associated with the biological

availability of Se, meaning the amount of dietary Se that reaches the systemic circulation.

Sulphur for example, has been reported to decrease the Se availability in sheep(20). In both

simple models of this study it was however positively associated with Se Aiv. An explanation

for this may be that a larger amount of S-containing amino acids may provide more binding

sites for Se, in the case of organically bound (selenomethionine and selenocysteine) dietary

Se. Also, the incorporation of DM and CP digestibility (+) in the complex equations may be

Chapter 3b


105

caused by a higher amount of Se that becomes available from digested amino acids. A

reduction in digestibility may result from an increase in dietary collagen(21), which increases

ash concentration, and thus, may be the cause for inclusion of ash (-) in the complex

equation for extruded diets. Lysine is involved in the Maillard reaction(22), which decreases

the protein quality(23). A reduction in protein quality may reduce the release of protein-

bound Se, which may be a reason for lysine (-) to be in the complex predictive equations.

The inclusion of CP (-) in the simple equations cannot be explained in this context, but

again, the relationship between the factors in the models and Se Aiv might not be causal.

The in vivo Se bioavailability of diets is expected to be somewhat lower than the Se Aiv.

This is because in vitro digestion mimics in vivo gastro-intestinal digestion, but does not

contain all facets, such as luminal absorption. Nevertheless, this study provided useful tools

to predict differences in availability of Se from dry pet foods.

3b.6 References


2. van Vleet JF (1975) Experimentally induced vitamin E-selenium deficiency in the growing dog. Journal of the American Veterinary Medical Association 166166166166, 769-774.

3. Levander O (1986) Selenium. In Trace elements in human and animal nutrition, 5th ed., pp. 209-279 [W Mertz, editor]. Orlando: Academic Press.


nutrition series. Washington D.C.: National Academies Press. 5. Pet Food Manufacturers' Association (2015) Market Data.

http://www.pfma.org.uk/market-data (accessed 8 August 2015). 6. Reilly C (2006) Selenium in food and health. 2nd ed. New York: Springer Science and

Business Media LLC. 7. van Zelst M, Hesta M, Alexander LG et al. (2015) In vitro selenium accessibility in dog

foods is affected by diet composition and type. British Journal of Nutrition 113113113113, 1888-1894.


9. Thiry C, Ruttens A, de Temmerman L et al. (2012) Current knowledge in species-related bioavailability of selenium in food. Food Chemistry 130130130130, 767-784.

10. Hervera M, Baucells MD, Blanch F et al. (2007) Prediction of digestible energy content of extruded dog food by in vitro analyses. Journal of Animal Physiology and Animal Nutrition 91919191, 205-209.



12. ISO (1999) Animal Feeding Stuffs - Determination of fat content (ISO 6492). Geneva, Switzerland: International Organization for Standardization.

106 Chapter 3b


13. ISO (2007) Determination of selected elements by inductively coupled plasma optical

emission spectrometry (ICP-OES, ISO 11885). Geneva, Switzerland: International Organization for Standardization.

14. Commission Directive (1998) Establishing Community methods of analysis for the determination of aminoacids, crude oils and fats, and olaquindox in feedingstuffs and amending, Directive 71/393/EEC. Official Journal of the European Communities L 257L 257L 257L 257, 14-28.

15. Henderson JW, Ricker RD, Bidlingmeyer BA et al. (2000) Rapid, acurate, sensitive and reproducible HPLC analysis of amino acids. In Application note 5980-1193E: Agilent Technologies.

16. Prosky L, Asp NG, Furda I et al. (1985) Determination of Total Dietary Fiber in Foods and Food Products - Collaborative Study. Journal of the Association of Official Analytical

Chemists 68686868, 677-679. 17. Spears JK, Fahey GC (2004) Resistant Starch as Related to Companion Animal Nutrition.

Journal of AOAC International 87878787, 787-791. 18. Tran QD, van Lin CGJM, Hendriks WH et al. (2007) Lysine reactivity and starch

gelatinization in extruded and pelleted canine diets. Animal Feed Science and Technology 138138138138, 162-168.

19. Hendriks WH, Emmens MM, Trass B et al. (1999) Heat processing changes the protein quality of canned cat foods as measured with a rat bioassay. Journal of Animal Science 77777777, 669-676.

20. Netto AS, Zanetti MA, Correa LB et al. (2014) Effects of dietary selenium, sulphur and copper levels on selenium concentration in the serum and liver of lamb. Asian-

Australasian Journal of Animal Sciences 27272727, 1082-1087. 21. El SN (1995) Evaluating protein quality of meats using collagen content. Food Chemistry

53535353, 209-210. 22. Moughan PJ, Rutherfurd SM (1996) A New Method for Determining Digestible Reactive

Lysine in Foods. Journal of Agricultural and Food Chemistry 44444444, 2202-2209. 23. Rutherfurd SM, Moughan PJ (1997) Application of a New Method for Determining

Digestible Reactive Lysine to Variably Heated Protein Sources. Journal of Agricultural and

Food Chemistry 45454545, 1582-1586.

Association between selenium bioavailability

______________________________________________________________

ssociation between diet typeavailability and -activity

_____________________________________________________________________________________________________________

Chapter 4

diet type and activity in dogs

_______________________________________________

"Selenium digestibility and bioactivity in dogs: what the can can, the kibble

can’t"

Mariëlle van Zelst1, Myriam Hesta1, Kerry Gray2, Karen Beech2, An Cools1, Lucille G.

Alexander2, Gijs Du Laing3, Geert P.J. Janssens1*






Submitted to PLOS ONE

Chapter 4

SSSSelenium bioavailabilityelenium bioavailabilityelenium bioavailabilityelenium bioavailability and and and and ----activityactivityactivityactivity

109

4.1 Abstract

There is a growing concern for the long-term health effects of selenium (Se) over- or

underfeeding. The efficiency of utilization of dietary Se is subject to many factors. Our study

in dogs evaluated the effect of diet type (canned versus kibble) and dietary protein

concentration on Se bioavailability and -activity. Canned and kibble diets are commonly

used formats of dog food, widely ranging in protein concentration. Twenty-four Labrador

retrievers were used and four canned and four kibble diets were selected with crude

protein concentrations ranging from 10.1 to 27.5 g/MJ. Crude protein concentration had no

influence on the bioavailability of Se in either canned or kibble diets, but a lower Se

bioavailability was observed in canned compared to kibble diets. However, the biological

activity of Se, as measured by whole blood glutathione peroxidase, was higher in dogs fed

the canned diets than in dogs fed the kibble diets and decreased with increasing crude

protein intake. These results indicate that selenium recommendations in dog foods need to

take diet type into account.

4.2 Introduction

Selenium (Se) is an essential trace element, primarily to protect against oxidative stress(1).

Current dietary recommendations include a safety margin to avoid loss of antioxidant

protection at low concentrations(2) and the risk of Se toxicity at high concentrations(3). In

addition, data on the importance of Se intake on long-term health are emerging that give

rise to other concerns. There are indications that Se is involved in the prevention of

diseases such as cancer(4-7) and cardiovascular diseases(8-10) and impaired immune function(11-

12), etc. On the other hand, the Se and vitamin E cancer prevention trial (SELECT) reported an

increased risk of diabetes mellitus type 2 in humans with a high Se intake (200 µg

selenomethionine/day)(13). The effects of selenium intake on the health and longevity of

companion animals such as the dog is now also coming under scrutiny(14-15). The current

recommended allowance for Se in dog food takes into account a bioavailability factor, but

is not diet type specific(16), despite several studies reporting a large variation in Se

bioavailability between diet types(17-20). We have previously shown in 20 canned and 23

kibble diets, the average amount of Se was higher in canned than kibble diets (34.8 vs. 22.5

µg/MJ, respectively). Also, apparent crude protein (CP) digestibility had a significant effect

on in vitro Se accessibility(20), i.e. the dietary Se fraction in the filtrate after in vitro digestion.

The reasons for the differences in Se bioavailability between canned and kibble diets may

be due to the way the diets are processed or the raw materials used during manufacture.

The raw materials used for pet foods vary greatly in their Se concentration(21-22) and

110 Chapter 4


availability(23). Canned diets typically contain higher concentrations of meat and/or meat

by-products, whilst kibble diets contain mainly cereal grains and cereal grain by-products(22).

Wedekind et al.(18) tested the availability of Se in several pet food ingredients and found a

range of 9% in mackerel to 38% in beef spleen when compared to the availability of

sodium selenite. Due to the differences in ingredients, it is likely that the chemical form of Se

in the diet will also be different between canned and kibble diets, which is known to be an

important factor for the bioavailability of Se(24-25).

Canned and kibble diets also differ in the way of processing. Kibble diets are extruded and

canned diets are retorted, resulting in differential effects of heat, pressure and shear which

may also impact on Se bioavailability. The differences between canned and kibble diets

may not only cause a variation in Se bioavailability between diet types, but also the

bioactivity, defined as the amount of Se that can be used for the incorporation into

selenoproteins. The antioxidant glutathione peroxidase (GPx) is often used as a measure of

Se bioactivity(26-30). In addition, serum isoprostanes (IsoPs) are a measure for lipid

peroxidation(31) and are used as an indicator of total antioxidant status. Selenium is not only

incorporated into GPx, but also into iodothyronine deiodinases, of which type I and II are

involved in the transformation of thyroxine (T4) into triiodothyronine (T3)(32-33). Olivieri et al.(33)

has shown that selenium status was positively correlated with the ratio of T3:T4 in humans

and Wedekind et al.(29) have reported similar results in puppies.

Based on the in vitro results(20), it was hypothesized that canned diets have a lower Se

bioavailability than kibble diets and that CP concentration (as the main factor for the

ingested amount of digestible protein) is positively associated with Se bioavailability in

kibble diets and negatively in canned diets in vivo. To test this hypothesis, two different

diet types (canned and kibble) and four different concentrations of CP per diet type were

selected.

4.3 Experimental methods

4.3.1 Study design

The study consisted of four feeding periods with six transition days and experimental

periods of between 29-43 days. Four groups of each six dogs were selected. Each dog

group was fed four of the eight experimental diets (2 canned, 2 kibble) in a randomised

incomplete cross-over design. Blood, urine and faecal samples were taken at the end of

every feeding period. This study (HO0647) was approved by the WALTHAM Centre for Pet

Nutrition Animal Welfare and Ethical Review Body and was conducted under Home Office

Project License authorisation.

Chapter 4


111

4.3.2 Dogs

Twenty-four adult Labrador retrievers, of which 15 female (14 neutered, 1 entire) and 9 male

(all neutered), were selected for this study. At the start of the study, the average age of the

dogs was 4.2 years (range 2.0 - 8.1) and the average body weight (BW) was 27.9 kg (range

23.7-32.7 kg). The dogs were fed twice daily (8:30 and 15:00) to maintain BW and body

condition score (BCS) at ideal (D) on the S.H.A.P.E™ (Size, Health And Physical Evaluation)

BCS-scale(34). BW and BCS were recorded weekly and food intake daily. Dogs had access to

fresh drinking water at all times. Dogs were divided into four treatment groups of six dogs,

with age, BW and gender as blocking factors. Dogs were housed in triplets with others of

the same diet group indoors with continuous access to a concrete outside pen. Between

9:00 and 14:30, the dogs also had access to a concrete outside paddock and they were

socialized at least once a day (e.g. toy play or walk). During the last six days of each

feeding period, dogs were housed individually with access to the outside paddock during

the day. The average age, body weight and energy intake in kJ/kg BW0.75 per dog group

are shown in Table 4.1.

Table 4.1 Gender, age, body weight and energy intake of the study dogs per dog group

Age (years) Body Weight (kg)

Energy Intake

(kJ/kg BW0.75)

Group n ♂ ♀ Mean Min Max Mean Min Max Mean Min Max

A 6 3 3 4.7 2.8 8.1 28.6 26.3 32.7 440 360 561

B 6 2 4 3.7 2.3 5.9 28.3 26.6 32.2 443 360 645

C 6 1 5 5.2 3.4 6.7 27.3 23.7 31.5 414 360 578

D 6 3 3 3.3 2.0 5.0 27.3 23.7 31.0 443 360 722

kJ, kilojoule; BW0.75, metabolic body weight; n, number of animals; ♂, male; ♀, female; Min, minimum;

Max, maximum

4.3.3 Diets

Eight commercially available diets were selected that varied in diet type (canned or kibble)

and protein concentration (Table 4.2). Commercial diets were chosen for practical relevance

of the study, and the difference in Se and other nutrient concentrations between the

canned and kibble diets are considered inherent to the diet types. For every diet type,

diets with an estimated (from the pet food labels) crude protein concentration of 9.6, 14.3,

19.1 and 23.9 g CP/MJ ME (40, 60, 80 and 100 g CP/1000 kcal ME, resp.) were included.

Four different protein concentrations were selected, rather than diets with different protein

112 Chapter 4


Table 4.2 Analysed chemical composition (g/MJ ME, except where specified), dry matter (DM) and metabolisable energy concentration (ME) of four canned and four kibble single batch dietsa with differing protein concentrations

Canned Kibble

Calculated protein concentration* 9.6b,d,h 14.3c,f 19.1b,d,f 23.9c,e,g 9.6c,i 14.3 19.1 23.9

DM (g/100g as is) 36.1 30.2 24.9 26.9 92.8 91.0 91.4 91.0

Crude protein 10.1 15.0 21.5 27.5 11.3 14.3 18.5 23.2

Crude fat 9.2 11.0 11.3 13.0 9.2 7.6 10.1 8.1

Total dietary fibre† 4.7 4.3 4.5 6.1 4.5 4.6 4.0 26.6

Crude ash 2.8 5.5 3.4 3.7 2.8 4.3 4.7 4.7

Selenium (µg/MJ ME) 34.8 39.3 47.5 40.7 13.9 25.5 19.4 30.3

Selenium (µg/100 g DM) 60.7 69.0 83.9 74.1 24.9 42.9 34.0 35.3

Iodine (µg/MJ ME) 255.7 138.6 74.1 14.3 219.6 374.4 300.4 348.9

d-α-tocopherol (mg/MJ ME) 24.0 21.6 20.7 21.1 29.0 31.6 32.4 83.5

ME (MJ/kg DM)‡ 17.5 17.5 17.7 18.2 17.9 16.8 17.5 11.6

Amino acids

Methionine 0.24 0.41 0.38 0.57 0.23 0.29 0.49 0.41

Cysteine 0.13 0.11 0.18 0.18 0.18 0.20 0.27 0.36

Lysine 0.41 0.94 1.18 1.51 0.49 0.71 0.83 0.92

Fatty acids

Linoleic acid 2.78 1.23 1.53 1.95 1.84 1.26 1.55 1.61

Arachidonic acid 0.04 0.20 0.18 0.14 0.04 0.04 0.04 0.05

Alpha-linolenic acid 0.09 0.10 0.13 0.15 0.16 0.10 0.13 0.14

Docosahexaenoic acid 0.01 0.17 0.20 0.32 0.05 0.03 0.03 0.07

Eicosapentaenoic acid 0.00 0.28 0.31 0.49 0.08 0.06 0.07 0.17

MJ, megajoule; ME, metabolisable energy a All diets were supplemented with 516 mg choline/dog/day, Choline chloride 78% solution, Taminco BVBA, Gent, Belgium b Supplemented with 89.7 mg magnesium/dog/day, Super magnesium, Metabolics Ltd, Eastcott, Wiltshire, England c Supplemented with 206.3 mg magnesium/dog/day, Super magnesium, Metabolics Ltd, Eastcott, Wiltshire, England d Supplemented with 2.2 mg copper/dog/day, Copper citrate, Metabolics Ltd, Eastcott, Wiltshire, England e Supplemented with 4.4 mg copper/dog/day, Copper citrate, Metabolics Ltd, Eastcott, Wiltshire, England f Supplemented with 3.8 µg vitamin D/dog/day, Pet-Cal™, Pfizer Animal Health, New York, USA g Supplemented with 0.43 mg iodine/dog/day, Iodine 11, Metabolics Ltd, Eastcott, Wiltshire, England h Supplemented with 1.19 g methionine/dog/day, synthetic methionine, Evonik Industries, Essen, Germany i Supplemented with 1.03 g methionine/dog/day, Evonik Industries, Essen, Germany * The diets are from left to right: Royal Canin® Canine Veterinary Diet Hepatic, Sensitivity Control

Duck & Rice, Canine Veterinary Care Nutrition Senior Consult Mature, Canine Veterinary Diet Recovery, Renal, Gastro Intestinal moderate calorie, Canine Veterinary Care Nutrition Pediatric Junior Large Dog and Canine Veterinary Diet Satiety Weight Management,

† Obtained from the pet

food producer, ‡ Calculated using predictive equations for ME(22).

Chapter 4


113

digestibility coefficients. This resulted in a range of the absolute amount of digestible

protein, as the limited differences in protein digestibility in commercially available diets are

usually overruled by the protein concentration per se. All diets were single-batch, to

prevent differences in nutrients over time due to variations in ingredients. Selected canned

diets were Royal Canin® Canine Veterinary Diet Hepatic, Sensitivity Control Duck & Rice,

and Recovery and Canine Veterinary Care Nutrition Senior Consult Mature. Selected kibble

diets were Royal Canin® Canine Veterinary Diet Renal, Gastro Intestinal Moderate Calorie,

and Satiety Weight Management and Canine Veterinary Care Nutrition Pediatric Junior

Large Dog. The canned diets contained Se solely from the ingredients, whereas Se in the

form of sodium selenite was supplemented in all kibble diets before processing (standard,

not specifically for this study). Supplemented amounts of sodium selenite were 0.05, 0.08,

0.08, and 0.08 mg/kg, respectively. As veterinary diets were used in this study, some of the

diets were supplemented with additional nutrients in order to be nutritionally complete and

balanced for healthy adult dogs with energy requirements of 397 kJ (95 kcal)/kg BW0.75.

Details of the supplementation are included in Table 4.2. Each dog was fed individually to

maintenance requirements.

4.3.4 Blood samples

At the end of every feeding period, blood samples (13ml) were taken at 13:00 h from each

dog by jugular venipuncture using a 21 gauge needle. Blood was put into 2 Microvette®

500 µl lithium-heparin tubes, one 300 µl fluoride-heparin tube, one 200 µl Tri-Kalium-EDTA

tube and 2 Vacuette® z serum clot activator tubes (1× 9 ml and 1× 4 ml). One of the lithium-

heparin tubes was used for whole blood GPx analysis immediately after collection. The

remaining heparin tubes were centrifuged (accuSpin™ Micro R, Thermo Fisher Scientific Inc.)

immediately after collection at 1680 g and 4°C for 10 min. Lithium-heparin plasma was

analysed for general biochemistry parameters and fluoride-heparin plasma for glucose

(Spectrophotometry, Olympus AU400). Tri-Kalium-EDTA tubes were placed on a roller at room

temperature until analysis for haematology parameters (Mythic 18 Vet analyser, Orphée

S.A.). Biochemistry and haematology measurements were used as a general health check to

confirm that all dogs were in good health. Serum tubes were incubated for 30 min on ice

and then centrifuged (Sigma 6K15, rotor 11150, cups 13550, Sigma GmbH) for 10 min. at

2000 ×g and 4°C. Serum samples were divided into centrifuge tubes and stored at -80°C for

analysis of Se, IsoPs and triiodothyronine (T3) and thyroxine (T4) at the end of the study.

114 Chapter 4


4.3.5 Urine samples

Free catch urine was collected in the section of the day between meals at the end of each

feeding period, using a Uripet urine collection device (Rocket Medical plc., Watford,

England). 1 ml of urine was stored in at -80°C and analysed for creatinine (CT) within one

month after sampling (IDEXX laboratories, UK). The rest of the sample was stored at -20°C

and analysed for Se content (ICP-MS).

4.3.6 Faeces samples

During the last six days of each feeding period, titanium dioxide (TiO2) was added to the

diets as a digestibility marker at a concentration of 0.45 g/day (mean 1.5 g/kg DM, range

0.67-2.14 g/kg DM). Total faeces were collected during four consecutive days at the end of

each diet phase. Faeces were homogenised with a mixer (Kenwood professional PM900,

Kenwood LTD) for 1 minute and a sample of approximately 350 g (mean 346, range 245-410

g) was taken. Samples were freeze-dried (SuperModulyo®, Thermo Fisher Scientific Inc.) until

a stable weight, at -50°C. Faeces were analysed for TiO2, DM, ash, nitrogen (N) and Se. Se

and N were used for the calculation of apparent Se bioavailability and CP (N×6.25)

digestibility coefficients, respectively, with the use of TiO2 as a marker by means of the

following formula:

Apparentdigestibility/bioavailability%= 100 − U100 × V %markerindiet%markerinfaeces× %nutrientinfaeces%nutrientindiet XY

4.3.7 Chemical analyses

Biochemistry and glucose analyses were carried out using spectrophotometry (Olympus

AU400, Olympus Inc.) with Beckman Coulter reagents (Beckman Coulter Biomedical LTD)

within 20 mins of sampling. Whole blood GPx was also analysed within 20 mins of sampling

using the Ransel kit (Randox laboratories LTD) on the Olympus AU400 spectrophotometer. A

4-point calibration curve was used as control. Whole blood GPx analysis had an average

CV of 1.4% in the samples and the 4-point calibration curve showed a recovery of 83.1,

93.7, 98.5, and 96.4%, respectively. Haematology parameters were analysed using a Mythic

18 Vet analyser (Orphée S.A.). Thyroid hormone analyses (T3 and T4) were performed

according to the method of Darras et al.(35) and the IsoPs were analysed using an enzyme-

linked immunosorbent assay (8-isoprostane EIA kit, Cayman Chemical Co.). Serum, urine and

faeces samples were prepared for total Se analyses with closed vessel microwave

destruction as described in van Zelst et al.(20). Se was analysed using inductively coupled

Chapter 4


115

plasma-MS (ICP-MS, Elan DRC-e, PerkinElmer), as described by Lavu et al.(36). Urine CT was

determined using a creatinine kit based on the Jaffe reaction (OSR6178, Beckman Coulter

Biomedical LTD, IDEXX Laboratories, UK). Faeces samples were analysed for DM and crude

ash by drying to a constant weight at 103°C and combusting at 550°C, respectively. The

Kjeldahl method (ISO 5983-1, 2005)(37) was used to determine CP (N×6.25). TiO2 was analysed

according to the method of Myers et al.(38).


