1

Antioxidant Capacity of breast-milk taken by patientswith kwashiorkor

A thesis submitted for the degree of Master of Scienceat the University of Aberdeen

by

Vesna Markoska

August, 2000

2

Declaration Page

I declare that this thesis has been composed entirely by myself and it has not

been accepted in any previous application for a degree. The work, of which it is a

record, has been done by myself. Quotations have been distinguished by quotation

marks, and sources of information have been specifically acknowledged.

3

Acknowledgements

I would like to thank my supervisor and teacher Michael Golden for showing me lotsof sunny and important windows.

My MSc and this project was performed with the support of MSF-Holland, and Iwould like to thank everyone in this organisation especially to all my lovely friends"Sans Frontieres" for being with me through the all loves and tears.

Special thanks to the Local MSF-Holland Kisangani team without who this projectwould not have been possible.

I am particularly grateful to Dr Ferko Ory for his big understanding and help.

Thanks to Dr John Arthur and Dr Garry Duthie who were very helpful during thechemical analysis part of my study and made me so feel welcome in the RowettResearch Institute.

Thanks to Janet Kyle, Fergus Nicol, Dr.Peter Gardner for their friendly approach andsupport.

I would also like to thank Dr. Geraldine McNeill for her gentleness, patience and bigoptimism.

I am thankful to my parents for their support.

The "seven angels" from the flat FS-2000, made this year to be remembered byunusual harmony love and peace.

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Summary............................................................................................................................................ 6

1. Introduction.............................................................................................................7Hypothesis…………………………………………………………………………………. 10

2.Methods and Materials........................................................................................112.1.Experimental design…………………………………………………………………… 11

2.3. Subject selection……………………………………………………………………….12

2.4. Procedures……………………………………………………………………………...13

2.5.Ethical considerations…………………………………………………………………..14

2.6. Biochemical methods…………………………………………………………………. 152.6.1.Selenium analysis of human milk .......................................................................................... 152.6.2.Glutathione Peroxidase Assay................................................................................................ 162.6.3. HPLC determination of carotenoids, tocopherols and retinol in human milk...................... 172.6.4. Total Phenol Compound Concentrations in Human Milk..................................................... 182.6.5.Assesment of Antioxidant capacity of Human Milk by Electron Spin Resonance (ESR)Spectroscopy................................................................................................................................... 192.6.7.Statistical methods ................................................................................................................. 19

3. Results…………………………………………………………………………….20

3.1.Selenium and Glutathion Peroxidase (GSH-Px)……………………………………… 20

3.2. α -Tocopherol & γ-Tocopherol……………………………………………………….. 22

3.3. Carotenoids and Retinol………………………………………………………………. 24

3.4.Total Phenolic compound of the milk…………………………………………………. 27

3.5.Total Antioxidant Capacity of the milk………………………………………………...29

4.Discussion……………………………………………………………..………..31

Conclusion.................................................................................................................39References: .................................................................................................................................. 40

5

List of Tables & Figures

Table 2.1: Characteristics of the patients and controls. 14

Table 3.1: Selenium concentration in µg/l and GSH-Px U/ml

in breast-milk from kwashiorkor and control subjects 20

Table 3.2: Estimates of the concentrations of α -Tocopherol

& γ-Tocopherol in breast-milk 22

Table 3.3: The carotenoids in breast milk samples 27

Table 3.4: The total phenolic compounds in breast-milk samples 28

Table 3.5: The total antioxidant capacity of milk-samples. 29

Figure3.1:The relationship between Se and GPX in breast milk

samples from mothers with children who have

kwashiorkor (K), marasmus (M) or are healthy (S) 23

Figure 3.2: The relation between ESR signal strength and α -Tocopherol 25

Figure 3.4: Total phenolic content versus total antioxidant capacity of milk 38

Figure 3.5: The total antioxidant capacity from each milk sample. 30

Graph 3.1: Reference Se concentration in mature human milk and

measured Se concentration in the milk of the study population 21

Graph3.2: α Tocopherol concentrations 23

Graph3.3: Mean retinol concentrations of mature human milk-

international and studied values 27

Graph3.4: Mean carotenoids concentrations of mature human milk-

international and studied values 27

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Summary

Some children with kwashiorkor are being breast fed. It is unclear why the breast-

milk does not protect these children from severe malnutrition. There is strong

evidence of a derangement of antioxidants as part of the pathogenic mechanism for

this illness. In this thesis I tested the hypothesis that the breast-milk being taken by

children with kwashiorkor was deficient in antioxidants and there precursors.

The study took place in Kisangani, Democratic Republic of Congo. Breast milk was

taken from the mothers of 7 children with kwashiorkor, 2 with marasmus and 7

healthy infants. The samples were frozen and transported to Aberdeen where

selenium, vitamin E, vitamin A, carotenoids and the Total Antioxidant Capacity were

measured.

There were no significant differences between the milk taken by the children with

kwashiorkor or marasmus and the healthy controls. All the samples had very low

selenium concentrations – lower than any others reported in the literature. Vitamin E

was normal. The level of the carotenoids and vitamin A was very low. Most of the

samples had no antioxidant capacity at all.

The results indicate that there is widespread selenium and vitamin A deficiency in

DRC. The milk being consumed by all the children studied in this study is deficient in

anti-oxidants and is therefore unlikely to adequately protect the infants from oxidative

stress. It is concluded that to have healthy infants the diet of lactating mothers cannot

be neglected. This particularly applies to type I nutrients which can be deficient

without any anthropometric changes in either the mother or the child.