Data were analysed using linear mixed effect models in RStudio (version 0.98.507,

RStudio,Inc.)(39) using the nlme package(40). Two primary response variables were measured:

GPx and urinary Se:CT ratio relative to the Se intake (relative urinary Se:CT ratio). To

account for two primary response variables, the statistical test level was corrected to

0.05/2=0.025 for these two parameters. The secondary response variables in this study were:

Energy intake, Se intake, serum Se, serum IsoP, serum T3:T4 ratio, Se bioavailability, apparent

CP digestibility, bioavailable Se intake (the absolute amount of bioavailable Se), digestible

CP intake and urinary Se:CT ratio relative to the bioavailable Se intake. For these response

variables a p-value of <0.05 was used as the threshold for significance.

The model contained a fixed effects structure of actual CP intake (g/kg BW0.75), diet type

and their interaction and a random structure of dog. If the interaction was not significant

(p≥0.05), the model was reduced to CP intake and format as fixed effects and dog as

random effect. For energy, Se, bioavailable Se and digestible CP intake, the model consisted

only of diet type as fixed, and dog as random effect. Distributional assumptions were

checked by visual inspection of residuals. Results are reported as means (± SEM).

Table 4.3 Energy, selenium and crude protein intakes per diet type of four canned

and four kibble diets

Canned Kibble p-value

mean SEM mean SEM diet type

Energy intake (kJ/ kg BW0.75) 428.8 11.7 442.9 11.7 0.325

Se intake (µg/kg BW0.75) 17.3 0.4

10.1 0.6

<0.001

Bioavailable Se intake (µg/kg BW0.75) 7.7 0.4

6.6 0.4

0.043

Digestible CP intake (g/kg BW0.75) 6.6 0.5 6.5 0.3 0.860

SEM, standard error of the mean; BW0.75, metabolic body weight; Se, selenium; CP, crude protein.

116 Chapter 4

SSSSelenium bioavailabilityelenium bioavailabilityelenium bioavailabilityelenium bioavailability and and and and ----

To assess the impact of time between a dog’s last meal and urine collection on the relative

urinary Se:CT ratio, a likelihood ratio test was carried out by including time

effects structure of the model. No significant differences were found when time was

included as a parameter; consequently, time was omitted from the structure of the mixed

effects model for relative urinary Se:CT ratio.

Figure 4.1 Selenium bioavailability (A), serum selenium (B), serum isoprostanes (C) and

serum T3:T4 ratio (D) in dogs in relation to crude protein intake of four canned and four

kibble diets. Black squares and solid line are canned diets, open triangles and dashed line

kibble diets. Symbols represent the means and error bars indicate their standard errors, based on the

raw data. BW0.75, metabolic body weight;

hormones.

----activityactivityactivityactivity


urinary Se:CT ratio, a likelihood ratio test was carried out by including time



effects model for relative urinary Se:CT ratio.

enium bioavailability (A), serum selenium (B), serum isoprostanes (C) and


Black squares and solid line are canned diets, open triangles and dashed line

Symbols represent the means and error bars indicate their standard errors, based on the

, metabolic body weight; T3:T4 ratio, ratio between triiodothyronine and thyroxine


urinary Se:CT ratio, a likelihood ratio test was carried out by including time into the fixed



enium bioavailability (A), serum selenium (B), serum isoprostanes (C) and


Black squares and solid line are canned diets, open triangles and dashed line are

Symbols represent the means and error bars indicate their standard errors, based on the

T3:T4 ratio, ratio between triiodothyronine and thyroxine

Chapter 4


117

4.4 Results

All biochemistry and haematology results were within the normal range for healthy dogs

(data not shown). The average Se concentration of the kibble diets was 22.3 µg/MJ (range

13.9 - 30.3) and 40.6 µg/MJ (range 34.8 - 47.5) for canned diets. Energy intake did not differ

between the diet types, hence the Se intake was approximately two-fold lower on kibble

than canned diets (Table 4.3).

Apparent CP digestibility showed a significant diet type by CP intake interaction (p<0.001).

Apparent CP digestibility increased with increasing CP intake, but only in canned diets. The

digestible CP intake did not differ between diet types (Table 4.3). Kibble diets had a higher

Se bioavailability on average than canned diets (p<0.001) and Se bioavailability did not

significantly change with CP intake (p=0.753, Figure 4.1A). On average, Se bioavailability

was 62.3% (± 1.5) in kibble and 44.9% (± 1.9) in canned diets. The average bioavailable Se

intake was lower in kibble than canned diets (Table 4.3).

The GPx response (U/g Hb) was lower on average in kibble compared to canned diets

(p<0.001) and decreased with an increasing CP intake (p<0.001, Figure 4.2). Serum Se

concentrations did not change significantly with diet type (p=0.158), but did with CP intake,

whereby serum Se concentrations decreased with increasing CP intake (p<0.001, Figure

4.1B). No association of diet type (p=0.069) or CP intake (p=0.216) on IsoPs was found (Figure

4.1C), nor with the T3:T4 ratio (p=0.102 and 0.960, respectively, Figure 4.1D).

The relative urinary Se:CT ratio was higher on average in kibble diets than in the canned

diets (p<0.001). No association of CP intake with urinary Se:CT ratio was found (p=0.059,

Figure 4.3). When urinary Se excretion was expressed per absolute amount of bioavailable

Se, dogs on kibble diets excreted more Se in their urine than dogs on canned diets

(p=0.001) and excretion decreased with increasing CP intake (p=0.017, data not shown).

4.5 Discussion

This study showed that the availability of Se through digestion and metabolism changed

considerably with diet type. The higher apparent Se bioavailability in kibble compared to

canned diets suggests that the use of a single recommendation for Se inclusion levels in pet

foods for both diet types needs reconsideration. Previous in vitro findings showed that Se in

canned diets was more susceptible to a decrease in Se accessibility, i.e. the amount that is

available for absorption in the gastro-intestinal tract, due to processing(20). The average Se

accessibility was also lower in canned than kibble diets(20). In the in vitro study(20), canned

diets had an average Se accessibility of 58% (± 4.01, n=20), while the Se accessibility of

118 Chapter 4


Figure 4.2 Whole blood glutathione peroxidase responses in dogs in

relation to crude protein intake of four canned and four kibble diets.

Black squares and solid line are canned diets, open triangles and dashed

line are kibble diets. Symbols represent the means and error bars indicate

their standard errors, based on the raw data. Hb, hemoglobin; BW0.75,

metabolic body weight.

kibble diets was 72% (± 3.95, n=23). Accordingly, in the present study canned diets showed

a lower Se bioavailability than kibble diets. The difference in apparent Se bioavailability

observed between the diet types may be caused by the Se species used in these diets. The

meat and meat-by-products that are used in canned diets, mainly contain organically

bound Se (e.g. selenomethionine)(41). Therefore, it may be that the dietary sulphur-containing

amino acids (methionine and cysteine) compete for absorption with the organically bound

Se(42). Sodium selenite, which is often used (as in the kibble study diets) to supplement pet

foods that do not comply to the recommended allowance (mainly kibble diets), is absorbed

through diffusion(43) and thus does not have to compete for absorption with organically

bound sulphur.

Interestingly, bioactivity in this study measured as GPx (U/g Hb), was higher in canned than

kibble diets. This may be explained by a higher amount of bioavailable Se in canned diets.

However, this finding should be interpreted with caution, as the range in dietary CP

concentration may also have affected the Hb concentration(44), which in turn may affect the

GPx results. Even though the apparent Se bioavailability of canned diets was much lower

than kibble diets (44.9% vs. 62.3%, respectively), the absolute amount of bioavailable Se was

still higher in canned diets (7.7 ± 0.4 vs. 6.6 ± 0.4 µg/kg BW0.75, respectively). Both this study

325

350

375

400

425

450

475

500

525

550

0 2 4 6 8 10 12 14 16 18

Glu

tath

ione p

ero

xidase

(U/g

Hb)

Crude protein intake (g/kg BW0.75)

0

Chapter 4


119

Figure 4.3 Urinary selenium to creatinine ratio relative to selenium

intake in dogs in relation to crude protein intake of four canned and

four kibble diets.

Black squares and solid line are canned diets, open triangles and dashed

line are kibble diets. Symbols represent the means and error bars indicate

their standard errors, based on the raw data. BW0.75, metabolic body weight.

and the in vitro study(20) were performed with commercially available diets and in both

studies a higher dietary Se concentration was found in canned than in kibble diets. In the

in vitro work, canned diets contained on average 34.8 µg Se/MJ (± 6.25, n=20) and kibble

diets 22.5 µg Se/MJ (± 2.42, n=23)(20). In this study, similar contents were measured (canned

diets: 40.6 µg Se/MJ, kibble diets: 22.3 µg/MJ). This indicates that the ingredients for canned

diets tend to contain a higher amount of Se, which suggests this may be inherent to this diet

type.

In addition to the higher amount of bioavailable Se in canned diets, the urinary Se

excretion relative to Se intake was lower in canned compared to kibble diets, even per

absolute amount of bioavailable Se. This suggests that the percentage of retained Se per

bioavailable Se is higher in canned than kibble diets. Consequently, a higher amount of

bioavailable Se from the canned diets can be used for either non-specific incorporation into

body proteins, or incorporation into the Se-dependent antioxidant GPx. In the latter

situation, this results in a higher bioactivity of Se from canned diets, as demonstrated in this

study (at least in U/g Hb). A difference in speciation is the most likely explanation for the

differences between the diet types. Unfortunately, efforts to determine Se speciation were

0

20

40

60

80

100

120

0 2 4 6 8 10 12 14 16 18

Urinary

Se:C

T rel.

to S

e inta

ke

Crude protein intake (g/kg BW0.75)

120 Chapter 4


unsuccessful due to detection limit issues. However, it is known that the kibble diets in this

study are supplemented with inorganic sodium selenite while the Se in the canned diets in

this study derives solely from the ingredients, which consist mainly of meat and meat by-

products, and thus it is primarily organically bound(41).

It should be acknowledged that apparent digestibility/bioavailability was calculated in this

study. Some of the absorbed Se is excreted via the bile into the faeces(45), which may cause

an underestimation of the Se bioavailability. However, it is not known whether there was a

difference in biliary Se excretion between the diet types. Given the higher dietary fat

concentration in canned diets, it may be assumed that bile excretion is higher in canned

compared to kibble diets, but this does not necessarily mean a higher Se excretion via bile.

Gregus et al.(46) suggest that Se excretion via bile is enhanced by binding to an organic

acid. So, if there was a difference in the amount of Se excreted via the bile, this may also

be due to a difference in Se speciation.

Taking blood GPx concentration as a measure for bioactivity may suggest a superior

antioxidant function of canned diets, but this was not associated with a higher

concentration of the oxidative stress parameter, isoprostanes. It cannot be excluded that a

difference between diet types may be found if other parameters of oxidative stress were

measured or if IsoP’s were measured during a longer period.

Like GPx, iodothyronine deiodinases are also Se-dependent proteins. Several studies have

shown that a higher dietary Se concentration has a positive influence on the T3:T4 ratio in

humans(33), kittens(17; 32) and dogs(17; 47). In this study, no effect on T3:T4 ratio was found and

they were shown to be within a clinically normal range(48). This may be because the

difference in bioavailable Se was not large enough between diet types. Remarkably, no

interactions were found for any of the parameters measured in this study. However, there

was a negative association between CP intake and both GPx and serum Se, irrespective of

diet type. It is unlikely that Se intake was responsible for these results as there was no

negative, but actually a positive association between Se and CP intake. Se bioavailability

was also not significantly associated with CP intake, therefore, unlikely to be responsible for

the negative association between bioactivity and CP intake. Furthermore, urinary Se

excretion corrected for bioavailable Se decreased with increasing CP intake, which suggests

a higher retention with increasing CP intake. The higher amount of retained Se was not

incorporated into GPx, but left in the blood stream as serum Se or excreted via the urine.

Therefore, taken together it is very likely that a part of the Se from canned diets was

incorporated into body proteins as selenomethionine instead of methionine. The negative

Chapter 4


121

association between CP intake and GPx (U/g Hb) may also have been attributed by a

positive effect of dietary CP on the Hb concentration of the blood.

To clarify whether the effects found in this study were attributed to the absolute amount of

Se or the Se species, semi-purified diets could be used in the future. However, it is a

challenge and maybe even impossible to manufacture diets with two different processing

types, using exactly the same ingredients. Furthermore, such a study would not be relevant

for the Se bioavailability and -activity of commercially available diets.

In conclusion, the diet type, whether this is caused by processing, Se speciation, or

otherwise, causes canned diets to have a lower apparent Se bioavailability, but a higher

amount of the bioavailable Se is retained in the body, which possibly enhanced Se

bioactivity (at least in U/g Hb). So the can can, what the kibble can't. This study suggests

that the recommendation for Se inclusion levels in pet foods should take diet type into

account. Further studies are warranted to quantify the exact origin of the difference

between the diet types, but this requires valid and sensitive parameters to monitor Se

adequacy. The impact of processing conditions and protein sources on Se bioavailability

and -activity is another topic that warrants more investigation. It should be noted that it is

not sufficient to solely measure Se bioavailability, because bioactivity and urinary excretion

measurements may give a different picture. Therefore, the choice of parameters can be

crucial for the conclusion when studying nutritional modulation of Se status in animals.

4.6 Acknowledgements

The authors express their special thanks to Joachim Neri of the Department of Applied

Analytical and Physical Chemistry at the Faculty of Bioscience Engineering of Ghent

University for total Se analyses and to Daniel Vermeulen of the Division of Livestock,

Nutrition and Quality at the Science, Engineering and Technology group of the Catholic

University of Leuven for the thyroid hormone analyses.

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45. McConnell KP, Martin RG (1952) Biliary excretion of selenium in the dog after administration of sodium selenate containing radioselenium. Journal of Biological

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48. Kantrowitz LB, Peterson ME, Melián C et al. (2001) Serum total thyroxine, total triiodothyronine, free thyroxine, and thyrotropin concentrations in dogs with nonthyroidal disease. Journal of the American Veterinary Medical Association 219219219219, 765-769.

Biomarkers of _______________________________________________________

Biomarkers of selenium status in dogs_____________________________________________________________________________________________________________

Chapter 5

selenium status in dogs______________________________________________________

"Biomarkers of selenium status in dogs"

Mariëlle van Zelst1, Myriam Hesta1, Kerry Gray2, Ruth Staunton2, Gijs Du Laing3, Geert P.J.

Janssens1*

1 Department of Nutrition, Genetics and Ethology, Faculty of Veterinary Medicine, Ghent





Submitted to BMC Veterinary Research

Chapter 5

Biomarkers of seleniumBiomarkers of seleniumBiomarkers of seleniumBiomarkers of selenium statusstatusstatusstatus

127

5.1 Abstract

Inadequate dietary selenium (Se) intake in humans and animals can lead to long-term

health problems, such as cancer. In view of the owner’s desire for healthy longevity of

companion animals, the impact of dietary Se provision on long-term health effects warrants

investigation. Little is currently known regards biomarkers, and rate of change of such

biomarkers in relation to dietary selenium intake in dogs. In this study, selected biomarkers

were assessed for their suitability to detect changes in dietary Se in adult dogs within eight

weeks.

Twenty-four dogs were fed a semi-purified diet with an adequate amount of Se (46.1 µg/MJ)

over an 8 week period. They were then divided into two groups. The first group remained

on the adequate Se diet, the second were offered a semi-purified diet with a low Se

concentration (6.5 µg/MJ; 31% of the FEDIAF minimum) for 8 weeks. Weekly urine and blood

was collected and hair growth measurements were performed. The urinary Se to creatinine

ratio and serum Se concentration were significantly lower in dogs consuming the low Se

diet from week 1 onwards, by 84% and 7% respectively. Serum and whole blood

glutathione peroxidase were also significantly lower in dogs consuming the low Se diet from

weeks 6 and 8 respectively. None of the other biomarkers (mRNA expression and serum

copper, creatine kinase, triiodothyronine:thyroxine ratio and hair growth) responded

significantly to the low Se diet over the 8 week period.

This study demonstrated that urinary Se to creatinine ratio, serum Se and serum and whole

blood glutathione peroxidase can be used as biomarkers of selenium status in dogs. Urinary

Se to creatinine ratio and serum Se concentrations responded faster to decreased dietary

Se than the other parameters. This makes these biomarkers candidates for early screening

of long-term effects of dietary Se provision on canine health.

5.2 Introduction

Selenium (Se) is an essential trace element in dogs(1). It is involved in many aspects of canine

physiology, such as anti-oxidant protection(2), thyroid hormone metabolism(3), and immune

function(4). Although no naturally occurring clinical cases of Se deficiency or toxicity have

been reported in dogs(1), an inadequate Se status is associated with long-term health effects,

such as calcium oxalate calculi(5-6) and cancer(7-8) formation.

Considering the importance dog owners place on the healthy longevity of their pets(9-10), the

health effects of dietary Se intake should be studied in more detail. Measuring only the

dietary Se content is not sufficient to study effects of Se on metabolism as the bioactivity of

Se, defined as the amount of dietary Se that can be incorporated into selenoproteins such

128 Chapter 5


as glutathione peroxidase (GPx), is affected by many factors(11). Long-term studies are costly

and difficult to perform, and therefore biomarkers are needed which can identify Se-

induced changes in metabolism at an early stage.

There is currently no literature on specific and sensitive biomarkers of dietary Se intake in

dogs, which confounds the accurate assessment of Se status. The main tissues and body

fluids for minimally-invasive measurement of Se concentration are whole blood, plasma,

serum, erythrocytes, urine, hair, and nails(12). In this study biomarkers were selected to assess

their sensitivity to a manipulation of dietary Se concentration in adult dogs.

Glutathione peroxidase (GPx) is the biomarker most often measured to estimate Se

bioactivity(13-15). GPx is a selenoprotein that acts as an anti-oxidant(16) and is currently used as

a proxy of selenium status, although has never been verified as the gold standard. GPx

measurements in chicks receiving different concentrations of Se supplement(17) was used to

inform the existing European minimum recommendation for the Se concentration in dog

foods, which is 17.9 µg/MJ(18).

Secondly, Levander et al.(19) reported that urinary Se excretion in humans consuming a low

Se diet (intake of approximately 35 µg/day) stabilised after 12 days. In cats, urinary Se

concentrations increased dose-dependently within 2 days of Se supplementation with either

sodium selenite or Se yeast at 1.5 and 2.0 mg Se/kg dry matter (DM) compared to 0.45 and

1.0 mg Se/kg DM(20).

The mRNA expression of one of the isoforms of GPx (GPx1) from liver tissue has also been

reported to be a biomarker of Se status in rats(21-22), together with selenoprotein H (SelH),

selenoprotein W (SepW1), thioredoxin reductase 1 (TrxRd1), thioredoxin reductase 2 (TrxRd2),

iodothyronine deiodinase 1 (DIO1), selenoprotein K (SelK), selenoprotein T (SelT), and 15 kDa

selenoprotein (Sep15)(21-22). Although selenoprotein P is the most abundant selenoprotein(23)

and is important in the transport of Se throughout the body(24), its mRNA has been shown to

not be significantly down-regulated in Se deficiency(25).

Data from rats indicates that mRNA from whole blood can be used to measure mRNA

biomarkers for Se status(26). RNA isolated from whole blood for the determination of GPx1

mRNA was expressed at levels comparable to the levels found in liver, kidney and heart

mRNA(26). The expression of GPx1 mRNA in rats on a Se deficient torula yeast diet (0.007 µg

Se/g diet) was only 14% of the GPx1 mRNA expression in rats on a diet supplemented with

0.2 µg sodium selenite/g diet, which was comparable to the fall in liver mRNA(26).

Additionally, hair growth can be included as a biomarker because it has been reported to

be reduced in beagles consuming a dietary Se concentration of 0.09 mg/kg DM (=

approximately 5.5 µg/MJ) after 11 weeks(27). Other indirect measures of Se status, such as

Chapter 5


129

serum creatine kinase (CK) and the thyroid hormones triiodothyronine (T3) and thyroxine (T4)

were also included. Se is involved in the conversion of T4 into the active form T3(28) and the

T3:T4 ratio has been reported to decrease in puppies(29) and kittens(30) fed low compared to

adequate Se concentrations. Finally, serum CK was increased in piglets fed either a diet

containing no Se, although vitamin E levels were also manipulated in these studies(31).

5.3 Experimental methods

5.3.1 Study design

A longitudinal, controlled and blinded study was performed using 24 adult Labrador

retrievers. The dogs were assigned into 2 groups of 12 dogs, with age, gender and hair

colour as blocking criteria. To ensure an adequate and stable selenium status in both

groups at the start of the experimental period, the dogs received a Se adequate diet for 8

weeks before the experimental period. During this adaptation period, they were sampled

three times (at weeks 2, 5 and 8) for baseline levels of blood and urine parameters and

weekly hair growth measurements were performed. After the adaptation period, one group

continued to receive the adequate Se diet and the other group received a diet with a low

Se concentration for further 8 weeks. Weekly blood and urine samples were taken. In

addition, weekly hair growth was measured. This study was approved by the WALTHAM®

Centre for Pet Nutrition Animal Welfare and Ethical Review Body and was conducted under

UK Home Office Project Licence authorisation.

Table 5.1 Gender*, age, body weight and energy intake of the study dogs per dog group

n

♂

♀

Age (years)

Body weight (kg)

Energy intake (kJ/kg BW0.75)

mean min max mean min max mean min max

Group A 12 6 6 4.6 2.4 6.3 26.7 23.3 30.6 471 361 611

Group B 12 4 8 4.1 2.4 6.3 26.1 22.6 31.2 424 346 525 kJ, kilojoule; BW0.75, metabolic body weight; n, number of dogs; ♂, male; ♀, female; min, minimum;

max, maximum * All dogs were spayed/neutered. Both dog groups received the adequate Se diet in the 8-week pre-feeding period. Dog group A continued to receive the adequate Se diet in the 8-week experimental period and dog group B switched to the low Se diet.

5.3.2 Dogs

Twenty-four adult Labrador retrievers (14 female and 10 male) were selected for this study.

The average age, body weight (BW) and energy intake in kJ/kg metabolic body weight

(BW0.75) per dog group are shown in Table 5.1. All dogs had an ideal body condition score

130 Chapter 5


Table 5.2 Analysed* chemical composition of two semi-purified diets with an adequate or low selenium concentration

Component (g/MJ, except where specified)

Adequate Se diet

Low Se diet

Dry matter (g/100g as is) 92.8 92.7

Crude protein 14.9 14.9

Crude fat 5.8 5.8

Crude fibre 1.6 1.5

Nitrogen free extract† 35.5 35.4

Starch 13.6 13.1

Crude ash 2.5 2.4 Metabolisable energy (MJ/kg DM)‡ 16.6 16.7

Amino acids

Lysine 0.36 0.38

Methionine 0.32 0.36

Cysteine 0.36 0.36

Fatty acids

Linoleic acid 1.35 0.96

α-Linolenic acid (mg/MJ ME) 91 113

Arachidonic acid (mg/MJ ME) 22.6 9.3

EPA & DHA (mg/MJ ME) 6.9 6.5

Minerals (mg/MJ ME)

Calcium 383 388

Phosphorus 299 304

Zinc 6.5 6.5

Iron 3.4 3.9

Copper 0.48 0.68

Iodine 0.08 0.08

Selenium (µg/MJ ME) 46.1 6.5

Selenium (µg/100 g DM) 76.5 10.8

Vitamins (mg/MJ ME)

d-α-tocopherol 1.8 1.8

Riboflavin 0.45 0.44

Pyridoxine 0.14 0.22

Retinol 0.16 0.16 MJ, megajoule; ME, metabolisable energy; EPA, Eicosapentaenoic acid; DHA, docosahexaenoic acid * All analyses were performed by Eurofins Food Testing, Wolverhampton, UK

† Calculated by substracting the amount (as g/100g as is) of crude fat, protein, ash and fibre from the percentage dry matter and dividing this by the metabolisable energy concentration (MJ/kg as is) ‡ Calculated using predictive equations for ME(1) Both dog groups received the adequate Se diet in the 8-week pre-feeding period. Dog group A continued to receive the adequate Se diet in the 8-week experimental period and dog group B switched to the low Se diet.

Chapter 5


131

(BCS) of D on the S.H.A.P.E™ BCS-scale(32). Body weight and BCS was recorded before the

start of the adaptation period and thereafter every week. The dogs were fed individually,

once a day at 8:30 h, to energy requirements in order to maintain BW and BCS. They had

access to fresh drinking water at all times. Food intake was also recorded throughout the

study. Dogs were housed indoors in triplets with others of the same diet group with

continuous access to an outside pen and access to an outside paddock during the day.

5.3.3 Diets

Two semi-purified diets (Ssniff spezialdiäten GmbH, Soest, Germany) were used, one Se-

adequate and one containing a low amount of Se (Table 5.2). Diets were formulated to be

nutritionally complete for dogs with energy requirements of at least 397 kJ (95 kcal)/kg

BW0.75. Both diets had the same nutritional formulation, except for Se concentration. The Se

adequate diet contained 46.1 µg Se/MJ and the low Se diet 6.5 µg Se/MJ, which is 31% of

the FEDIAF recommended minimum for dogs with an energy intake of 95 kcal/kg BW0.75(18).

Se concentration from the ingredients was approximately 0.75 µg/MJ. Supplementary Se was

in the form of sodium selenite.

5.3.4 Blood samples

At weeks 2, 5 and 8 of the pre-feed period and weekly during the experimental period,

blood samples (10.5 ml) were taken from each dog by jugular venipuncture using a 21

gauge needle. Blood was collected into a 2.5 ml PAXgene tube, 2 Microvette® 500 µl

lithium-heparin tubes, one 300 µl fluoride-heparin tube, one 200 µl Tri-Kalium-EDTA tube

and two 4 ml Vacuette® z serum clot activator tubes (one of them filled with only 2.5ml of

blood). The PAXgene tubes were incubated at room temperature for 2 hours and stored at -

20°C for mRNA expression analysis. One of the lithium-heparin tubes was immediately

stored at -80°C and used for whole blood glutathione peroxidase (GPx) analysis at the end

of the study, which is considered to be more valid than analysing the fresh samples

immediately (see Supplement). The other heparin tubes were centrifuged (accuSpin™ Micro

R, Fisher Scientific™, Pittsburgh, PA, USA) immediately after collection at 1680 ×g and 4°C for

10 min. Lithium-heparin plasma was analysed for general biochemistry parameters and

fluoride-heparin plasma for glucose (Spectrophotometry, Olympus AU400). EDTA tubes were

placed on a roller at room temperature until analysis for haematology parameters (Orphée

Mythic 18 Vet analyser) within 4 hours of collection. Serum tubes were incubated for 30 min

on ice and then centrifuged (Sigma 6K15, rotor 11150, cups 13550, Sigma GmbH, Osterode

am Harz, Germany) for 10 min. at 2000 ×g and 4°C. Serum samples were divided into

132 Chapter 5


Eppendorfs and stored at -80°C for analysis of GPx, Se, triiodothyronine (T3), thyroxine (T4),

copper (Cu) and creatine kinase (CK) at the end of the study.

5.3.5 Urine samples

Free catch urine was collected weekly after feeding (between 8:30 and 16:00 h), using a

Uripet urine collection device (Rocket Medical plc. Watford, England). From all urine samples,

1 ml was stored in an Eppendorf at -80°C and analysed for creatinine (CT) within one month

after sampling (IDEXX laboratories, UK). The rest of the samples were stored at -20°C and

analysed for total Se content (ICP-MS, PerkinElmer DRC-e, Waltham, USA).

5.3.6 Hair growth measurements

Hair growth was measured according to a method adapted from Yu et al.(27). Due to

practical issues only chocolate and black Labradors were used for the hair growth

measurements (5 black, 1 chocolate for the adequate Se diet and 6 black, 1 chocolate for

the low Se diet). At the start of the pre-feed and experimental period, an area in the groin

of approximately 5 x 5 cm was shaved with a 40 mm blade (Andis Super AGR+ cordless

clipper; Andis Company Corporate, Sturtevant, Wisconsin, USA). Directly after shaving, the

area was marked with a Duramark permanent marker. The shaved area was covered with a

glass slide with a ruler attached to it, and a picture was taken using a Canon EOS 1000D

digital camera with EF50mm f/2.5 compact-macro lens (Canon Inc., Melville, New York, USA)

and Hoya PRO1 Digital Polarising Filter (HOYA Optics, Milpitas, California, USA). The glass

slide was also used to flatten the hairs, when it had grown back in the following weeks to

accurately measure them. Pictures were taken weekly during the pre-feed and experimental

periods and were analysed with ImageJ analysing software(33). Hair growth (mm/week) was

measured as the difference of the average length of the hairs within the marked box

between two consecutive pictures.

5.3.7 Chemical analyses

Biochemistry and glucose analyses were carried out using spectrophotometry (Olympus

AU400, Olympus Inc.) with Beckman Coulter reagents (Beckman Coulter Biomedical) within

20 mins of sampling. Haematology parameters were analysed using a Mythic 18 Vet

analyser (Orphée S.A.). Serum and urine samples were prepared for total Se analyses with

closed vessel microwave destruction as described in van Zelst et al.(34). Se was analysed

using inductively coupled plasma-MS (ICP-MS, Elan DRC-e, PerkinElmer), as described by

Chapter 5


133

Lavu et al.(35). Urine CT was determined using a creatinine kit based on the Jaffe reaction

(OSR6178, Beckman Coulter Biomedical, IDEXX Laboratories, London, UK).

Serum Cu and CK were analysed with the Randox copper and CK NAC-activated kit

(Randox laboratories, London, UK), respectively, as per the manufacturer’s instructions.

Thyroid hormones were analysed with canine T3 and T4 enzyme-linked immunosorbent

assay (ELISA) kits (Cusabio, Wuhan, China). Whole blood GPx was analysed using the Ransel

kit (Randox laboratories) and an Olympus AU400 spectrophotometer as per the

manufacturer’s instructions. A 4-point calibration curve was used as control, with a 1:11, 1:21,

1:41 and 1:61 dilution. Whole blood and serum GPx analysis of the samples had an average

coefficient of variation of 1.9% and 2.0%, respectively. The 4-point calibration curve showed

a recovery of 74.5, 87.1, 98.6, and 100.6%, respectively.

mRNA expression was performed for the three baseline samples and the samples of weeks

2, 4, 6, and 8 of the experimental period. Total RNA, for mRNA expression, was isolated from

the PAXgene tubes using the PAXgene blood RNA kit (Qiagen, Manchester, UK) as per the

manufacturer’s instructions. The RNA was eluted in 2 x 40 µl of the elution buffer. Further

DNase digestion of the RNA solution was carried out using RQ1 RNase-Free DNase

(Promega, Southampton, UK) as per the manufacturer's instructions with the sample incubated

for 30 min at 37°C. In order to remove the DNase and reaction buffer from the purified

RNA, it was passed through the RNeasy Mini Kit using the RNA clean-up protocol and was

eluted in 2 x 40µl of elution buffer (10mM Tris HCl, pH 8.4). The RNA concentration in the

eluate was measured using the Qubit RNA Assay Kit (Invitrogen, Paisley, Scotland).

Primers and probes were designed using Primer3(36) and M-Fold using the canine specific

GenBank (37) sequences for GPx1, SelH, SepW1, TrxRd1, TrxRd2, DIO1, SelK, SelT, Sep15,

tumor necrosis factor alpha (TNF-α), and nuclear factor kappa-light-chain-enhancer of

activated B cells (NFκB) as described by Peters et al.(38). The assays for the potential

housekeeper genes were the same as described in Peters et al.(39).

Synthesis of complementary DNA (cDNA) was carried out with 750ng of random hexamers

using the ImProm-II Reverse Transcription System (Promega, Southampton, UK) and 750 ng of

total RNA (as measured by the Qubit) in a final volume of 30 µl. All reactions were

prepared according to the manufacturer’s instructions giving a final magnesium chloride

concentration of 3 mM. The cDNA synthesis was carried out by mixing the RNA with the

random primers in a reaction tube. Samples were heated to 70°C for 5 min in the PTC-200

DNA engine (Bio-Rad Laboratories, Hemel Hempstead, UK) before cooling to 4°C for 5 min.

Tubes were placed in a cold block before addition of the reaction buffer, deoxynucleotides

(dNTP’s), magnesium chloride, reverse transcriptase enzyme mix and water to make a total

134 Chapter 5


Ta

ble

5.3

Prim

er and p

robe s

equence

s use

d for positive

control and q

PCR a

ssays

Pro

du

ct

Siz

e

114

113

148

116

60

95

84

65

79

84

124

99

90

123

qPCR, quantita

tive

poly

mera

se c

hain

reaction; G

Px1

, glu

tath

ione p

ero

xidase

1; SelH

, se

lenopro

tein

H; SepW

1, se

lenopro

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thio

redoxi

n reducta

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; Trx

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; DIO

1, io

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, se

lenopro

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, se

lenopro

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T; Sep15,

15 k

Da s

ele

nopro

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; TN

F-α

, tu

mor necro

sis

facto

r alp

ha; N

FκB

1, nucle

ar fa

cto

r kappa-lig

ht-chain

-enhancer of activa

ted B

cells

; B2M

, beta

-2-

mic

roglo

bulin; G

APDH, gly

cera

ldehyde 3

-phosp

hate

dehydro

genase

; HPRT1, hypoxa

nth

ine p

hosp

horibosy

ltra

nsfera

se 1

Pro

be

AA

GTTG

GG

CTCG

AA

CC

CG

CC

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AG

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CG

TG

GG

GG

CC

AA

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AA

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GG

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GG

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GG

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GA

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TA

CCA

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GCTTTG

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GG

AG

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TC

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AG

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CTG

GA

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AA

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GA

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AA

GG

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Re

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Pri

me

r

TTCA

CC

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AA

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AA

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CC

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CC

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TTG

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GG

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TG

GG

TG

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ACC

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TG

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GG

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GG

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GG

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ATT

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CTCA

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AG

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GA

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ne

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1

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B2M

GA

PDH

HPRT1

Chapter 5


135

volume of 20 µl. Reverse transcription (RT) was undertaken by heating the samples to 25°C

for 5 min, 42°C for 30 min and finally 75°C for 10 min in the PTC-200 DNA engine (Bio-Rad

Laboratories, Hemel Hempstead, UK). Duplicate RT reactions were performed for each RNA

sample. All cDNAs were diluted 1:5 (v/v) for the genes of interest and housekeeper genes

using EB Buffer (10mM Tris-HCl pH-8.4, Qiagen, Manchester, UK) and then stored at -20°C for

future use. No template controls were performed by addition of nuclease-free water in

place of RNA.

Quantitative polymerase chain reaction (qPCR) was performed using GoTaq Colourless

Master Mix (Promega, Southampton, UK) as described by Van de Velde et al.(40). The absence

of genomic contamination of the RNA samples was confirmed prior to the RT reactions and

none of the samples showed evidence of amplifiable genomic DNA with the succinate

dehydrogenase complex, subunit A qPCR assay. One qPCR reaction was run for each RT

repeat resulting in two threshold values (Ct) for each RNA sample. A mean Ct value was

calculated for each sample using the two measured Ct values for each dog for each of the

potential housekeeper genes. The mean Ct value was converted to a relative copy number

value using the EδCt method (E: reaction efficiency as determined from a standard curve) with

δCt values calculated relative to the sample with the largest Ct (fewest gene copies). The

geNorm visual basics for applications applet for Microsoft Excel was used to determine the

most stable genes from the set of tested genes(41). The three most stable housekeeper genes

for the blood samples were beta-2-microglobulin (B2M), glyceraldehyde 3-phosphate

dehydrogenase (GAPDH), and hypoxanthine phosphoribosyltransferase 1 (HPRT1). The

primer and probe sequences of the genes of interest and these housekeeper genes are

shown in Table 5.3.

Relative copy number expression values were calculated for each sample and normalised

against the housekeeper gene results using the qBase applet for Microsoft Excel which

employs the methodology described by Vandesompele et al.(41). The sample with the fewest

gene copies (latest Ct value) is given a relative copy number of 1 and all other samples are

given values relative to this sample.

In order to assess reaction efficiency of the newly designed assays, a set of primers were

designed for the gene target to amplify a larger fragment, which included the portion

amplified by the qPCR assay. These assays were tested against a cDNA obtained from

RNA extracted from canine blood. Products were separated by 2% agarose gel

electrophoresis, purified by NucleoSpin Extract II kit (Macherey-Nagel, Düren, Germany) and

then quantified by QBit (Invitrogen, Paisley, Scotland). The number of copies per µl of

purified product was calculated and then a 1:10 dilution series from 107 to 1 copy per qPCR

136 Chapter 5


Table 5.4 Serum biomarker concentrations in dogs fed a diet with a low or adequate selenium concentration

diet adequate-Se low-Se week n mean SE n mean SE p-value

Serum Se (µg/L) 0 12 263 5.3 12 263 4.7 0.987

1 12 271 5.8 11 252 6.1 0.030

2 12 263 3.6 12 245 6.0 0.029

3 12 266 4.5 12 249 6.1 0.050

4 12 261 4.5 12 236 3.5 0.000

5 12 247 5.1 12 240 8.3 0.853

6 12 248 4.6 12 230 6.4 0.024

7 11 254 5.8 12 230 5.7 0.000

8 12 243 4.7 12 224 4.1 0.013

Serum GPx (U/L) 0 12 14884 383 12 15027 323 0.778

1 12 14754 355 11 14175 251 0.228

2 12 15274 353 12 14563 380 0.101

3 12 14726 309 12 14066 296 0.143

4 12 14215 315 12 13644 304 0.250

5 12 14421 304 12 14330 255 0.998

6 12 14968 287 12 14007 313 0.012

7 11 15052 349 12 14459 232 0.232

8 12 15755 375 12 14579 271 0.001

Serum CK (U/L) 0 12 76 5.6 12 75 6.1 0.912

1 10 79 6.3 10 75 5.2 1.000

2 12 73 7.2 12 72 6.7 1.000

3 12 84 10.7 11 75 5.9 0.965

4 12 61 7.7 12 58 3.3 1.000

5 12 45 4.4 12 54 5.2 0.837

6 12 60 8.8 12 51 4.6 0.858

7 11 52 3.8 11 56 4.5 1.000

8 12 52 2.9 12 54 4.0 1.000

Serum T3:T4 0 12 0.031 0.001 12 0.029 0.001 0.093

1 11 0.031 0.001 11 0.029 0.001 0.998

2 12 0.031 0.001 12 0.031 0.001 0.987

3 12 0.034 0.001 11 0.033 0.001 1.000

4 12 0.038 0.001 12 0.033 0.001 0.144

5 12 0.034 0.001 12 0.033 0.001 0.999

6 12 0.034 0.001 12 0.031 0.001 0.900

7 11 0.035 0.001 12 0.033 0.001 0.997

8 12 0.036 0.001 12 0.035 0.001 0.996

Serum Cu:Cu intake 0 12 15.8 1.0 12 19.3 1.2 0.033

1 7 32.1 2.5 8 22.8 3.5 0.985

2 8 21.4 1.5 11 18.2 1.0 1.000

3 12 23.2 1.6 10 20.0 1.0 0.944

4 11 26.7 2.2 12 20.2 1.3 0.919

5 12 17.4 0.8 12 14.8 0.7 0.999

6 11 16.4 1.0 10 13.5 0.7 1.000

7 8 17.5 1.0 6 13.1 1.0 1.000

8 12 19.6 1.3 11 15.4 0.7 1.000 Se, selenium; n, number of dogs; SE, standard error; GPx, glutathione peroxidase; CK, creatine kinase; T3, triiodothyronine; T4, thyroxine; Cu, copper. Values at weeks zero indicate the average baseline values. Means and standard errors are based on the raw data.

Chapter 5


137

was analysed in duplicate using the qPCR assay and the reaction efficiency calculated

using the MxPro software.


The two primary parameters were whole blood GPx and urinary Se:CT ratio. The secondary

parameters in this study were: Se intake, serum Se, serum GPx, serum CK, serum T3:T4 ratio,

serum Cu relative to Cu intake, hair growth, and mRNA expression for GPx1, SelH, SepW1,

TrxRd1, TrxRd2, DIO1, SelK, SelT, Sep15, TNF-α, and NFκB.

The average of the three baseline measurements were analysed between dog groups with

two-sample Student's t-tests. Using the entire dataset, each parameter was analysed

univariately with a linear mixed effects model in R version 3.1.1 using the nlme package (R

Foundation for Statistical Computing, Vienna, Austria). The fixed effects were diet, week, diet

x week interaction, and baseline of the parameter (the average of the dogs’ three pre-feed

measurements). The random effect was dog. The assumption of normality was assessed for

each model by visual inspection of the residuals and urinary Se:CT, serum Cu and all mRNA

parameters were log transformed. Between diet contrasts were applied in each week and

between week contrasts were applied within each diet. Contrasts on the two primary

parameters were Bonferroni corrected to an overall significance level of 5% (i.e. threshold p-

value of 0.025).

5.4 Results

Se intake during the pre-feed period was similar in the two experimental groups (Group A:

average 22.9 µg/kg BW0.75, range 19.0 - 29.5 µg/kg BW0.75, Group B: average 20.6 µg/kg

BW0.75, range 16.8 - 25.2 µg/kg BW0.75). During the experimental period, average Se intake

for the dogs on the adequate Se diet was 21.7 µg/kg BW0.75 (range 16.6 - 28.2 µg/kg BW0.75)

and for the dogs on the low Se diet was 2.7 µg/kg BW0.75 (range 2.2 - 3.4 µg/kg BW0.75)

which was significantly different comparing each week (p<0.001). There were no significant

differences between the groups for any of the parameters at baseline, except for the serum

Cu:Cu intake ratio (p=0.033).

Urinary Se:CT ratio significantly decreased in dogs on the low Se diet from week 1 onwards

and on average decreased by 84% (Figure 5.1). Whole blood GPx activity was significantly

lower (by 7%) in dogs fed the low Se diet at week 8 (Figure 5.2). Cumulative hair growth

results are shown in Figure 5.3 and no differences between the diets were found. An

overview of the results of all serum measurements is given in Table 5.4. Serum Se

significantly decreased by 7% in dogs fed the low compared to the adequate Se diet from

138 Chapter 5


Figure 5.1 Urinary selenium to creatinine ratio of dogs fed a low or

adequate selenium diet

The low selenium diet contained 6.5 µg/MJ and the adequate selenium diet

46.1 µg/MJ. Black triangles are dogs on the adequate selenium diet, open

squares are dogs on the low selenium diet. Values at week zero indicate the

average baseline values. Symbols represent the means and error bars indicate

their standard errors, based on the raw data.

Figure 5.2 Glutathione peroxidase activity (U/L) in whole blood of dogs

fed a low or adequate selenium diet

The low selenium diet contained 6.5 µg/MJ and the adequate selenium diet

46.1 µg/MJ. Black triangles are dogs on the adequate selenium diet, open


average baseline values. Symbols represent the means and error bars indicate

their standard errors, based on the raw data.

0

5

10

15

20

25

30

35

40

45

0 1 2 3 4 5 6 7 8 9

urinary

sele

niu

m:cre

atinin

e ratio

week

60000

62000

64000

66000

68000

70000

72000

74000

76000

0 1 2 3 4 5 6 7 8 9

whole

blo

od G

Px

act

ivity (U/L

)

week

0

Chapter 5


139

Figure 5.3 Cumulative hair growth (mm) of dogs fed a low or adequate

selenium diet

Hair growth was measured weekly in the groin of Labrador retrievers fed a

diet containing a low (6.5 µg/MJ) or adequate (46.1 µg/MJ) concentration of

selenium. Black triangles are dogs on the adequate selenium diet, open


average baseline values at weeks 2, 5 and 8 of the pre-feed period, where

hair had been removed at week 0. Symbols represent the means and error

bars indicate their standard errors, based on the raw data.

week 1 onwards. A significant difference in serum GPx was detected from week 6 (6%

mean decrease from first significance).

None of the mRNA measures were significantly changed by the consumption of the low Se

diet (see Table 5.5). Interestingly, there was an upregulation in both groups in mRNA

expression of SelK, SelT, and Sep15 during the second half of the experimental period

(weeks 2 & 4 vs. weeks 6 & 8) and a downregulation in the expression of SelH, TrxRd1,

TrxRd2, TNF-α, and NFκB (p<0.05).

5.5 Discussion

This study indicated that there was a potential difference in the reaction of biomarkers to

changes in dietary Se concentration. Some of the measured biomarkers reacted within one

week, whilst others did not significantly change within the 8 week study period. The

estimated biomarker reaction time will be dependent on the power for each biomarker and

so, more biomarkers might have been identified as significantly reacting if more dogs were

0

2

4

6

8

10

12

14

16

0 1 2 3 4 5 6 7 8 9

cum

ula

tive

hair g

row

th (m

m)

week

140 Chapter 5


Table 5.5 Relative� mRNA expression in dogs fed a diet with a low or adequate selenium

concentration


GPx1 0 12 5.52 0.90 12 6.29 1.17 0.611

2 12 5.93a 1.15 12 6.03a 1.35 0.956

4 12 4.80a 0.77 12 4.62a 0.59 1.000

6 12 4.42a 0.49 12 5.92a 1.24 0.950

8 12 5.30a 0.48 12 6.27a 1.28 0.992

SelH 0 12 2.68 0.19 12 3.12 0.23 0.154

2 12 3.58a 0.36 12 4.08a 0.26 0.972

4 12 3.05a 0.29 12 3.38a 0.46 0.993

6 12 2.20b 0.27 12 2.37b 0.19 1.000

8 12 2.27b 0.36 12 2.20b 0.18 0.954

SepW1 0 12 2.16 0.24 12 2.62 0.35 0.286

2 12 2.41a 0.31 12 2.41a 0.26 0.870

4 12 2.48a 0.41 12 2.41a 0.44 0.581

6 12 2.07a 0.26 12 2.05a 0.20 0.868

8 12 2.61a 0.72 12 2.03a 0.30 0.468

TrxRd1 0 12 2.00 0.09 12 1.96 0.06 0.721

2 12 5.54a 0.23 12 4.77a 0.38 0.259

4 12 3.97b 0.26 12 3.89a 0.37 0.995

6 12 1.89bc 0.15 12 1.67b 0.11 0.681

8 12 1.64bc 0.09 12 1.65b 0.15 1.000

TrxRd2 0 12 2.86 0.19 12 3.01 0.20 0.615

2 12 4.04a 0.40 12 3.84a 0.21 1.000

4 12 3.75a 0.40 12 3.61ac 0.42 0.993

6 12 2.25b 0.27 12 2.48bc 0.24 0.910

8 12 3.17ab 0.64 12 2.31b 0.26 0.452

SelK 0 12 13.10 0.78 12 13.72 0.77 0.575

2 12 2.78a 0.25 12 2.95a 0.23 0.998

4 12 2.68a 0.25 12 2.80a 0.33 1.000

6 12 11.42b 0.58 12 12.89b 1.17 0.982

8 12 17.96c 2.01 12 15.91b 1.61 0.538

SelT 0 12 9.72 1.17 12 10.25 1.11 0.746

2 12 4.27a 0.52 12 4.72a 0.52 0.921

4 12 4.85a 0.63 12 5.05a 0.67 0.998

6 12 7.12b 0.74 12 7.97b 1.07 1.000

8 12 7.57b 0.86 12 7.92b 0.85 1.000

Sep15 0 12 2.37 0.15 12 2.47 0.19 0.693

2 12 1.90ac 0.14 12 1.97a 0.11 0.996

4 12 1.87a 0.20 12 1.71a 0.15 0.788

6 12 2.44b 0.16 12 2.66b 0.21 0.962

8 12 2.42bc 0.25 12 2.67b 0.31 0.953

Chapter 5


141

Table 5.5 continued


TNF-αααα 0 12 3.28 0.30 12 3.54 0.38 0.597

2 12 6.73a 0.91 12 7.03a 0.67 0.990

4 12 6.73a 0.54 12 6.95a 0.66 1.000

6 12 2.89b 0.40 12 3.31b 0.51 0.909

8 12 3.29b 0.34 12 2.91b 0.29 0.681

NFκκκκB 0 12 2.07 0.11 12 2.17 0.12 0.543

2 12 5.18a 0.35 12 5.19a 0.21 1.000

4 12 6.55a 0.34 12 6.26a 0.47 0.946

6 12 1.72b 0.09 12 1.68b 0.13 0.982

8 12 1.87b 0.16 12 1.71b 0.12 0.821 � Samples with the lowest number of gene copies for each gene of interest had a value of 1 with

all other sample values relative to that. Values at weeks zero indicate the average baseline values. Weeks within one gene and diet with a common letter in superscript (mean column) do not significantly differ (p>0.05). qPCR, quantitative polymerase chain reaction; Se, selenium; n, number

of dogs; SE, standard error; GPx1, glutathione peroxidase 1; SelH, selenoprotein H; SepW1, selenoprotein W; TrxRd1, thioredoxin reductase 1; TrxRd2, thioredoxin reductase 2; DIO1, iodothyronine deiodinase 1; SelK, selenoprotein K; SelT, selenoprotein T; Sep15, 15 kDa selenoprotein; TNF-α, tumor necrosis factor alpha; NFκB1, nuclear factor kappa-light-chain-

enhancer of activated B cells; B2M, beta-2-microglobulin; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HPRT1, hypoxanthine phosphoribosyltransferase 1.

included in the study. The number of dogs in this study (n=24) was selected to ensure 80%

power to detect differences between the diets in the primary measures: urinary Se:CT and

whole blood GPx. This is considered a realistic number to be repeated at other dog

facilities, as larger numbers of dogs are rarely found in research facilities. This study did not

evaluate the effects of Se on dog health, but does give an indication on which parameters

may be used to assess Se associated long-term health effects.

Urinary Se:CT ratio dramatically decreased within one week after changing to the low Se

diet. Urine is the main route of excretion of excess Se(42-43). 24-hour urinary Se concentrations

have previously been demonstrated to correlate well with Se intake in humans(43-44), cats(15; 20),

and dogs(15) and this study demonstrated that the more practical single void sample,

corrected for CT concentration, is a valuable indicator of dietary Se concentration in dogs.

Urinary Se:CT ratio has previously been validated as a proxy measure of 24-hour urinary Se

excretion in humans(45) but has never been measured in diseased dogs (e.g. with cancer), so

there is no dog specific estimate of a safe urinary Se:CT concentration. However, in humans

there are indications that urinary Se reflects Se requirements, as excretion decreases in

healthy children, pregnant women and in people with cancer, where it is likely that Se

requirements are higher compared to healthy individuals(43). This makes urinary Se:CT ratio

142 Chapter 5


an important biomarker to measure changes in dietary Se intake, but the safe minimum level

of urinary Se:CT ratio that is associated with the prevention of long-term health effects

warrants further research.

Serum Se concentrations also decreased within one week after switching the dogs to the

low Se diet. The average serum Se concentrations of the dogs in this study was 257 µg/L

(range 213-310 µg/L) in the adequate and 238 µg/L (range 172-298 µg/L) in the low Se

group, and fell within the range reported by Forrer et al.(46) (150-340 µg/L) for healthy dogs.

However, dogs in the study of Forrer et al. were healthy at the time of sampling, but were

not monitored during a longer period, so disease may have developed at a later stage.

Pilarczyk et al.(47) found a significantly lower (p<0.01) serum Se concentration in dogs with

malignant neoplasm (range 103-265 µg/L) compared to healthy dogs (range 208-346 µg/L).

This association reinforces the need to verify whether this relationship is causal.

GPx measurements were taken as an indicator of Se bioactivity. Se requirements are often

based on GPx concentrations reaching a plateau(29; 48). However, we do not know if optimal

selenoprotein concentrations are desirable for optimal health. In humans, a blood Se

concentration of 79-95 µg/L is considered sufficient to maximize GPx activity(48). This seems to

be higher in dogs, as the average serum Se concentration of dogs in the low Se group in

the last week of the study was 224 µg/L, where both serum and whole blood GPx

concentrations were significantly lower than in the adequate Se group, and thus not

maximised.

In this study, inorganic Se (sodium selenite) was used as the primary source of Se. This can

be converted to selenocysteine and incorporated into selenoproteins(49). Selenomethionine

cannot be synthesized from inorganic selenium, but may be non-specifically incorporated

into body proteins(50), so using inorganic Se prevents selenomethionine mixing with the

methionine pool and incorporation of selenomethionine into body proteins where it has no

Se specific role(49). The use of selenomethionine (organically-bound Se) would have likely

resulted in a higher retention of Se in the body (i.e. lower excretion) without being

bioactive, and a higher dietary Se requirement may thus apply for organic compared to

inorganic Se species in order to maximize selenoprotein activity.

The body stores of Se may also have had an impact on all biomarkers measured in this

study. In the first part of the study, Se may still have been present in body proteins and

during protein turn-over may have been released to become available for selenoprotein

incorporation (in this study measured as GPx), while in the last few weeks body stores of Se

may have become depleted. It is likely that the amount of stored Se is animal species

specific, as Todd et al.(15) found that cats retain less Se than dogs. This may be linked to the

Chapter 5


143

evolutionary "feast and famine" feeding approach of dogs, as described by Bosch et al.(51), in

which dogs gorge feed, and during the famine stage, stored Se may be used for

physiological processes.

Serum GPx, in this study, significantly reacted two weeks earlier to a change in dietary Se

compared to whole blood GPx. Similar findings were reported in cats by Todd et al.(20).

Changes in plasma GPx were observed after feeding a diet containing 2, 1.43 and 0.98 mg

sodium selenite/kg DM after 16, 32 and 24 days, respectively, compared to a basal diet

containing 0.45 mg Se/kg DM. No changes in whole blood GPx were observed within the

32-day lasting study of Todd et al.(20). This difference may simply be caused by the higher

level and variation of GPx activity in whole blood compared to serum. GPx activity of dogs

in whole blood is approximately five times higher than in serum. Another possible reasoning

is that the surplus of tissue GPx is effluxed into the plasma, as indicated by the high

correlation between tissue and plasma GPx concentrations(52). The difference in reaction time

between serum and whole blood GPx activity may also be explained by different isoforms

of GPx. Plasma is known to contain mainly the plasma GPx (GPx3) enzyme(53), while whole

blood may additionally contain other forms of GPx. However, the GPx activity measurement

does not discriminate between various forms of GPx and it may be that the activity of serum

GPx is more quickly affected by a reduction in dietary Se than other forms of GPx. In rats, it

has been determined that plasma GPx3 activity is more highly regulated by dietary Se

concentration than, for example, muscle GPx4(54). The same authors(54) have shown that

although GPx1 mRNA expression was highly regulated by dietary Se concentration in rats,

dietary Se requirements based on tissue (plasma, red blood cell, liver, kidney, and muscle)

GPx activity were higher than when based on mRNA expression (i.e. mRNA expression

plateaus at lower dietary Se concentrations). This is in accordance with the findings of the

present study. The fact that GPx is affected, but the selenoprotein mRNA levels are not,

indicates that the bioactive Se fraction of the low Se diet (total Se concentration: 6.5 µg/MJ

or 0.11 mg/kg DM) is sufficient to maintain mRNA expression of the measured

selenoproteins. Although mRNA expression is the basis of protein formation, the actual

formation of the active selenoproteins were not maximized by the low Se diet. This may be

due to effects on translation, like the structure of specific stem-loops which bind proteins(55).

Also, post-translational modifications (which affect the actual activity of the selenoproteins,

e.g. protein folding, phosphorylation status, etc.) may have played a role in the difference in

reaction of GPx1 mRNA expression and GPx activity to a change in dietary Se

concentration. Therefore, GPx activity may be a more reliable biomarker for Se status than

the mRNA expression of selenoproteins. However, it may be that maximal selenoprotein

144 Chapter 5


activity is not necessary for the prevention of disease on the long-term. Selenius et al.(56), for

example, found that TrxRd1 mRNA expression was increased with increasing addition of

sodium selenite concentrations (range 2.5–10 µM) to lung cancer cells, while TrxRd activity

decreased at high selenite concentrations, which indicates the impairment of selenoprotein

formation. Therefore, it is hypothesized that selenoprotein mRNA expression may be a useful

biomarker in the detection of high dietary Se concentrations, but that GPx activity is more

useful in the estimation of minimum Se requirements.

The fact that the SelK, SelT, and Sep15 mRNA markers were increased and SelH, TrxRd1,

TrxRd2, TNF-α, and NFκB were decreased in the second half compared to the first half of

the study, point towards an event of inflammation during the first part of the study, as TNF-α,

and NFκB are inflammation markers. The study did not include induction of inflammation

and there are no indications of such events during or before the study, all circumstances

stayed the same. This reinforces the idea that other factors than dietary Se have more

impact on the selenoprotein mRNA expression than dietary Se concentration.

The dietary Se concentration did not result in a significant difference in hair growth

between the diets. Previous studies which have used hair growth as a marker of Se status(27)

used beagles, so there may be a breed variation in hair growth. Although no literature on

speed of hair growth between different breeds could be found, there is evidence for

genetic differences in coat formation between breeds(57). However, the most likely

explanation for the lack of difference in hair growth is that the current study has used

sodium selenite as Se source and in the study of Yu et al.(27), selenomethionine was

supplemented. Methionine is often a limiting amino acid and selenomethionine can be non-

specifically incorporated into body proteins such as hair (50). Therefore, it may be that the

decrease in hair growth in the study of Yu et al. is not due to a reduction in Se, but in

methionine.

As there is no clear adequate range of dietary Se for adult dogs, the low Se diet may have

contained sufficient Se (6.5 µg/MJ or 0.11 mg/kg DM) to maintain health. In this 8 week

study it did not have a negative effect on thyroid hormone metabolism (T3:T4). The

conversion of T4 into the active form T3 has been reported to increase with increasing

dietary Se concentrations in puppies within a range of 0-0.52 mg/kg(29). Also Cu is indicated

to be involved in thyroid hormone metabolism, as a lower GPx activity and liver

selenodeiodinase activity was found in Cu deficient rats compared to a control group(58).

These results indicate that the low Se diet is not deficient for thyroid hormone metabolism of

adult dogs. Although the serum Cu:Cu intake ratio was significantly different between the

groups at baseline, this is unlikely to have affected the results as including the baseline

Chapter 5


145

intake as a covariate in the analysis will normalize any differences between groups. Further

studies are required to determine the long-term health effects associated with a change in

urinary Se:CT ratio, serum Se and serum GPx concentrations. This can be done by a

retrospective study to determine adequate ranges of these biomarkers and to establish

which set of biomarkers from our study is needed to provide a sufficient prediction of the

risk of Se-induced disease. Consecutively, these ranges can be used in a study to

determine the minimal Se requirement of dogs.

5.6 Conclusion

There were variations in the reaction time of Se biomarkers to a reduction in dietary Se

concentration in dogs. This study has demonstrated that urinary Se:CT ratio, serum Se, and

serum GPx activity react most quickly. These may be useful biomarkers in future long-term

studies to evaluate the minimum requirements for optimal health in dogs.

5.7 Acknowledgements

The hair growth measurements were done by Robyn Bednall and Tim O'Brine of the

WALTHAM® Centre for Pet Nutrition, which was very much appreciated. The authors also

express their thanks to Joachim Neri of the Department of Applied Analytical and Physical

Chemistry at the Faculty of Bioscience Engineering of Ghent University for the Se analyses.

Donna Vanhauteghem of the Laboratory of Animal Nutrition at Ghent University is thanked

for the Cu, CK, T3 and T4 analyses and Iain Peters of the Innovation Centre (Exeter, UK) is

gratefully acknowledged for his expertise and analysis of the mRNA samples.

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40. Van de Velde H, Janssens GPJ, de Rooster H et al. (2013) The cat as a model for human obesity: insights into depot-specific inflammation associated with feline obesity. British

Journal of Nutrition 110110110110, 1326-1335. 41. Vandesompele J, De Preter K, Pattyn F et al. (2002) Accurate normalization of real-time

quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biology 3333, research 0034.

42. Francesconi KA, Pannier F (2004) Selenium Metabolites in Urine: A Critical Overview of Past Work and Current Status. Clinical Chemistry 50505050, 2240-2253.

43. Sanz Alaejos M, Díaz Romero C (1993) Urinary selenium concentrations. Clinical Chemistry 39393939, 2040-2052.

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44. Hawkes W, Richter BD, Alkan Z et al. (2008) Response of Selenium Status Indicators to Supplementation of Healthy North American Men with High-Selenium Yeast. Biological

Trace Element Research 122122122122, 107-121. 45. Hojo Y (1981) Evaluation of the expression of urinary selenium level as ng Se/mg

creatinine and the use of single-void urine as a sample for urinary selenium determination. Bull Environm Contam Toxicol 27272727, 213-220.

46. Forrer R, Gautschi K, Lutz H (1991) Comparative determination of selenium in the serum of various animal species and humans by means of electrothermal atomic absorption spectrometry. Journal of Trace Elements and Electrolytes in Health and Disease 5555, 101-113.

47. Pilarczyk B, Tomza-Marciniak A, Pilarczyk R et al. (2013) Relationship between serum Se concentration in dogs and incidence of some disease conditions. Central European Journal

of Biology 8888, 527-533. 48. Thomson CD (2004) Assessment of requirements for selenium and adequacy of selenium

status: a review. European Journal of Clinical Nutrition 58585858, 391-402. 49. Suzuki KT (2005) Metabolomics of Selenium: Se Metabolites Based on Speciation Studies.

Journal of Health Science 51515151, 107-114. 50. Schrauzer GN (2000) Selenomethionine : A Review of Its Nutritional Significance ,

Metabolism and Toxicity. Journal of Nutrition 130130130130, 1653-1656. 51. Bosch G, Hagen-Plantinga EA, Hendriks WH (2015) Dietary nutrient profiles of wild wolves:

insights for optimal dog nutrition? British Journal of Nutrition 113113113113, S40-S54. 52. Combs Jr GF, Combs SB (1986) Biochemical functions of selenium. In The Role of Selenium

in Nutrition, pp. 205-263. New York: Academic Press. 53. Avissar N, Ornt DB, Yagil Y et al. (1994) Human kidney proximal tubules are the main

source of plasma glutathione peroxidase. American Journal of Physiology 266266266266, C367-C375. 54. Barnes KM, Evenson JK, Raines AM et al. (2009) Transcript analysis of the selenoproteome

indicates that dietary selenium requirements of rats based on selenium-regulated selenoprotein mRNA levels are uniformly less than those based on glutathione peroxidase activity. Journal of Nutrition 139139139139, 199-206.

55. Hesketh JE, Vasconcelos MH, Bermano G (1998) Regulatory signals in messenger RNA: determinants of nutrient–gene interaction and metabolic compartmentation. British

Journal of Nutrition 80808080, 307-321. 56. Selenius M, Fernandes AP, Brodin O et al. (2008) Treatment of lung cancer cells with

cytotoxic levels of sodium selenite: Effects on the thioredoxin system. Biochemical

Pharmacology 75757575, 2092-2099. 57. Cadieu E, Neff MW, Quignon P et al. (2009) Coat Variation in the Domestic Dog Is

Governed by Variants in Three Genes. Science 326326326326, 150-153. 58. Olin KL, Walter RM, Keen CL (1994) Copper deficiency affects selenoglutathione

peroxidase and selenodeiodinase activities and antioxidant defense in weanling rats. American Journal of Clinical Nutrition 59595959, 654-658.

_____________________________________________________________________________________________________________

General discussion_____________________________________________________________________________________________________________

Chapter 6

General discussion_____________________________________________________________________________________________________________

Main findings of the studies reported within this thesis

1. Diet type and crude protein digestibility have a significant impact on Se accessibility

in vitro, where canned diets showed a negative association with crude protein

digestibility and kibble diets a postitive association (Chapter 3a).

2. The Se accessibility from dry pet foods can be estimated by predictive equations

(Chapter 3b).

3. Canned diets contain on average more Se than kibble diets (Chapter 3a & 4).

4. The bioavailability of Se is higher from kibble than from canned diets (Chapter 4).

5. Of the biomarkers measured, urinary Se to creatinine ratio, serum Se and serum

glutathione peroxidase reacted most quickly to a decrease in dietary Se

concentration in dogs (Chapter 5).

Definitions used:

Accessibility: the amount of dietary selenium that is potentially available for absorption after in

vitro digestion.

Bioavailability: the fraction of the dietary selenium that reaches the systemic circulation.

Availability: when accessibility and bioavailability are discussed together.

Bioactivity: selenium that is incorporated into selenoproteins.

Chapter 6

General discussionGeneral discussionGeneral discussionGeneral discussion

151

6.1 Methods to measure selenium availability

When considering how to measure Se availability, either in vitro or in vivo digestibility

measurements can be performed. In vitro studies give an estimation of the accessibility; the

amount that is available for absorption. However, in vitro studies do not inform us of the

actual luminal absorption, and therefore will likely overestimate the apparent in vivo

digestibility. To the author's knowledge, there are no in vitro data available that measures

Se accessibility of pet foods. Therefore, it is not possible to compare our results with

literature. Using the diets of Chapter 3a and 4, the average apparent Se bioavailability

coefficients and the average apparent DM and CP digestibility coefficients were calculated

(Figure 6.1). According to these data, in vitro digestibility overestimates the apparent DM

and CP digestibility and apparent Se bioavailability with 15, 14 and 13%, respectively (all

p<0.001). No significant interaction for diet type (canned vs. kibble) x study type (in vitro vs.

in vivo) was found for apparent DM digestibility and Se bioavailability (p=0.449 and 0.481,

respectively), indicating that there was no difference in the overestimation of in vivo data

with in vitro data between canned and kibble diets. The extent to which apparent in vivo

Se bioavailability was overestimated was also independent of dietary Se concentration

(p=0.545). There was a diet type x study type interaction for CP digestibility, with a higher

overestimation of in vivo data in canned compared to kibble diets (p=0.008).

Not only were the in vitro and in vivo study performed with a different set and number of

diets, but overestimation of apparent in vivo CP digestibility by an in vitro digestibility

method also increases with increasing CP digestibility, as reported in kibble diets by

Hervera et al.(1). One of the limitations of an in vivo study is that apparent digestibility is

measured. This includes endogenous losses, such as enzymatic and biliary Se, that were

actually bioavailable, leading to an underestimation of true bioavailability. The extent of

underestimation is not known exactly and is likely to differ between diets. To overcome this

limitation, dogs should be ileally cannulated, a practice that causes ethical debate. In vitro

studies, on the other hand, do not cause ethical discussion and the Se originating from the

enzyme solutions can be measured separately (in a blank sample) to correct the in vitro Se

accessibility with. Therefore, in vitro studies may give a better estimation of the actual

amount of available Se. In addition, in vitro studies allow to test a larger number of samples

in a shorter amount of time than in vivo studies.

152 Chapter 6

General discussioGeneral discussioGeneral discussioGeneral discussionnnn

Figure 6.1 In vitro and in vivo selenium availability and

apparent dry matter and crude protein digestibility

coefficients

Open bars with black and grey line represent the in vitro results

of canned (n=16) and kibble (n=19) diets, respectively. Black and

grey filled bars stand for the in vivo results of canned (n=4) and

kibble (n=4) diets, respectively. Error bars represent the standard

error. Diets from the in vitro study (Chapter 3a) and in vivo

bioavailability study (Chapter 4) were used in this figure.

6.2 Factors affecting selenium availability

Many factors affect the availability of Se, as described in paragraph 1.7. In this paragraph,

we will only discuss those factors that affected Se availability in the studies reported in this

PhD thesis.

6.2.1 Dietary selenium concentration

In the in vitro Se accessibility (Chapter 3a) and in vivo Se bioavailability (Chapter 4) studies,

the dietary Se concentration was higher in canned than kibble diets. We know from the in

vitro study that the Se concentration per se does not affect the percentage of availability,

but the dietary Se concentration can influence the absolute amount of available Se.

Figure 6.2 shows the association between dietary Se and Se availability. The FEDIAF

minimum recommendation of dietary Se concentration is depicted by the black line. The

dietary Se concentration, together with the amount of dietary intake determines the Se

0

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selenium availability

dry matter digestibility

crude protein digestibility

Av

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stib

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y (

%)

Chapter 6


153

intake, and thus affects the potential absolute amount of available Se. Dietary intake may

be decreased as a therapy for obesity. Worldwide, 22 to 40% of the pet dogs suffer from

obesity(2). Together with the reduction in caloric intake, Se intake may also be reduced.

Linder et al.(3) found that diets specifically produced for weight loss often do not comply

with the current minimum recommendations for Se. In our studies, one canned and one

kibble weight loss diet were included, which both contained more Se than minimally

recommended by FEDIAF (canned 1.58 and kibble 0.56 mg/kg DM). In addition, weight loss

diets commonly contain high fibre concentrations(4), which may reduce the bioavailability of

Se from the diet and thus may increase the requirement (per MJ) of dietary Se. Nevertheless,

the in vitro study did not identify fibre as an important contribution to the variation in Se

accessibility. Considering the fact that clinical cases of Se deficiency are not reported, it can

be concluded that it is unlikely to develop Se deficiency when commercially available diets

are fed. In addition, it can be seen from Figure 6.2 that although the dietary Se

concentration may be below the FEDIAF minimum recommendation or well above the legal

upper limit for Se supplemented diets (assumed to be a "safe upper intake level"), the

majority of the diets are still in the "safe range" of Se availability.

The lower cut-off for the "safe-range" of available Se (green field in Figure 6.2) is based on

a study by Wedekind et al.(5). This limit can be questioned, as selenomethionine was used

as the Se source in this study. Unlike inorganic Se species, selenomethionine may be non-

specifically incorporated into body proteins, by which it is unavailable for incorporation into

selenoproteins. As the breakpoint reported in this study was based on GPx activity, the Se

requirements may have been overestimated.

The upper cut-off is based on the legal upper limit for Se supplemented diets. However, no

information is available on the basis of this legislation. It is known from studies by

Wedekind et al.(5) and Yu et al.(6) that 5 mg SeMet/kg diet may result in adverse effects in

adult dogs. Although, in these studies, Se was supplemented after processing and was thus

likely to have a higher availability than when Se would have been processed as part of the

diet. This suggests that the dietary Se concentrations reported in this thesis are unlikely to

induce adverse effects, but that Se availability cannot be ignored as an important factor

determining Se provision to dogs. In order to allow a fair estimation of the available Se in

dog foods, the Se accessibility models reported in Chapter 3b may be useful for the

formulation of diets.

154 Chapter 6


Figure 6.2 Dietary selenium and apparent

availability coefficients of canned and kibble pet foods

† In vitro selenium accessibility coefficients were corrected for a 13% overestimation of

selenium bioavailability, as presented in Figure 6

digested kibble diets (n=19) and blue filled triangles the

open squares signify in vitro

digested canned diets (n=4). The black line is the minimum requirement for adult dogs (FEDIAF)

and the grey line is the legal upper limit for Se supplemented diets. The lower cut

green field is based on a study of Wedekin

legal upper limit for supplementation.

6.2.2 Selenium speciation

Some diets, in particular those that contain an insufficient amount of Se from their

ingredients, are supplemented to reach the FEDIAF minimum recommendation. Most

commonly, inorganic Se supplements (sodium selenate and sodium selenite) are used

When collecting pet foods for the

European regulation No 767/2009

on the label. Pet food producers where allowed to market pet foods without stating the

additives on the packaging until the

to remain on the market until stocks were exhausted (454/2010 art.2)

Dietary selenium and apparent in vivo and corrected† in vitro

availability coefficients of canned and kibble pet foods

selenium accessibility coefficients were corrected for a 13% overestimation of

bility, as presented in Figure 6.1. Blue open triangles represent

digested kibble diets (n=19) and blue filled triangles the in vivo digested kibble diets (n=4). Red

in vitro digested canned diets (n=16) and red filled squares the


and the grey line is the legal upper limit for Se supplemented diets. The lower cut

on a study of Wedekind et al.(5) and the upper cut-off is based on the

legal upper limit for supplementation.


ents, are supplemented to reach the FEDIAF minimum recommendation. Most

commonly, inorganic Se supplements (sodium selenate and sodium selenite) are used

When collecting pet foods for the in vitro study (chapter 3a), many did not yet comply with

No 767/2009(7), which states that any Se additives should be declared


ves on the packaging until the 31st of August 2011 and these products were permitted

to remain on the market until stocks were exhausted (454/2010 art.2)(8).

in vitro selenium

selenium accessibility coefficients were corrected for a 13% overestimation of in vivo

.1. Blue open triangles represent in vitro

digested kibble diets (n=4). Red

led squares the in vivo


and the grey line is the legal upper limit for Se supplemented diets. The lower cut-off of the

off is based on the


ents, are supplemented to reach the FEDIAF minimum recommendation. Most

commonly, inorganic Se supplements (sodium selenate and sodium selenite) are used(4).

), many did not yet comply with

, which states that any Se additives should be declared


of August 2011 and these products were permitted

Chapter 6


155

When retrievable, Se addition of the diets used in the in vitro study (chapter 3a), together

with the in vivo bioavailability study diets (chapter 4), were used to calculate the amount of

Se derived from the ingredients. Se supplementation was not specified in any of the canned

diets (12 out of 20 retrieved). In kibble diets, 15 of the 18 retrieved diets (out of 23)

contained supplemented inorganic Se. The amount specified on the package is the amount

of the sodium selenite salt (in mg/kg diet) and not the actual addition of the Se mineral.

Therefore, a standard Se concentration of 45.6% in sodium selenite supplements(4) is used to

calculate the absolute amount of supplemented Se. The amount of supplementation ranged

from 0.01 to 0.15 mg Se/kg DM, which is 3 to 28% of the total dietary Se concentration

(Figure 6.3). The amount of Se from the ingredients in kibble diets therefore ranged between

0.19 and 1.15 mg Se/kg DM. In canned diets this was 0.17 to 2.12 mg Se/kg DM (assuming

that no declaration means no supplementation). Two of the kibble diets were supplemented

above the legal maximum for Se supplemented diets. The pet food producers may have

used estimated values for the formulation of the diets and may not have been aware of the

high Se concentrations (or variability per batch) in the ingredients. This reflects the difficulty

for pet food manufacturers to produce pet foods within the minimum recommended and

legal maximum range. The analysis of dietary Se for every batch of pet food is not a

standard procedure, due to the costs involved. To avoid unnecessary supplementation, it

would however be advisable to analyse the Se concentration of the diets on regular basis.

The legislation on additive specification on pet food labels was made for clarification of pet

food ingredients to customers, but may be misleading, as the amount of sodium selenite

supplement is mentioned and not the absolute amount of Se added, nor the total dietary Se

concentration. The additional amount of Se generally seems to be a small percentage of the

total dietary Se concentration and may give customers a wrong indication of the dietary Se

concentration. Additionally, the specified amount on the package may actually be 40%

lower or even 300% higher, as long as it does not exceed the legal maximum(9), which may

question the relevance of this regulation even more.

The addition of selenite in kibble diets tended to increase the apparent Se availability

coefficients of the diets reported in this thesis (p=0.086, Figure 6.4). The limited amount of Se

supplementation of the total dietary Se concentration may be the explanation for the lack of

a significant effect. However, the diets used in these studies should be considered a random

market sample, so apparently the fraction of supplemented Se represents only a small

percentage of the total analysed dietary Se. Therefore, the differences in Se accessibility

and bioavailability between canned and kibble diets cannot be merely attributed to the

dietary Se species.

156 Chapter 6


Figure 6.3 Supplemented selenite and selenium from ingredients

in kibble diets (n=18)

Filled bars represent the selenium from the ingredients and open bars

are the amount of supplemented selenite. The black line indicates the

recommended minimum Se concentration and the grey line the legal

maximum when Se is supplemented. Diets from the in vitro study (Chapter

3a) and in vivo bioavailability study (Chapter 4) were used in this figure.

Figure 6.4 Effect of selenite supplementation on apparent in vivo

and corrected† in vitro selenium availability coefficients in kibble

diets (n=18)

† In vitro selenium accessibility coefficients were corrected for a 13%

overestimation of in vivo selenium bioavailability, as presented in Figure

6.1. R2, coefficient of determination. P-value was calculated with linear

regression.

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Se

len

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(m

g/k

g D

M)

Diet

R² = 0.173

0

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0 5 10 15 20 25 30

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Added selenite per total dietary selenium (%)

p = 0.086

Chapter 6


157

6.2.3 Processing conditions

The difference in processing conditions may be another factor that can explain the higher

Se availability from kibble compared to canned diets. The negative impact of retorting on

the in vitro Se accessibility from canned diets was demonstrated in Chapter 3a. Todd et

al.(10) found similar results in a canned cat food, of which the apparent Se absorption

(bioavailability) of the diet was only 37% in dogs, but when sodium selenite or selenium

yeast was supplemented after processing the Se absorption increased more than two-fold

(83 and 79%, respectively), indicating that the negative effect of retorting can be overcome

by supplementing Se after processing. Interestingly, we have also demonstrated that the in

vitro Se accessibility from kibble diets was not affected by extrusion, which emphasizes the

difference between diet types. This means that there are Maillard products or other

complexes formed by the retortion process that renders the Se unavailable.

A large part of the Se in pet foods is protein-bound, therefore, the CP digestibility may have

an influence on the availability of Se. The in vitro study (Chapter 3a) showed that there was

a positive association between Se accessibility and CP digestibility in kibble diets but a

negative association in canned diets. This paradoxical finding can be explained by the net

effect of two factors; protein digestibility as such, and a potential higher susceptibility of

highly digestible protein for cross-linking during retorting. Formation of cross-linkages

require free amino groups(11-12), which are more abundant from highly digestible protein

sources, such as hydrolysed protein, than from protein sources with a low digestibility. The

cross-linkages may bind a large proportion of protein-bound Se. In kibble diets, protein

digestibility appeared to be a more important factor to affect Se accessibility.

When expressed as amount of digestible CP (g/kg DM; Figure 6.5), the effects of CP

digestibility are unclear because dietary CP concentration – as factor in the equation for

digestible CP – causes variability. As such, dietary CP concentration is the main determinant

for the dietary digestible CP concentration, because of the relatively limited range in CP

digestibility within commercially available pet foods versus the large range in dietary CP

concentration (demonstrated in Chapter 4). Yet, the data show that the digestibility rather

than the amount of digestible CP determines Se accessibility: if a particular amount of Se is

bound to protein, it can be logically explained that protein digestibility determines the

proportion of Se that becomes accessible. In this case, the level of protein in the diet does

not contribute to Se accessibility.

The in vivo study revealed no effect of digestible CP on apparent Se bioavailability. In

theory, this might be the result of neglecting shifts in endogenous Se losses. This is however

unlikely, because the data from the in vivo trial fit nicely in the data cloud of the in vitro

158 Chapter 6


trial, as shown in Figure 6.5. A more feasible explanation is that the test range in digestible

CP in the in vivo trial was much narrower than in the in vitro trial, so that the residual

variation became relatively larger. This likely hindered the demonstration of a significant

relationship between digestible CP and apparent Se bioavailability within the test range.

This emphasises that the impact of diet type is more important for Se availability than

digestible CP.

Figure 6.5 Effect of digestible crude protein on in vitro selenium

accessibility and apparent in vivo selenium bioavailability

Blue open triangles represent in vitro digested kibble diets (n=19) and

blue filled triangles the in vivo digested kibble diets (n=4). Red open

squares signify in vitro digested canned diets (n=16) and red filled

squares the in vivo digested canned diets (n=4). Diets from the in vitro

study (Chapter 3a) and in vivo bioavailability study (Chapter 4) were used

in this figure.

6.3 Bioavailability versus bioactivity

Bioavailability was used as an indication of the ability of Se to incorporate into

selenoproteins (=bioactivity). However, the in vivo bioavailability study (Chapter 4) showed

that there were large differences in urinary Se excretion between diets and that the urinary

excretion reacts quickly to a change in dietary Se. This indicates that the retention, and

thus the amount available for bioactivity is different between diet types. Therefore, it is

recommended to measure urinary Se excretion in combination with the bioavailability, as

0

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100

120

0 200 400 600 800 1000

sele

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)

digestible crude protein (g/kg DM)

Chapter 6


159

this will give a better picture of the amount available for Se bioactivity. Bioactivity is what

makes Se essential for the dog, so that should be the main determinant for the amount of

dietary Se. In addition, the Se excreted in the urine may be considered potentially harmful

to the dog. Therefore, not only bioactivity, but also bioavailability and urinary Se excretion

(as a measure for retention) should be studied, since this might determine the amount of Se

circulating in the body without "safe" incorporation into selenoproteins.

It is clear that determination of the Se speciation would provide mechanistic insights in the

above mentioned issues. For instance, to determine the association between urinary Se

metabolites and Se toxicity and/or deficiency, it is convenient to know which metabolites

are excreted in the urine by dogs. It is known from a pilot study for the studies in this thesis,

that dog urine can contain selenosugars. In 3 out of 10 urine samples of dogs fed a

commercially available dry diet containing 0.32 mg Se/kg diet, selenosugar 2 could be

detected with liquid chromatography high-resolution mass spectrometry (LC-HR-MS)

(unpublished data). TMSe was not identified in any of the samples. The selenosugar

concentration accounted for approximately 1 to 11% of the total urinary Se in samples in

which it could be detected. However, the high detection limit (50 ng/ml) and complex

urinary matrix, made it difficult to quantify other Se species in dog urine. For more insight

into Se metabolism of dogs, future studies may focus on urinary Se speciation between

canned and kibble diets and at different dietary Se concentrations.

6.4 Factors affecting measurement of glutathione peroxidase activity

In Table 6.1, the dietary Se intake and a set of biomarkers are compared between the diets

used in the bioavailability study (Chapter 4) and the adequate diet of the biomarker study

(Chapter 5). It should be noted that these data came from different studies including

different diets and different dogs. Se intake was significantly higher in the biomarker study

compared to both the canned (primarily organic Se) and kibble (partly inorganic Se) diets,

which was due to the high Se concentration of the semi-purified diet (completely inorganic

Se). Interestingly, the difference between the semi-purified diet and the commercial diets did

not result in an increased whole blood GPx activity, suggesting that GPx activity may have

reached a plateau, or was less effectively increased by the sodium selenite used in the

semi-purified diets compared to the Se in the commercially available diets. If the latter is

true, and the difference in canned and kibble diets (Chapter 4) is caused by the Se species

in the diets, kibble diets may need to be supplemented with organic Se instead of sodium

selenite. However, that does not necessarily mean that bioactivity is increased when organic

Se is non-specifically incorporated into body proteins.

160 Chapter 6


6.4.1 Expressing glutathione peroxidase activity

GPx expressed per gram of haemoglobin (Hb, to correct for dilution) did show a significantly

higher level in canned compared to semi-purified diets, but not in kibble compared to the

semi-purified diets. This was attributed to a lower Hb concentration in the blood of dogs fed

canned diets compared to dogs fed the semi-purified diet (p<0.001). One of the factors

causing this, are preservatives that may be used in canned dog foods, such as propylene

glycol(4). Propylene glycol is reported to induce oxidative damage and decrease the

erythrocyte half-life(13), for which it is prohibited from use in cat foods.

When erythrocytes and their Hb are broken down, bilirubin is formed and excreted via the

bile, which causes the typical green to brown colour. In addition to bilirubin, bile consists of

water, bile acids, fats, and minerals(14). The average lifespan of canine erythrocytes is 110

days(15). After that, the Hb from the cytoplasm of erythrocytes is broken down into globin

and heme(14). The globin moiety may contain SeMet(16) and its amino acids are reused for

protein synthesis(14). The heme moiety is divided into iron and porphyrin, which is in turn

excreted in the bile as conjugated bilirubin(14).

It is likely that bile contains higher concentrations of Se when canned diets are fed to dogs.

Canned diets contain on average more fat, which triggers the cholecystokinin secretion by

mucosal epithelial cells, which in turn increases biliary secretion(17). In addition, a dose

dependent biliary Se excretion has been determined for selenite(18-19). If all Se species are

dose dependently excreted in the bile, the higher average Se concentrations of canned

diets will cause a higher biliary Se excretion. Next to the overall higher Se concentration,

canned diets contain on average more organically bound Se, which may be converted to

Selenotaurine(20). More than 99% of bile acids are taurine conjugates(4). Therefore, it is

plausible that the amount of Se excreted via bile is higher from canned than kibble diets.

This results in the conclusion that there is a potential bias in the interpretation of in vivo

apparent Se bioavailability between diet types. Future research could analyse Se-

containing bile acids in the faeces of dogs fed canned and kibble diets to gain a better

insight into the amount of biliary Se excretion and the effect of diet type.

Table 6.1 also shows that a higher amount of the Se from the semi-purified diet compared

the to canned and kibble diets was excreted in the urine. Serum Se and the thyroid

hormones were also increased by the semi-purified diet. The thyroid hormones may have

been increased by a higher iodine and copper intake. Although both were lower in the

semi-purified compared to the canned and kibble diets (I 0.08 and average 0.22 mg/MJ; Cu

0.48 and average 0.81 mg/MJ, respectively), the availability of the minerals may have been

higher in the semi-purified diet. The serum Se concentration is still within the range of 150-

Chapter 6


161

340 µg/L by Forrer et al.(21). It is not clear what the biological relevance of the increase in

serum Se may be. Serum Se should possibly be seen as a short term Se storage. The Se may

be available for GPx activity when necessary (apparently not in this case), or gradually

excreted in the urine.

The apparent higher GPx activity (U/g Hb) in canned compared to kibble diets (Chapter 4)

may be due to the Hb concentration after canned diets were fed to the dogs, similarly to

the difference in GPx activity (U/g Hb) between canned and semi-purified diets. For

example, Todd et al.(10) did not find a difference in GPx activity (U/L) in dogs fed a

commercially available canned diet supplemented with Se yeast or selenite. However, the

lack of a difference in the latter study may also have been caused by a plateau in GPx

activity, as supplements were added to a commercially available, non-Se deficient (0.6

mg/kg DM), canned diet. The studies that did report a higher bioactivity of selenite

compared to SeMet either used growing rats(22) or an amount of dietary Met (2.4 g/kg DM)

in rats(23) considered deficient for adult dogs (FEDIAF minimum recommendation 4 g/kg DM),

suggesting that the SeMet is used as protein building block rather than for the

incorporation in GPx.

6.4.2 Serum versus whole blood glutathione peroxidase activity

In the biomarker study (Chapter 5), GPx activity in dogs was found to be 5 times higher in

whole blood than serum. In humans erythrocyte GPx activity is reported to be 25 to 100

times higher than plasma(24). This difference is likely not due to the difference between

plasma and serum, as plasma only contains clotting proteins (e.g. fibrinogen) extra compared

to serum. No reports have been found that clotting proteins contain GPx, but if they did, that

would only increase the difference between dog and human. GPx concentrations (and thus

activity) can be affected by erythrocyte leakage due to hemolysis(24). However, no sign of

hemolysis was found in the serum samples. The most likely explanation for the larger

difference in erythrocyte and plasma GPx activity in humans compared to whole blood and

serum GPx activity in dogs, is the dilution factor from erythrocyte to whole blood, as whole

blood also contains serum, with a lower GPx concentration than erythrocytes. GPx may also

be used to store Se(25), which suggests that maximal GPx levels are not necessary for optimal

health.

If serum GPx activity would have been measured in the bioactivity study instead of whole

blood GPx, it may have resulted in an even larger difference, due to the lower variability of

serum GPx activity compared to whole blood. Striking is that GPx activity was only

162 Chapter 6


Ta

ble

6.1

Effect

of die

t ty

pe o

n s

ele

niu

m inta

ke a

nd b

iom

ark

ers

p k

ibble

vs

sem

i-

purified

<0.001

0.0

03

0.2

17

0.9

40

<0.001

<0.001

<0.001

<0.001

<0.001

SE, standard

error; G

Px, g

luta

thio

ne p

ero

xidase

; Hb, hem

oglo

bin

; T3, triiodoth

yro

nin

e; T4, th

yro

xine; Se, se

leniu

m; CT, cre

atinin

e.

P-v

alu

es

are

calc

ula

ted w

ith a

tw

o-sam

ple

t-test. Poole

d d

ata

per dog o

f both

in v

ivo s

tudie

s ("Canned" and "Kib

ble

" of Chapte

r 4 a

nd "Sem

i-

purified" of C

hapte

r 5) w

ere

use

d for th

is table

, exc

ept fo

r th

e low

-Se d

iet of th

e b

iom

ark

er study.

canned v

s se

mi-

purified

<0.001

0.1

11

0.2

54

<0.001

<0.001

<0.001

<0.001

<0.001

<0.001

Sem

i-purified (n=2

4)

SE

0.5

3

1308

6

0.0

15

0.7

0.0

01

1.3

4.7

mean

21.3

261

68407

428

1.0

17

33.3

0.0

31

22.9

92.2

Kib

ble

(n=2

4)

SE

0.3

4

860

7

0.0

38

1.1

0.0

02

0.5

3.8

mean

10.5

244

66429

429

0.5

31

23.2

0.0

23

8.1

68.0

Canned (n=2

4)

SE

0.2

6

1105

7

0.0

36

0.8

0.0

01

0.5

2.4

mean

17.2

251

66398

462

0.4

85

24.0

0.0

21

7.3

36.5

Sele

niu

m inta

ke (

µg/k

g B

W0.7

5)

Seru

m s

ele

niu

m (

µg/L

)

Whole

blo

od G

Px

(U/L

)

Whole

blo

od G

Px

(U/g

Hb)

Seru

m T

3 (ng/m

l)

Seru

m T

4 (ng/m

l)

Seru

m T

3:T4 ratio

Urinary

Se:C

T ratio

Urinary

Se:C

T p

er Se

inta

ke

Chapter 6


163

significantly different between the low and adequate semi-purified diets (Chapter 5) after 6

and 8 weeks in serum and whole blood, respectively; the whole blood GPx activity between

the canned and kibble diets (Chapter 4) was significantly different after 29 (or 43) days. This

reinforces the suggestion that diet type (ingredients, processing conditions) is a major factor

in the bioactivity of Se, considering the fact that the difference in dietary Se concentration

in the semi-purified diets (86%) was much larger than between the canned and kibble diets

(average 55%).

6.5 Other factors to consider when assessing selenium requirements of dogs

The current FEDIAF minimum recommendations are based on a study in kittens, but it has

been shown that dogs have a higher Se retention than cats(10). This may be linked to the

evolutionary feast and famine feeding approach of dogs, in which dogs gorge feed(26).

During the famine stage, stored Se can be used for physiological processes. However, it is

believed that inorganic Se cannot be stored in the body, which makes this theory untrue for

inorganic Se. The higher urinary Se:CT excretion per Se intake in the semi-purified (close to

100% inorganic Se) diets compared to the kibble (approximately 20-25% inorganic Se) and

canned diets (Table 6.1) and in the kibble diets compared to the canned diets (Chapter 4)

confirms this. Therefore, the supplementation of pet foods with inorganic Se may be

interesting for research purposes, but pet food manufacturers should be aware that the

susceptibility to a suboptimal Se status in dogs increases with increasing percentage of

inorganic Se per total dietary Se. Therefore, organic Se may be a better option for

supplementing pet foods.

6.5.1 Gender

Sexual dimorphisms of Se metabolism has been reported in many human and rodent

studies(27), but has not yet been verified in dogs. Brown and Burk(28) have demonstrated that

female rats had higher blood and liver Se concentrations after a selenite injection than

males. A study by Paβlack et al.(29) did not show significant gender differences in Se

concentrations of liver, renal cortex and renal medulla in dogs. However, numerically bitches

had higher average Se concentrations of these tissues (liver: dog 258, bitch 634; renal

cortex: dog 531, bitch 933; renal medulla: dog 163, bitch 334 µg/kg).

In this thesis, serum Se was higher in bitches than in dogs (all spayed/neutered, except one

bitch, p=0.033, Table 6.2). GPx and thyroid hormones were not affected by gender. Daily Se

intake and bioavailability were similar between sexes, and the urinary selenium excretion

did also not differ between dogs and bitches (Table 6.2). However, when the urinary Se:CT

164 Chapter 6


excretion was corrected for the amount of bioavailable Se, excretion was higher in bitches

compared to dogs, indicating a higher retention in dogs. These results, together with the

findings of Paβlack et al.(29) suggest that dogs require more Se for processes in other tissues

than whole blood, liver and kidneys. The testes are known to require Se for

spermiogenesis(30), however, that cannot be the reason for the differences in these dogs, as

all dogs were neutered. Another explanation may be in the fact that dogs have a larger fat-

free body mass compared to bitches(31), which includes tissue in which Se may be non-

specifically incorporated (muscle, organs). More research is warranted into the gender

differences in Se metabolism and gender should be taken into account in future studies into

the Se requirements of dogs.

Table 6.2 Effect of gender* on selenium parameters

Dogs (n=19) Bitches (n=29)

mean SE Mean SE p

Selenium intake (µg/day) 220 14 192 10 0.097

Selenium bioavailability† (%) 55.5 2.9 53.5 1.7 0.539 Serum selenium (µg/L) 247 4.4 258 3.0 0.033

Whole blood GPx (U/L) 66516 1054 68179 1116 0.317

Serum T3 (ng/ml) 0.763 0.069 0.780 0.049 0.839

Serum T4 (ng/ml) 28.4 1.4 28.6 1.2 0.896

Serum T3:T4 ratio 0.026 0.002 0.027 0.001 0.754

Urinary Se:CT ratio 17.1 2.3 14.2 1.5 0.277

Urinary Se:CT per Se intake 74.5 6.7 73.9 4.5 0.943 Urinary Se:CT per bioavailable Se† 8.4 0.4 9.8 0.4 0.037 SE, standard error; GPx, glutathione peroxidase; T3, triiodothyronine; T4, thyroxine; Se, selenium; CT, creatinine. * All dogs were neutered/spayed, except for one bitch.

† Selenium bioavailability is only measured in the bioavailability study reported in Chapter

4. Therefore, selenium bioavailability and urinary selenium excretion per bioavailable selenium are based on 9 dogs and 15 bitches. P-values are calculated with a two-sample t-test. Pooled data per dog of both in vivo studies (Chapters 4 and 5) were used for this table, except for the low-Se diet of the biomarker study.

6.5.2 Age

Paβlack et al.(29) reported an age difference in tissue Se concentrations in dogs, with

significantly lower tissue (liver, renal medulla and renal cortex) Se concentrations in the age

range of 1.5-4 years compared to dogs with an age between 4.5 and 7 years. In this thesis,

age in the range of 2 to 8 years had no effect on the parameters measured in both in vivo

studies (Figure 6.6). These differences in findings may be explained by the unknown dietary

Chapter 6


165

Se concentrations in the study of Paβlack et al.(29), which may be different between the 1.5-4

years and 4.5-7 years group.

Figure 6.6 Effect of dog age on serum selenium (A), whole blood GPx (B), serum T3:T4

ratio (C) and urinary selenium:CT ratio (D)

R2, coefficient of determination; GPx, glutathione peroxidase; U, unit; T3, triiodothyronine; T4,

thyroxine; CT, creatinine. Pooled data per dog (n=48) of both in vivo studies (Chapters 4 and 5)

were used for these figures, except for the low-Se diet of the biomarker study. P-values were

calculated with linear regression.

6.5.3 Breed

Dog breed could also have had an effect on the results reported in this thesis. All in vivo

studies were performed with Labrador retrievers. There are no studies reported that

investigated dog breed differences in Se metabolism. However, it has been reported by

Weber et al.(32) that larger dog breeds have a higher nutrient digestibility. Energy

requirements of Great Danes have been reported to be approximately 60% higher than

other breeds and that of New Foundland dogs 20% lower(33). Large breeds are known to

age faster(4), by which the need for anti-oxidants, and thus Se, may increase. Also highly

R² = 0.004

175

195

215

235

255

275

295

1 2 3 4 5 6 7 8 9

Se

rum

se

len

ium

(µ

g/L

)

R² = 0.025

0

20000

40000

60000

80000

100000

1 2 3 4 5 6 7 8 9

Wh

ole

blo

od

GPx (

U/L

) B

R² = 0.006

0.000

0.005

0.010

0.015

0.020

0.025

0.030

0.035

0.040

1 2 3 4 5 6 7 8 9

Se

rum

T3

:T4

ra

tio

Age (years)

C

R² = 0.001

0

5

10

15

20

25

30

35

40

1 2 3 4 5 6 7 8 9

Uri

na

ry s

ele

niu

m:C

T r

ati

o

Age (years)

D

0

A

p = 0.686 p = 0.293

p = 0.603 p = 0.883

166 Chapter 6


active dog breeds, such as Border Collies, sled dogs (e.g. Husky's), or racing dogs (e.g.

greyhounds) are likely to have increased oxidative stress and thus a higher anti-oxidant

requirement. The Labrador retreiver is a medium sized dog and is the most commonly kept

dog breed in the UK(34). These potential breed effects are worth being further investigated,

but given the general kind of the studies in this thesis, it is dared to

assume that the general ideas from these results are widely applicable

across dog breeds.

6.5.4 Metabolic changes associated with selenium status

When determining Se minimum requirements or a legal upper limit in dogs, it is necessary

to account for changes in Se metabolism. Significant metabolic changes due to Se

deficiency have been shown in a metabolomics study in mice(35). The Se deficient mice had

a serum Se concentration of 81 µg/L compared to a serum Se concentration of 430 µg/L in

mice fed an adequate Se diet (dietary Se concentration not stated). Se deficiency resulted

in an increase in serum serine, threonine, phenylalanine, methionine, creatine, fructose,

sorbose, sucrose, proline, glycine, isoleucine and pyruvate(35). This indicates an increased

protein turnover in Se deficiency, which may be caused by increased protein degradation

due to oxidative stress. The increase in methionine may also reflect a dysregulation of the

methionine-homocysteine cycle, which is also involved in the transamination of SeMet to

SeCys for incorporation into selenoproteins. It is, however, not known whether the change in

metabolism remains when Se status is restored to adequate levels.

There are indications that animals can adapt to high Se intakes, which also suggests a

metabolic change to high concentrations of Se. Hintze et al.(36) reported that kidney Se

concentrations of steers from seleniferous areas were lower on a high Se diet (11.9 mg/kg)

than in steers from non-seleniferous areas. In rats, males have been reported to adapt better

to high Se intakes than female rats, due to an increase in volatile Se metabolites that are

excreted via the respiratory tract(37). Therefore, it is important to determine the baseline Se

status of the animal when assessing Se requirements.

Chapter 6


167

6.6 Future perspectives

Although many studies have been performed on Se during the last decades, the

recommendation of Se for maintenance in healthy adult dogs is still not determined

accurately. This thesis showed evidence for the need to separate the minimum requirements

for canned and kibble diets and the potential sex differences in Se requirements. The

accuracy of the Se biomarkers, urinary Se:CT, serum Se and serum GPx, as biomarkers for

prevention of Se related diseases can actually only be verified when Se deficiency is

triggered. The actual Se requirements of dogs to prevent long-term Se related diseases can

possibly only be determined by following up groups of dogs in a longitudinal study (during

early adulthood into the geriatric life stage) that are fed a range in dietary Se (from below

0.1 to above 0.56 mg/kg DM) and look at the onset of Se related diseases. However, there

are still other factors that may influence the requirements, such as genetic predisposition,

oxidant triggering (for example, due to an increased exercise level(38) or obesity(39)), etc.

Therefore, the assumption should be made that the reported biomarkers are really related

to the onset of Se related diseases. An estimation of an adequate range in urinary Se:CT,

serum Se and serum GPx can be determined using epidemiological studies in healthy and

diseased (cancer, diabetes, etc) dogs.

These ranges can be used as an endpoint (to prevent disease development in the study

dogs) in studies into the minimum Se requirements. Minimum requirement studies can be

performed by feeding dogs a pre-feed containing merely inorganic Se, to deplete Se body

stores, as organically-bound Se can be non-specifically stored in body proteins, from which

it can be gradually freed to potentially become bioactive.. A study using stable isotopes

should be done to determine the time necessary to deplete Se body stores in dogs, but the

maximum of 10 months as found in humans(40) could be used as a guidance. After the pre-

feeding period, dogs should be devided into groups with equal numbers of dogs and

bitches. Each group should be fed one experimental diet, that is either a canned or kibble

diet with a Se concentration of the diets ranging between 0 and 0.3 mg/kg DM. It is known

from the study of Van Vleet(41) that clinical Se deficiency signs appeared after 6 to 8 weeks

with a diet containing 0.01 mg Se/kg and no vitamin E. The biomarker study also showed

differences in urinary Se:CT, serum Se and serum GPx within 8 weeks. Therefore, 8 weeks

should be a sufficient study length to determine the minimum Se requirements of dogs. The

Se concentration that does not cause biomarker concentrations to drop below the lower

limit of the range determined in the epidemiological study, can be considered the minimum

requirement of healthy adult dogs.

168 Chapter 6


Se requirements of dogs in other life stages (e.g. lactating, geriatric), from different breeds, or

with another physical status (e.g. obesity) may be different, for example, due to a higher

level of oxidative stress. Minimum requirements of these dogs can be determined in the

same way.

In addition, more research is required to get a better understanding of the Se metabolism of

dogs. This can be done by the use of inorganic and organically-bound stable isotopes, for

example, to determinethe turn over time of Se body stores or the difference in the amount

of biliairy Se excretion between the Se species. Analysis of urinary Se species between

canned and kibble diets at low, adequate and high Se concentrations, and the

measurement of Se containing bile acids in the faeces are other ways to get a better insight

into the Se metabolism of dogs.

The effect of specific dietary components on the effect of Se bioavailability and -activity

also require more research. Fibre was not identified as an important factor for Se

accessibility of kibble diets in the in vitro study, but it did significantly decrease in vitro Se

accessibility of canned diets. It would be interesting to investigate whether different fibre

types (e.g. guar gum, sugar beet fibre, cellulose) have a different effect on the Se

bioavailability of canned diets. Also, the interaction of Se with other anti-oxidants, such as

vitamin E and C, should be investigated in more detail, to verify for which dietary range in

these anti-oxidants, the dietary Se recommendation accounts.

6.7 Conclusions

The studies reported in this thesis have demonstrated that diet type and everything that is

associated with it (ingredients, heat processing, Se source) has a major impact on the

availability of Se. Therefore, the minimum requirements for Se inclusion levels in dog foods

are more variable than the current recommendation of FEDIAF allows for. The studies in this

thesis and the determined biomarkers, urinary Se:CT ratio, serum Se and serum GPx, are a

first step towards the actual minimum requirements of healthy adult dogs to prevent long-

term Se related diseases. Based on these requirements, more specific recommendations on

Se inclusion levels in dog foods could be defined.

Chapter 6


169

6.8 References

1. Hervera M, Baucells MD, González G et al. (2009) Prediction of digestible protein content of dry extruded dog foods: comparison of methods. Journal of Animal Physiology and

Animal Nutrition 93939393, 366-372. 2. German AJ (2006) The Growing Problem of Obesity in Dogs and Cats. The Journal of

Nutrition 136136136136, 1940S-1946S. 3. Linder DE, Freeman LM, Morris P et al. (2012) Theoretical evaluation of risk for nutritional

deficiency with caloric restriction in dogs. Veterinary Quarterly 32323232, 123-129. 4. National Research Council (2006) Nutrient requirements of dogs and cats. [Rev. ed, Animal

nutrition series. Washington D.C.: National Academies Press. 5. Wedekind KJ, Kirk CA, Yu S et al. (2002) Defining the safe lower and upper limit for

selenium (Se) in adult dogs. FASEB Journal 15151515, A992-A993. 6. Yu S, Wedekind KJ, Kirk CA et al. (2006) Primary hair growth in dogs depends on dietary

selenium concentrations. Journal of Animal Physiology and Animal Nutrition 90909090, 146-151. 7. European Commission (2009) Regulation (EC) No 767/2009 of the European parliament

and of the council of 13 July 2009 on the placing on the market and use of feed, amending European Parliament and Council Regulation (EC) No 1831/2003 and repealing Council Directive 79/373/EEC, Commission Directive 80/511/EEC, Council Directives 82/471/EEC, 83/228/EEC, 93/74/EEC, 93/113/EC and 96/25/EC and Commission Decision 2004/217/EC. Official Journal of the European Union L 229L 229L 229L 229, 1-28.

8. European Commission (2010) Commission regulation (EU) No 454/2010 of 26 May 2010 on transitional measures under Regulation (EC) No 767/2009 of the European Parliament and of the Council as regards the labelling provisions for feed. Official Journal of the

European Union L 128L 128L 128L 128, 1-2. 9. FEDIAF (2011) Code of Good Labelling Practice for Pet Food. Brussels: European Pet Food

Industry Federation. 10. Todd SE, Thomas DG, Bosch G et al. (2012) Selenium status in adult cats and dogs fed high

levels of dietary inorganic and organic selenium. Journal of Animal Science 90909090, 2549-2555. 11. Singh H (1991) Modification of food proteins by covalent crosslinking. Trends in Food

Science & Technology 2222, 196-200. 12. Zhang Q, Ames JM, Smith RD et al. (2009) A Perspective on the Maillard Reaction and the

Analysis of Protein Glycation by Mass Spectrometry: Probing the Pathogenesis of Chronic Disease. Journal of Proteome Research 8888, 754-769.

13. Bauer MC, Weiss DJ, Perman V (1992) Hematologic alterations in adult cats fed 6 or 12% propylene glycol. American Journal of Veterinary Research 53535353, 69-72.

14. Guyton AC, Hall JE (2011) Textbook of medical physiology. 11th ed. Philadelphia: Elsevier Inc.

15. Christian J, Rebar A, Boon G et al. (1993) Senescence of canine biotinylated erythrocytes: increased autologous immunoglobulin binding occurs on erythrocytes aged in vivo for 104 to 110 days. Blood 82828282, 3469-3473.

16. Combs Jr GF, Combs SB (1986) Biochemical functions of selenium. In The Role of Selenium

in Nutrition, pp. 205-263. New York: Academic Press. 17. Cummings JH, Wiggins HS, Jenkins DJ et al. (1978) Influence of diets high and low in

animal fat on bowel habit, gastrointestinal transit time, fecal microflora, bile acid, and fat excretion. Journal of Clinical Investigation 61616161, 953-963.

18. Symonds HW, Mather DL, Vagg MJ (1981) The excretion of selenium in bile and urine of steers: the influence of form and amount of Se salt. British Journal of Nutrition 46464646, 487-493.

170 Chapter 6


19. Gyurasics Á, Perjési P, Gregus Z (1998) Role of glutathione and methylation in the biliary excretion of selenium. the paradoxical effect of sulfobromophthalein. Biochemical

Pharmacology 56565656, 1381-1389. 20. National Research Council (1983) Selenium in Nutrition. Washington D.C.: National

Academies Press. 21. Forrer R, Gautschi K, Lutz H (1991) Comparative determination of selenium in the serum

of various animal species and humans by means of electrothermal atomic absorption spectrometry. Journal of Trace Elements and Electrolytes in Health and Disease 5555, 101-113.

22. Lane HW, Strength R, Johnson J et al. (1991) Effect of Chemical Form of Selenium on Tissue Glutathione Peroxidase Activity in Developing Rats. Journal of Nutrition 121121121121, 80-86.

23. Sunde RA, Gutzke GE, Hoekstra WG (1981) Effect of dietary methionine on the biopotency of selenite and selenomethionine in the rat. Journal of Nutrition 111111111111, 76-88.

24. Rucker RB, Fascetti AJ, Keen CL (2008) Trace Minerals. In Clinical Biochemistry of Domestic

Animals (Sixth Edition), pp. 663-693 [JJKWHL Bruss, editor]. San Diego: Academic Press. 25. Foster LH, Sumar S (1997) Selenium in health and disease: A review. Critical Reviews in

Food Science and Nutrition 37373737, 211-228. 26. Bosch G, Hagen-Plantinga EA, Hendriks WH (2015) Dietary nutrient profiles of wild wolves:

insights for optimal dog nutrition? British Journal of Nutrition 113113113113, S40-S54. 27. Schomburg L (2012) Variations in Selenium Metabolism in Males and Females. In Selenium

It's molecular biology and role in human health, pp. 419-432 [DL Hatfield, MJ Berry and VN Gladyshev, editors]. New York: Springer New York.

28. Brown DG, Burk RF (1973) Selenium Retention in Tissues and Sperm of Rats Fed α Torula Yeast Diet. Journal of Nutrition 103103103103, 102-108.

29. Paßlack N, Mainzer B, Lahrssen-Wiederholt M et al. (2015) Concentrations of strontium, barium, cadmium, copper, zinc, manganese, chromium, antimony, selenium, and lead in the liver and kidneys of dogs according to age, gender, and the occurrence of chronic kidney disease. Journal of Veterinary Science 16161616, 57-66.

30. Flohé L (2007) Selenium in mammalian spermiogenesis. In Biological Chemistry, vol. 388, pp. 987-995.

31. Laflamme DP (1997) Development and validation of a body condition score system for dogs. Canine Practice 22222222, 10-15.

32. Weber M, Martin L, Biourge V et al. (2003) Influence of age and body size on the digestibility of a dry expanded diet in dogs. Journal of Animal Physiology and Animal

Nutrition 87878787, 21-31. 33. Kienzle E, Rainbird A (1991) Maintenance Energy Requirement of Dogs: What is the

Correct Value for the Calculation of Metabolic Body Weight in Dogs? The Journal of

Nutrition 121121121121, S39-S40. 34. The Kennel Club (2007) Registration statistics for all recognised dog breeds - 2005 and

2006. http://web.archive.org/web/20090904034036/http://www.thekennelclub.org.uk/cgi-bin/item.cgi?id=926&d=pg_dtl_art_news&h=238&f=0 (accessed 30-11 2015).

35. Mickiewicz B, Villemaire M, Sandercock L et al. (2014) Metabolic changes associated with selenium deficiency in mice. Biometals 27272727, 1137-1147.

36. Hintze KJ, Lardy GP, Marchello MJ et al. (2002) Selenium Accumulation in Beef: Effect of Dietary Selenium and Geographical Area of Animal Origin. Journal of Agricultural and Food

Chemistry 50505050, 3938-3942.

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171

37. Combs Jr GF, Combs SB (1986) Effects of selenium excesses. In The Role of Selenium in

Nutrition, pp. 463-525. New York: Academic Press. 38. Kanter M (1998) Free radicals, exercise and antioxidant supplementation. Proceedings of

the Nutrition Society 57575757, 9-13. 39. Furukawa S, Fujita T, Shimabukuro M et al. (2004) Increased oxidative stress in obesity

and its impact on metabolic syndrome. Journal of Clinical Investigation 114114114114, 1752-1761. 40. Patterson BH, Levander OA, Helzlsouer K et al. (1989) Human selenite metabolism: a

kinetic model. American Journal of Physiology 257257257257, R556-R567. 41. van Vleet JF (1975) Experimentally induced vitamin E-selenium deficiency in the growing

dog. Journal of the American Veterinary Medical Association 166166166166, 769-774.

glutathione peroxidase activity analysis __________________________________________________________

Supplement

Storage of whole blood forglutathione peroxidase activity analysis

_____________________________________________________________________________________________________________

Supplement

Storage of whole blood for glutathione peroxidase activity analysis

___________________________________________________

"Storage of heparinised canine whole blood for measurement of glutathione peroxidase activity"

Mariëlle van Zelst1, Myriam Hesta1, Kerry Gray2, Geert P.J. Janssens1




Submitted as short communication to Biological Trace Element Research

Supplement

Glutathione peroxidase storageGlutathione peroxidase storageGlutathione peroxidase storageGlutathione peroxidase storage

175

S.1 Abstract

Glutathione peroxidase activity is used as a biomarker of selenium status in dogs. Freshly

collected blood samples are usually measured, due to the lack of knowledge on the effect

of storing the samples. This study investigated if analysis of glutathione peroxidase activity

in whole blood collected from dogs was affected by storage time, between 5 and 164 days.

Results indicated that glutathione peroxidase activity was more variable in the freshly

analysed samples compared to the stored samples. Although the mean differences between

fresh and stored samples were not always encompass zero, this is thought to be caused by

the variability of reagent preparation rather than by storage, as no consistent increase or

decrease in glutathione peroxidase activity was found. Therefore, it can be concluded that

heparinised dog blood samples can be successfully stored up to 164 days before analysis

of glutathione peroxidase activity.

S.2 Introduction

Glutathione peroxidase (GPx) is an intra-cytoplasmic selenium-dependent antioxidant that

protects the body against free radicals and other oxidative damage. As such, it is an

important and frequently used biomarker of selenium status in mammals(1-3). Eighty percent of

the enzymatic activity of GPx is confined to the cell membrane of erythrocytes and only

some activity can be found in plasma(4). GPx activity is often analysed immediately after

sampling, because the effects of storage of canine whole blood on GPx activity is not well

documented. However, the limited shelf life of analysis kits and stability of reagents(5) makes

it inefficient to use for long-term trials with frequent sampling points. No data are available

on the suitability of long-term storage of canine whole blood for this analysis. Therefore, the

aim of this study was to investigate the effect of storage duration on the activity of

glutathione peroxidase measured in heparinised whole blood from dogs after storage at -

80°C for varying periods of time.

S.3 Experimental methods

These data were collected as part of a study examining selenium bioavailability by van

Zelst et al. (unpublished, Chapter 4). This study (HO0647) was approved by the WALTHAM

Centre for Pet Nutrition Animal Welfare and Ethical Review Body and was conducted under

Home Office Project License authorisation. Twenty-three adult Labrador retrievers were

divided into four groups. Dogs were fed four of eight commercially available diets (2

canned and 2 kibble) in a randomized order, for 29-42 days. Before the start of the study

and at the end of every diet change, 0.5 ml of fasted whole blood was taken for GPx

176 Supplement


activity analysis by jugular venipuncture (resulting in 23 dogs x 5 sampling occasions = 115

samples). Blood was transferred into a lithium heparin tube and stored on ice until

analysis/storage. GPx activity was measured using 25 µl of whole blood within 20 minutes of

sampling. The remaining sample was immediately stored in its lithium heparin tube at -80°C.

Storage duration was 5 to 164 days, and all samples were assayed as one batch at the end

of the trial (therefore blood samples were always their own control). GPx activity

measurements were performed in duplicate on each individual sample using the Ransel kit

(Randox Laboratories Ltd., UK), based on a method of Paglia & Valentine(6), and the Olympus

AU400 spectrophotometer (Olympus Inc., USA). This method is based on GPx catalysing the

oxidation of glutathione by cumene hydroperoxide. In the presence of glutathione

reductase and NADPH, the oxidised glutathione is immediately converted to the reduced

form with a concomitant oxidation of NADPH to NADP+. The decrease in absorbance is

measured photometrically at 340 nm. A 4-point calibration curve of the Ransel quality

control was used as control. Mean values of the duplicates were calculated and used for

statistical analysis. The mean coefficient of variation (CV, %) over the fresh and stored

measurements was calculated for each sample individually. A standard error of the mean

(SEM) was calculated over all samples for the fresh and stored samples separately. Mean

differences (fresh minus stored) and confidence intervals were obtained from the descriptive

statistics in SPSS 21.

S.4 Results and discussion

Twenty-seven samples did not contain enough blood for analysis on both fresh and stored

samples of GPx activity and were omitted from the study, resulting in a total of 88 analysed

samples of 23 dogs. The average CV of the GPx activity analysis within samples (duplicates)

was 1.4% in the fresh samples and 2.1% in the stored samples. The 4-point calibration curve

of the quality control dilutions 1:11, 1:21, 1:41 and 1:61, showed an average recovery of 83.4

± 1.2 (mean ± standard error), 93.5 ± 1.2, 98.5 ± 1.5, and 94.6 ± 2.7%, respectively in the fresh

samples. The average recovery was 81.8 ± 0.5, 93.9 ± 1.1, 98.2 ± 0.3, and 102.7 ± 1.7%,

respectively in the stored samples. Average results of the GPx activity measurements in fresh

and stored samples are shown in Table S.1 and mean differences between the fresh and

stored samples are plotted in Figure S.1. The confidence intervals of the mean differences

between fresh and stored samples did not encompass zero after 12, 40, 88, 95, and 130 and

all further days of storage. However, the mean CV of the fresh and stored samples always

remained below 10%, which is usually considered as the cut-off value for acceptance of

biological data. In this study the highest mean CV between fresh and stored samples was

Supplement


177

Table S.1 Mean glutathione peroxidase activity and coefficient

of variation of fresh and stored (-80°C) canine blood

Fresh (U/L) Storage

(days)

Stored (U/L) Mean CV†

(%) n Mean SE Mean SE

5 66317 2481 5 65944 2409 0.53

6 62426 1206 12 64275 1337 2.05

5 64929 2388 19 65280 2526 0.46

5 60835 1707 26 59792 1387 1.79

5 73954 918 40 67826 788 6.11

6 68544 1765 47 67669 1908 2.78

5 63875 2971 54 63746 2490 1.73

5 61330 2042 61 61861 2079 2.79

5 65845 1030 74 67276 951 1.76

6 68171 2285 81 66970 2405 1.71

4 60173 2228 88 65268 1963 5.79

3 58243 1574 95 61201 1242 3.52

5 63676 2147 123 65112 1640 2.07

6 63289 1159 130 66795 1582 3.77

6 62655 1397 137 68067 1671 5.84

6 68055 1874 157 64234 2000 4.32

5 62247 1840 164 59313 1584 3.39

°C, degrees Celsius; n, number of dogs; U, units; SE, standard error; CV,

coefficient of variation.

†coefficient of variation is calculated over the fresh and stored samples

per dog.

6.1%, which could be considered as interassay variation, where in other studies using the

same analytical kit, the interassay variation of merely fresh or stored samples was 7.5%(7)

and 9.9%(8), respectively, indicating that storage in this study did not increase interassay

variability. The biological assay is still of value for the measurement of GPx activity, but

depending on the power of the study, differences in GPx activity between groups could

overrule the interassay variation. The SEM of the fresh samples was 407 and of the stored

samples 296, indicating that variability was much larger in the fresh compared to the stored

samples. The variability in SEM may be due to variation in reagents caused by freshly

178 Supplement


Figure S.1 Mean difference in glutathione peroxidase activity (U/L)

between fresh and stored canine heparinised whole blood.

Error bars represent the 95% confidence interval.

preparing the reagents every sampling occasion. Therefore, it may be argued that the

significant differences found in the samples after 12 and 40 days of storage may also be

due to this variation. There was also not an obvious increase or decrease of GPx activity

after storage, indicating that storage did not specifically reduce the enzyme activity of GPx.

This strengthens the suggestion that analysis of GPx activity on different days using

different kits (and thus having to prepare the reagents multiple times) has more impact on

the accuracy of the measurements than storage of the blood. This leads to the conclusion

that heparinised dog blood samples can be successfully stored up to 164 days before

analysis of GPx activity, rather than preparing the reagents multiple times, in the

performance of long-term studies with multiple sampling points. In addition, it would be

valuable to increase the reproducibility and accuracy of the method by identifying the

0

10

20

30

40

50

60

70

80

90

100

110

120

130

140

150

160

170

180

-7500 -5000 -2500 0 2500 5000 7500

da

ys

of

sto

rag

e

Difference in glutathione peroxidase activity between fresh and stored samples (U/L)

Supplement


179

factors (e.g. reagens) responsible for the variability between GPx activity in fresh samples

and after storage.

S.5 References

1. Thomson CD, Robinson MF, Campbell DR et al. (1982) Effect of prolonged supplementation with daily supplements of selenomethionine and sodium selenite on glutathione peroxidase activity in blood of New Zealand residents. American Journal of

Clinical Nutrition 36363636, 24-31. 2. Wedekind KJ, Yu S, Combs GF, Jr. (2004) The selenium requirement of the puppy. Journal

of Animal Physiology and Animal Nutrition 88888888, 340-347. 3. Todd SE, Thomas DG, Hendriks WH (2011) Selenium balance in the adult cat in relation to


Physiology and Animal Nutrition 96969696, 148-158. 4. Nève J (1988) Biological functions of selenium. Proceedings of the Second International

Congress on Trace Elements in Medicine and Biology, 97-111. 5. Randox Laboratories (2005) Ransel Glutathione Peroxidase Manual. 6. Paglia DE, Valentine WN (1967) Studies on the quantitative and qualitative

characterization of erythrocyte glutathione peroxidase. Journal of Laboratory and Clinical

Medicine 70707070, 158-169. 7. Guemouri L, Artur Y, Herbeth B et al. (1991) Biological variability of superoxide dismutase,

glutathione peroxidase, and catalase in blood. Clinical Chemistry 37373737, 1932-1937. 8. Blankenberg S, Rupprecht HJ, Bickel C et al. (2003) Glutathione Peroxidase 1 Activity and

Cardiovascular Events in Patients with Coronary Artery Disease. New England Journal of

Medicine 349349349349, 1605-1613.

_____________________________________________________________________________________________________________

Summary_____________________________________________________________________________________________________________

Summary _____________________________________________________________________________________________________________

SummarySummarySummarySummary

183

Selenium is an essential trace mineral involved in many biological processes, such as anti-

oxidant, thyroid and immune function. Current recommendations for selenium inclusion in

dog foods lack knowledge on the requirements of adult dogs and the effect of various

factors on its bioavailability (i.e. the fraction of the dietary selenium that reaches the

systemic circulation) and bioactivity (i.e. the selenium that is incorporated into

selenoproteins). Therefore, the objective of this PhD project (Chapter 2) was to identify

factors that contribute to the biological utilisation of dietary selenium, as a first step towards

correct selenium inclusion levels in dog foods to prevent long-term selenium related

diseases.

First, an in vitro study was performed (Chapter 3a) to assess the effect of a large range of

factors, such as diet type; and dietary protein, methionine, cysteine, lysine and selenium

concentration, on in vitro selenium accessibility (i.e. the amount of dietary selenium that is

potentially available for absorption after in vitro digestion). Sixty-two pet foods (dog, n=52;

cat, n=10) were enzymatically digested: 54 were commercially available (kibble, n=20; pellet,

n=8; canned, n=17; raw meat, n=6; steamed meat, n=3) and eight were unprocessed (kibble,

n=4; canned, n=4) from the same batch as the corresponding processed diets. The canning

process (n=4) decreased in vitro selenium accessibility, whereas extrusion (n=4) did not.

Canned and steamed meat diets had a lower in vitro selenium accessibility than pelleted

and raw meat diets. A positive correlation between in vitro selenium accessibility and crude

protein digestibility was found for extruded diets (kibbles, n=19; r= 0.540) and a negative

correlation for canned diets (n=16; r= -0.611). Data from this in vitro study were also used for

analyses with stepwise linear regression. Predictive equations were successfully formulated

for dry pet foods (Chapter 3b), in order to predict selenium accessibility from diets.

The main factors identified in the in vitro selenium accessibility study (diet type and crude

protein) were evaluated in an in vivo study (Chapter 4) using four canned and four kibble

diets with crude protein concentrations ranging from 10.1 to 27.5 g/MJ. These diets were fed

to four groups of six Labrador retrievers in an incomplete cross over design. Selenium

bioavailability was higher from kibble than canned diets and was not affected by crude

protein intake from both diet types. However, the bioactivity of selenium, as measured by

whole blood glutathione peroxidase activity (U/g Hb), was higher in dogs fed the canned

diets than in dogs fed the kibble diets and decreased with increasing crude protein intake.

These results suggest that there were indeed differences in the bioavailability and -activity

of selenium from canned and kibble diets, which can be caused either by the difference in

ingredients used, by the selenium species or by the processing conditions.

184 SummarySummarySummarySummary

Glutathione peroxidase activity was used as a marker for the bioactivity of selenium. This is

a marker frequently measured in anti-oxidant studies. However, there is a lack of

knowledge on the effect of storing whole blood for this analysis, which is convenient when

performing longitudinal studies with frequent sampling points. Based on the study in

Chapter 4, a study has been performed (Supplement) in which the effect of storing canine

whole blood on the activity of glutathione peroxidase was assessed. Glutathione peroxidase

activity was measured in fresh samples and samples stored for 5 up to 164 days. There

were some significant differences in glutathione peroxidase activity between fresh and

stored samples, but no consistent increase or decrease was found. Results also indicated

that glutathione peroxidase activity was more variable in the freshly analysed samples

compared to the stored samples, which is thought to be caused by the variability from

reagent preparation rather than by storage. Therefore, it can be concluded that canine

blood samples can be successfully stored up to 164 days before analysis of glutathione

peroxidase activity.

Although glutathione peroxidase activity is one of many markers frequently used to

measure selenium status, no studies have been performed to assess whether this marker or

other biomarkers react most quickly to a change in dietary selenium intake. The study

described in Chapter 5 assessed selected biomarkers (urinary selenium:creatinine ratio;

whole blood glutathione peroxidase activity and mRNA expression of selenoproteins; serum

selenium, glutathione peroxidase activity, copper, creatine kinase, and

triiodothyronine:thyroxine ratio; and hair growth) on their suitability to detect a decrease in

selenium provision to adult dogs within eight weeks. Twenty-four dogs received a semi-

purified diet with adequate selenium (46.1 µg/MJ) for eight weeks, after which 12 of the

dogs remained on this diet for another eight weeks and the other 12 dogs received a semi-

purified diet with a low selenium concentration (6.5 µg/MJ; 31% of the FEDIAF minimum). The

urinary selenium:creatinine ratio and serum selenium concentration decreased within one

week after switching to the low selenium diet (84 and 7%, respectively). Serum and whole

blood glutathione peroxidase activity were also significantly lower in dogs consuming the

low selenium diet (6 and 7%, respectively) from weeks 6 and 8 respectively. None of the

other biomarker concentrations changed significantly in response to the low selenium diet

over the 8 week period. Therefore, urinary selenium:creatinine ratio, serum selenium and

serum glutathione peroxidase activity should be used in epidemiological trials, to determine

their association with the occurrence of long-term selenium related diseases and for their

eventual use in trials to determine the minimum selenium requirements in adult dogs.

SummarySummarySummarySummary

185

In conclusion, the studies included in this PhD thesis showed that diet type and dietary

crude protein concentration were associated with selenium bioavailability and bioactivity.

Selenium is generally more bioavailable from kibble diets, but selenium from canned diets

result in an absolute higher bioactivity. Future studies should use the urinary

selenium:creatinine ratio, serum selenium and serum glutathione peroxidase activity to

assess the selenium requirements of adult dogs.

_____________________________________________________________________________________________________________

Samenvatting_____________________________________________________________________________________________________________

Samenvatting _____________________________________________________________________________________________________________

SamenvattingSamenvattingSamenvattingSamenvatting

189

Selenium is een essentieel sporenelement betrokken bij vele biologische processen, zoals

anti-oxidantfunctie, schildklierfunctie en het immuunsysteem. Bij de huidige aanbevelingen

voor het seleniumgehalte in hondenvoeding ontbreekt kennis over de behoefte van

volwassen honden en het effect van verschillende factoren op de biologische

beschikbaarheid (d.i. de fractie van selenium in de voeding dat de systemische circulatie

bereikt) en biologische activiteit (d.i. selenium dat gebruikt wordt voor de vorming van

selenoproteïnen). Daarom was het doel van dit werk (Hoofdstuk 2) om factoren te

identificeren die bijdragen tot het biologische gebruik van selenium uit de voeding, als een

eerste stap op weg naar correcte seleniumconcentraties in hondenvoeding om selenium-

gerelateerde lange termijn ziekten te voorkomen.

Als eerste werd een in vitro onderzoek uitgevoerd (Hoofdstuk 3a) waarin een groot aantal

factoren; zoals dieet type; en de concentraties aan eiwit, methionine, cysteïne, lysine en

selenium gescreend werden op hun effect op potentiële in vitro selenium beschikbaarheid

(de hoeveelheid selenium uit de voeding dat potentieel beschikbaar is voor absorptie na in

vitro vertering). Tweeënzestig honden- en kattenvoeders (hond, n = 52; cat, n = 10) werden

in vitro enzymatisch verteerd: 54 waren commercieel verkrijgbaar (geëxtrudeerde diëten, n

= 20, geperste diëten, n = 8, blikvoer, n = 17; rauw vlees, n = 6, gestoomd vlees, n = 3) en acht

waren niet hitte-behandeld (geëxtrudeerde diëten, n = 4, blikvoer, n = 4) uit dezelfde partij

als de overeenkomstige commerciele voeding. Door de hitte behandeling van blikvoeders (n

= 4) daalde de potentiële in vitro selenium beschikbaarheid, terwijl extruderen (n = 4) geen

verschil opleverde. Blikvoeders en gestoomde vlees diëten hadden een lagere potentiële in

vitro selenium beschikbaarheid dan geperste- en rauw vleesdiëten. Een positieve correlatie

tussen potentiële in vitro selenium beschikbaarheid en eiwitverteerbaarheid was gevonden

voor geëxtrudeerde diëten (n = 19; r = 0,540) en een negatieve correlatie voor blikvoeders (n

= 16; r = -0,611). Gegevens uit deze in vitro studie werden ook gebruikt voor analyse met

lineaire regressie. Voorspellingsmodellen werden succesvol ontwikkeld voor de

geëxtrudeerde en geperste diëten (Hoofdstuk 3b), om daarmee de selenium

toegankelijkheid van voeders in te schatten.

De factoren die in de in vitro studie als meest belangrijk werden aangeduid (dieettype en

eiwit) werden in een in vivo studie (Hoofdstuk 4) getest. Vier blikvoeders en vier

geëxtrudeerde diëten werden geselecteerd met eiwitconcentraties tussen 10,1 en 27,5 g/MJ.

Deze diëten werden in een onvolledige cross-over opstelling gevoerd aan vier groepen

van zes Labrador retrievers. De biologische beschikbaarheid van selenium was hoger in

geëxtrudeerde diëten dan blikvoeders en werd bij beide dieettypen niet beïnvloed door

eiwitinname. De biologische activiteit van selenium, gemeten door glutathionperoxidase

190 SamenvattingSamenvattingSamenvattingSamenvatting

activiteit in bloed (U/g Hb), was hoger bij honden die het blikvoer aten dan bij honden die

de geëxtrudeerde diëten kregen en daalde met toenemende eiwitinname. Deze resultaten

suggereren dat er inderdaad verschillen zijn in de biologische beschikbaarheid en activiteit

van selenium tussen blikvoeders en geëxtrudeerde diëten. Dit kan worden veroorzaakt door

het verschil in gebruikte ingrediënten, de seleniumspecies of het type hitte-behandeling.

Glutathionperoxidase activiteit werd gebruikt als een merker voor de biologische activiteit

van selenium. Dit is een vaak gebruikte merker in studies naar anti-oxidanten. Het invriezen

van bloed voor deze analyse is handig bij het uitvoeren van longitudinale studies met

frequente staalnames, maar kennis over de impact van bewaring op de

glutathionperoxidase activiteit ontbreekt. Er is een studie uitgevoerd (Supplement),

gebaseerd op de studie beschreven in Hoofdstuk 4, waarbij het effect van het invriezen

van hondenbloed op de activiteit van glutathionperoxidase werd bepaald.

Glutathionperoxidase activiteit werd gemeten in verse stalen en stalen ingevroren bij -80°C

gedurende 5 tot 164 dagen. Glutathionperoxidase activiteit verschilde op een aantal

momenten tussen verse en ingevroren stalen, maar er werd geen consistente toename of

afname gevonden. De resultaten gaven ook aan dat glutathionperoxidase activiteit

variabeler is in de vers geanalyseerde stalen in vergelijking met de ingevroren stalen.

Vermoedelijk veroorzaakt het telkens opnieuw bereiden van de reagentia een grotere

variabiliteit dan het invriezen van de stalen. Hondenbloedstalen kunnen dus tot minstens

164 dagen worden ingevroren zonder nadelige invloed op de analyse van

glutathionperoxidase activiteit.

Hoewel glutathionperoxidase activiteit vaak gebruikt wordt als merker voor seleniumstatus,

zijn nog geen studies uitgevoerd om te beoordelen welke biologische merkers het meest

geschikt zijn om verandering in seleniuminname te meten. De in Hoofdstuk 5 beschreven

studie onderzocht geselecteerde biologische merkers (urinaire selenium:creatinine

verhouding; glutathionperoxidase activiteit en mRNA expressie van selenoproteïnen in

bloed; serum selenium, glutathionperoxidase activiteit, koper, creatinekinase, en

triiodothyronine:thyroxine verhouding; en haargroei) op hun geschiktheid om binnen acht

weken een daling in seleniuminname te detecteren bij volwassen honden. Vierentwintig

honden kregen een semi-synthetisch dieet met een adequate hoeveelheid selenium (46,1

µg/MJ) gedurende acht weken, waarna 12 honden dit dieet voor nog eens acht weken

kregen en de overige 12 honden werden overgeschakeld op een semi-synthetisch dieet

met een lage seleniumconcentratie (6,5 µg/MJ, 31% van de FEDIAF minimum aanbeveling).

De urinaire selenium:creatinine verhouding en serum selenium concentratie daalde binnen

één week na het overschakelen naar het lage selenium dieet (84 en 7% respectievelijk).

SamenvattingSamenvattingSamenvattingSamenvatting

191

Serum en bloed glutathionperoxidase activiteit waren ook significant lager bij honden op

het lage selenium dieet (6 en 7% respectievelijk) vanaf week 6 en 8 respectievelijk. Geen

van de andere biologische merkers reageerde binnen acht weken op de overschakeling

naar het dieet met de lage selenium concentratie. De urinaire selenium:creatinine

verhouding, serum selenium en serum glutathionperoxidase activiteit zouden gebruikt

moeten worden in een epidemiologische studie om te bepalen wat de associatie van deze

merkers is met het optreden van selenium-gerelateerde ziekten op de lange termijn.

Uiteindelijk kunnen ze dan gebruikt worden bij het bepalen van de minimale

seleniumbehoefte van volwassen honden.

Kortom, de studies in dit proefschrift toonden aan dat dieettype en eiwitconcentraties

invloed hebben op de biologische beschikbaarheid en activiteit van selenium. De

biologische beschikbaarheid van selenium is over het algemeen hoger bij geëxtrudeerde

diëten, maar selenium uit blikvoeders resulteert in een hogere biologische activiteit.

Toekomstige studies kunnen de urinaire selenium:creatinine verhouding, serum selenium en

serum glutathionperoxidase activiteit gebruiken om de seleniumbehoefte van volwassen

honden te achterhalen.

_________________________________________________________________

Curriculum _____________________________________________________________________________________________________________

Curriculum vitae ____________________________________________

Curriculum vitaeCurriculum vitaeCurriculum vitaeCurriculum vitae

195

Mariëlle van Zelst was born on the 19th of October 1984 in Utrecht, the Netherlands. In

2005, she started a BSc study in Animal Husbandry and Animal Health Care at HAS Den

Bosch, the Netherlands. During her research internships she investigated whale and dolphin

interactions (Atlantic Whale foundation, Tenerife) and alternative protein sources for

hypoallergenic pelleted dog foods (Wielink diervoeders, IJsselmuiden). Mariëlle was a

coordinator of many volunteers at the Atlantic Whale foundation and was a member of the

public relations team of HAS Den Bosch. For her graduation project she developed a

protocol for food preference tests in mink. In 2008, she obtained her bachelor's degree with

distinction (according to UGent standards with "great distinction").

Consecutively, she started a master's programme in Animal Sciences at Wageningen

University and became a member of the student association "De Veetelers" and a board

member and treasurer of the application course committee. Her master theses were about

the behavioural and glucocorticoid responses of assistance dogs during training and about

the effects of diet type on behaviour of dogs. She obtained the MSc degree in 2010

(according to UGent standards with "great distinction") with specialisation in animal nutrition

and ethology.

Fascinated by scientific research and animal nutrition, she started her PhD research in

October 2011 at the department of Nutrition, Genetics and Ethology of the Faculty of

Veterinary Medicine at Ghent University, in collaboration with the renowned pet food

research centre, WALTHAM® Centre for Pet Nutrition, UK. During her thesis, Mariëlle was

first author of several scientific papers in international peer-reviewed journals. She has also

participated actively in various international conferences and was awarded the

Waltham/ESVCN student award for best presentation at the 2015 ESVCN congress, for her

presentation titled "Selenium absorption and bioactivity in dogs are affected by dietary

format", In addition, she has supervised multiple master students during their theses and

successfully fulfilled the UGent doctoral training programme in life sciences and medicine.

196 Curriculum Curriculum Curriculum Curriculum vitaevitaevitaevitae

Mariëlle van Zelst werd geboren op 19 oktober 1984 te Utrecht, Nederland. In 2005 is zij

de Bachelor studie Dier- en Veehouderij gestart aan HAS Den Bosch, Nederland. Tijdens

deze studie heeft zij onderzoeksstages gedaan naar de interactie tussen walvissen en

dolfijnen (Atlantic Whale foundation, Tenerife) en naar alternatieve eiwitbronnen voor

hypoallergene gepelleteerde hondenvoeders (Wielink diervoeders, IJsselmuiden). Mariëlle

was coordinator van een groot aantal vrijwilligers van de Atlantic Whale foundation en

actief lid van het public relations team van HAS Den Bosch. Voor haar afstudeerproject

heeft Mariëlle een protocol voor voedingsacceptatietesten voor edelpelsdieren ontwikkeld.

Zij behaalde in 2008 het bachelor diploma met "genoegen" (volgens UGent richtlijnen "grote

onderscheiding").

Daarna startte zij de mastersopleiding Dierwetenschappen aan Wageningen Universiteit en

werd lid van de studentenvereniging "De Veetelers" en bestuurslid en penningmeester voor

de sollicitatie commissie. Met haar master theses heeft ze gekeken naar het gedrag en

glucocorticoïden reacties van hulphonden tijdens hun training en naar de effecten van

dieet type op het gedrag van honden. Ze behaalde het master diploma (volgens UGent

richtlijnen met grote onderscheiding) in 2010 met de specialisaties diervoeding en

ethologie.

Geboeid door het wetenschappelijk onderzoek en de diervoeding, startte zij in oktober

2011 een doctoraatsstudie bij de vakgroep Voeding, Genetica en Ethologie aan de

Faculteit Diergeneeskunde van Universiteit Gent. Deze studie werd uitgevoerd in

samenwerking met het gerenomeerde onderzoekscentrum WALTHAM® Centre for Pet

Nutrition, UK. Tijdens haar doctoraat was Mariëlle eerste auteur van meerdere artikels in

wetenschappelijke internationale tijdschriften, heeft zij actief deelgenomen aan

internationale congressen en was vereerd met het verkrijgen van de Waltham/ESVCN

student award voor beste presentatie tijdens het ESVCN congres in 2015 voor haar

presentatie getiteld "Selenium absorption and bioactivity in dogs are affected by dietary

format". Daarnaast heeft zij verschillende masterstudenten begeleid met hun masterproeven

en voldaan aan de eisen voor het behalen van het UGent doctoraatsdiploma in life

sciences and medicine.

___________________________________________________________

Bibliography_____________________________________________________________________________________________________________

Bibliography __________________________________________________

BibliographyBibliographyBibliographyBibliography

199

Publications in international peer-reviewed journals

van Zelst M, Hesta M, Alexander LG, Gray K, Bosch G, Hendriks WH, Du Laing G, De

Meulenaer B, Goethals K, Janssens GPJ. (2015) In vitro selenium accessibility in dog

foods is affected by diet composition and type. British Journal of Nutrition 113, 1888-

1894.

van Zelst M, Hesta M, Gray K, Beech K, Cools A, Alexander LG, Du Laing G, Janssens GPJ.

(2015) Selenium digestibility and bioactivity of dog foods: what the can can, the kibble

can't. Submitted to PLOS ONE, revisions.

van Zelst M, Hesta M, Gray K, Staunton, R, Du Laing, G., Janssens, GPJ. (2015) Biomarkers of

selenium status in dogs. Submitted to BMC Veterinary Research, revisions.

van Zelst M, Hesta M, Gray K, Goethals K, Janssens GPJ. Predictive equations of selenium

accessibility of dry pet foods. Submitted to Journal of Animal Physiology and Animal

Nutrition.

van Zelst M, Hesta M, Gray K, Janssens, GPJ. Storage of heparinised canine whole blood for

measurement of glutathione peroxidase activity. Submitted to Biological Trace

Element Research, revisions.

Abstracts presented at international conferences

van Zelst M, Hesta M, Alexander LG, Bosch G, Hendriks WH, Du Laing G, De Meulenaer B,

Janssens GPJ. (2013) Association between diet composition and in vitro selenium

bioaccessibility in pet foods. In: Proceedings of the 38th Animal Nutrition Research

(ANR) Forum, p.48. May 21st 2013, Roeselare, Belgium.


Goethals K, Janssens GPJ. (2013) Prediction models for in vitro selenium bioaccessibility

in dry pet foods. In: Proceedings of the 17th European Society of Veterinary and

Comparative Nutrition (ESVCN) congress, p.71. September 19th-21st 2013, Ghent,

Belgium.

200 BibliographyBibliographyBibliographyBibliography


Janssens GPJ. (2013) Association between diet composition and in vitro selenium

bioaccessibility in pet foods. In: Proceedings of the WALTHAM® International

Nutritional Sciences Symposium (WINSS), p.144. October 1st-4th 2013, Portland,

Oregon, USA.


Janssens GPJ. (2014) In vitro selenium bioaccessibility in pet foods. In: Proceedings of

the 15th International Symposium on Trace Elements in Man and Animals (TEMA

15), p.119. June 22nd-26th 2014, Orlando, Florida, USA.

van Zelst M, Hesta M, Gray K, Alexander LG, Beech K, Cools A, Du Laing G, Janssens GPJ.

(2015) Dietary format affects selenium absorption and bioactivity in dogs. In:

Proceedings of the 40th Animal Nutrition Research (ANR) Forum, p.45. May 22nd

2015, Merelbeke, Belgium.

van Zelst M, Hesta M, Gray K, Alexander LG, Beech K, Cools A, Du Laing G, Janssens GPJ.

(2015) Selenium absorption and bioactivity in dogs are affected by dietary format. In:

Proceedings of the 19th European Society of Veterinary and Comparative Nutrition

(ESVCN) congress, p.65. September 17th-19th 2015, Toulouse, France.

Awarded the Waltham/ESVCN student award for best presentation.

van Zelst M, Hesta M, Gray K, Staunton R, Du Laing G, Janssens GPJ. (2015) Preliminary data

on biomarkers for selenium status in dogs. In: Proceedings of the 19th European

Society of Veterinary and Comparative Nutrition (ESVCN) congress, p.129.

September 17th-19th 2015, Toulouse, France.

Doctoral Training Program__________________________________________________________________________________________

Doctoral Training Program__________________________________________________________________________________________

Doctoral Training Programme _____________________________________________________________________________________________________________

DoctoralDoctoralDoctoralDoctoral Training ProgramTraining ProgramTraining ProgramTraining ProgramTraining ProgramTraining ProgramTraining ProgramTraining Programmemememe

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Dat was het dan! Wat zijn de afgelopen vier jaar snel voorbij gegaan. Gedurende die vier jaar

heb ik met ontzettend veel plezier aan de studies in dit doctoraat gewerkt. Uiteraard was het

eindresultaat niet hetzelfde zonder de hulp van heel veel mensen die ik in deze

acknowledgements wil bedanken.

Allereerst wil ik graag mijn promotoren bedanken voor hun begeleiding. Geert en Myriam,

ontzettend bedankt voor de goede adviezen die u mij heeft gegeven op geschreven werk en

tijdens de vele meetings die we hebben gehad. Geert, ook bedankt voor het vertrouwen dat u

in mij had vanaf het eerste begin en het oppeppen van mijn zelfvertrouwen wanneer ik dat

nodig had.

I would also like to express my gratitude to the Waltham Centre for Pet Nutrition for giving me

the opportunity to conduct the studies reported in this thesis! I genuinely enjoyed executing

them, and it was a great honour to have been able to perform the in vivo studies at your

facilities in England. In particular, I would like to thank Kerry Gray and Lucy Alexander for their

support and enthusiastic guidance and for thoroughly reading through all my writings during

my PhD. Thank you very much!

Also, very valuable comments to improve the quality of this thesis were given by the jury

members. Therefore, I would like to thank Gijs Du Laing, Wouter Hendriks, Stefaan De Smet,

Evelyne Meyer, Jacques Debraekeleer, Marianne Diez, Kerry Gray and, the chair of the jury,

Ann van Soom.

Wouter Hendriks wil ik nog specifiek bedanken voor het mogen uitvoeren van de in vitro

studie in zijn laboratoria in Wageningen. Guido Bosch heeft mij tijdens de uitvoering van deze

studie heel erg geholpen. Guido, ik heb al kennis mogen maken met jouw intensieve

begeleiding tijdens mijn master thesis in Wageningen, waar ik erg veel van geleerd heb, en nu

mocht ik opnieuw een beroep doen op jouw kennis voor de in vitro studie. Jouw adviezen en

commentaar op het manuscript waren van onschatbare waarde en de hulp met het filtreren heel

erg gewaardeerd! Zoals ze in Vlaanderen zouden zeggen: een hele dikke merci! Ook ben ik

Saskia van Laar-van Schuppen en Jane-Martine Muylaert dankbaar voor hun hulp en adviezen

tijdens de chemische analyses van de in vitro studie.

Verder ben ik mijn collega's erg erkentelijk voor het mij wegwijs maken in België en op het labo

diervoeding. Ontzettend bedankt! Met name wil ik mijn bureaugenoten Daisy en Jia bedanken

voor de gezellige jaren waarin wij een bureau hebben mogen delen. Sarah, bedankt voor je

hulp bij de test van de in vitro studie. Herman en Donna hartelijk dank voor het uitvoeren van

een aantal van de chemische analyses. An, jou wil ik bedanken voor je expertise en hulp op

het gebied van de statistische analyses. Ruben en Jenny, hartelijk dank voor het afhandelen

van alle administratie die komt kijken bij het uitvoeren van een doctoraat. Marta, jou wil ik heel

208 AcknAcknAcknAcknowledgementsowledgementsowledgementsowledgements

erg bedanken voor het wegnemen van een hoop stress door tot in de late uurtjes nog samen te

zitten om de general discussion op orde te krijgen.

Verder wil ik Sofie Coelus bedanken voor haar uitleg van de aminozuur analyse en Joachim

Neri en Daniel Vermeulen voor het uitvoeren van Se en schildklierhormoon analyses. Iain Peters,

thank you very much for your explanation of mRNA expression and for the analysis of the

mRNA samples.

I would also like to thank all the people at Waltham that were involved in one or both in vivo

studies. The unit owners, unit specialists and care takers of the dog units Betsy (Dog 6), Endal

(Dog 8) and Apollo (Dog 9). A special thanks to Colleen Irvine who was involved in both in vivo

studies and did a very good job in making sure that everything was organised well. I would

like to thank Ruth Staunton and Karen Beech for their knowledge and help with the statistical

analysis of both in vivo studies. During the chemical analysis of the first in vivo study and the

optimisation of the GPx analysis, Wendy Tomlinson had an answer to all my questions and

helped me with every problem I encountered, many thanks! Jay and Emma were my support in

the lab during the biomarker study, thank you very much! Robyn Bednall has helped me

tremendously during the biomarker study. She kept the study going during my absence in

England and, together with Tim O'Brine, she performed the hair growth measurements, for which

I am very grateful. Robyn and Tim, thanks a lot! Of course, a special thanks to the stars of the

studies, the dogs: Arnold, Branston, Cinderella, Davina, Dermot, Eclipse, Ella, Elliot, Erin, Esme, Fern,

Flick, Ickle, Indigo, Jackson, Jinx, Keira, Krista, Larry, Leila, Lexie, Lois, Merlin, Morph, Mowgli,

Oasis, Olivia, Orchid, Pebbles, Pedro, Pepe, Pippa, Pixie, Quaver, Rita, Romany, Ronnie, Rosco,

Rupert, Tigger, Twiggy, and Zara.

Ik wil in deze acknowledgements ook graag de kans aangrijpen om de mensen te bedanken

die mijn interesse in de diervoeding hebben geïnitieerd; Hans Kruft en Marco Halff. Hans,

ontzettend bedankt voor de vele mogelijkheden en de grote hoeveelheid aan informatie die je

mij hebt gegeven in de tijd dat ik bij je in de dierenwinkel werkte (2002-2005) en voor de

vriendschap die er nog steeds is. Marco, jou wil ik bedanken voor de ontzettend interessante

dagen waarop ik met jou bij InterPet Products en Vobra Special Pet Foods mee heb mogen

lopen. Door jouw enthousiasme is mijn interesse in de diervoeding gegroeid en wist ik vanaf

toen zeker dat ik in dit vakgebied verder wilde.

Daarnaast wil ik mijn familie en schoonfamilie bedanken. Pap en mam, ontzettend bedankt voor

het altijd steunen van mijn studiekeuzes en voor het altijd klaar staan voor mij. Paul, bedankt

voor alle keren dat je Engelse teksten voor me hebt nagelezen als ik er weer eens onzeker over

was. Ik ben trots op je en op alles wat je op eigen kracht bereikt hebt. Pap en Paul, jullie ben ik

ook nog een bedankje verschuldigd voor het helpen verhuizen naar Merelbeke in 2011, op de

AcknowledgementsAcknowledgementsAcknowledgementsAcknowledgements

209

warmste dag in september. Jos, Mieke, Christian, Judith, Lucien en Daniëlle, bedankt voor de

hartelijke opname in de familie Koks! Ik kwam er als laatste schoondochter bij, maar ik heb me

vanaf het eerste moment bijzonder welkom gevoeld. Mijn vriendenclubje Nicoline, Albert, Lynn,

Wilbert, Lottie en Nelly wil ik bedanken voor alle gezellige uitjes en samenkomsten waardoor

mijn gedachten even weg waren bij selenium en hondenvoeding, wat ook wel eens goed is

(zeggen ze). Hoewel we elkaar door de afstanden niet heel vaak zien, hecht ik toch erg veel

waarde aan jullie vriendschap. Suus en Jorrit, ik heb het bijzonder gewaardeerd dat jullie me

kwamen opzoeken in Engeland. Super bedankt voor jullie vriendschap! Mathieu, jou wil ik

bedanken voor je mentale steun en motivatie gedurende het hele PhD traject. Je hebt er niet

voor gekozen dat ik 4 jaar lang het grootste deel van de tijd in het buitenland zou zijn, maar je

hebt me desondanks altijd enorm gesteund... waar waren wij geweest zonder Skype en

telefoon.. ;-) In Engeland kwam je me zoveel mogelijk opzoeken en de tijd dat ik in België zat,

was het elk weekend weer fijn thuiskomen bij jou in Weesp. Jij bent de beste echtgenoot die ik

me wensen kan!

During my stay in England I have also had a great time outside work, thanks to the Wade

family and their "gang". Sarah, I would like to thank you for always making me laugh, and for

introducing me to an amazing family. During my time in England I had the pleasure of staying

with the Wade family; Lynda, Trevor, Laura and Ryan. I have lived with them for almost a year

during the in vivo studies. I am extremely grateful for having the chance to know them. Ryan, I

wish you all the best with finishing your university and with your roller and ice hockey

tournaments! Laura, thanks a lot for lending me your room and best of luck with building your

career! Trevor, thank you for being such a genuine and friendly person! I really enjoyed our

trips to London, Leicester and the Lidl ;-) I wish you lots of happiness, peace and good times in

the future! Lynda became like a second mum to me, she was always there with advice when I

needed it. She was a lovely, intelligent, funny and truly remarkable person. Due to a terrible car

accident, Lynda passed away on Christmas day 2014, five days after we'd said "see you soon!".

Lynda, I'm very grateful for having known you and for the wonderful moments we've shared.

You will always be on my mind!

Mariëlle

Merelbeke, December 2015

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