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1. Introduction

Golden and Ramdath examined the evidence that kwashiorkor results from an

imbalance between the production of free radicals and their safe disposal. In children

who die from kwashiorkor, vitamin E and the selenium containing enzyme,

glutathione peroxidase (GPX), were particularly low and free iron was increased.

Glutathione itself was very low due to a very rapid rate of consumption, and the

NADPH/NADP ratio was low showing that the cells were oxidised. GPX activity of

below 17 U/g haemoglobin and a ferritin of above 250 mg/l was an accurate predictor

of the survival of the severely malnourished child.

Studies from Guatemala (Burk at al.1967) and Zaire (Fondu at al.1978)

confirm that Se itself is reduced in the blood of children with kwashiorkor in other

countries as well as Jamaica. Inefficient removal of organic peroxide will result in

production of toxic aldehydic products (Slater, 1984) that damage cell membranes.

Such damage has been found in kwashiorkor, in Nigeria, by Lichsenring’s group.

Furthermore, Forrester et al reproduced the electrolyte abnormalities of kwashiorkor

in normal cells by reduction of their glutathione content.

There is also evidence that plasma vitamin E is severely reduced in

kwashiorkor, related to prognosis McLaren at al.(1969) and useful in classification of

malnourished children.

The level of the antioxidants in kwashiorkor may be low because of their high

consumption, because of their low intake, or both. The level of these nutrients in the

breast-milk is dependant on the dietary intake of the mother. Mothers exposed to an

impoverished diet may provide poor antioxidant protection for their breastfed child.

Selenium deficiency is particularly likely in Democratic Republic of Congo

(DRC). This comes from a consideration of selenium chemistry. Se is easily leached

8

from soil where there is a high rainfall (similar to iodine), it is reduced to very

insoluble products when the ground is waterlogged or acid, and it forms a complex

with iron so that anywhere where the soil is red (lateritic, bauxitic etc) is likely to be

selenium deficient. DRC has all these features. There is an interaction between Se

and Iodine in that the conversion of T4 to T3 (by 5’thyroxine deiodinase) is selenium

dependent. This means that goitre is very much worse in areas of iodine deficiency

that are also selenium deficient. Such a situation is found in DRC, for this reason Se

nutrition has been studied in Zaire, now DRC and has been found in surveys to be

commonly deficient.

Golden and Grellety, 1997, collected data from 3024 severely malnourished

children age 6 to 36 months were collected from 22 nutritional centres from 11

African countries. One fourth of the kwashiorkor children were receiving breast-milk.

When the children were stratified by age the proportion of the children less then one

year of age who were being breastfed was the same for kwashiorkor and marasmus.

Of 65 children from 6 to 11 months age 9 were exclusively breastfed. From this

analysis Golden and Grellety concluded that the mother’s breast-milk was unable to

protect the child from developing kwashiorkor and thus must be lacking in one or

more of the nutrients that are protective against this illness. They also suggested that

the factor(s) are likely to a) vary in breast milk with the diet of the mother, which is a

characteristic of the type I nutrients, and b) be low in the breast-milk of mothers of

children who have kwashiorkor, and c) be important for the antioxidant defence of the

infant.

With type 1 nutrient deficiency the mother may appear well nourished

anthropometrically. Kwashiorkor has been observed (albeit rarely) in exclusively

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breastfed children, which demonstrates that breast milk can be sufficiently deficient to

cause kwashiorkor. Golden and Grellety’s other findings, that are relevant are that

breastfed malnourished children took a longer time to gain weight then non-breast-fed

children and that the mortality rate was the same for breastfed and weaned children.

The purpose of this study was to investigate the breast milk composition of

mothers who were breast feeding children with kwashiorkor.

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HYPOTHESIS

1) The antioxidants, particularly selenium and Vitamin E, are lower in the

breast-milk of mothers of children who develop kwashiorkor than mothers of children

who get marasmus or are normally nourished.

2) The antioxidants, particularly selenium and Vitamin E , are lower in the

breast-milk of mothers from DRC than the internationally published values for normal

lactating women in other parts of the world.

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2.Methods and Materials2.1.Experimental design

This study was designed as a prospective cross-sectional observational control study

of breast milk composition of mothers whose infants developed either kwashiorkor,

marasmus, or were healthy.

2.2. Location

The breast milk for this study was collected from the community and the five

Therapeutic Feeding Centres (TFCs) run by MSF-Holland (Medecins Sans Frontiers),

located in the suburb and the town of Kisangani (erstwhile Stanleyville), Democratic

Republic of Congo from April to May, 2000.

The famine has been subsequent to the active war which has been continuous for the

last two years. Initially, the study was delayed for one-week in order to obtain

authorisation from the local ethical committee. The sample collection was not

completed as suggested with the initial study design. This is because the war

encroached upon the town and there was active fighting. The study area became part

of the front line between the forces and the town itself (including the MSF buildings)

were subject to shelling and heavy machine gun fire so that the team were evacuated

during a lull in the fighting. Later it was found that 628 people were killed and over

3000 wounded during this action.

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2.3. Subject selection

Breast-milk was collected from three groups of freely lactating mothers. Seven

mothers whose children recently developed kwashiorkor participated as cases; two

whose children recently developed marasmus were first the control and seven mothers

of healthy children from the same community were second control.

All the mothers were healthy women aged 20 to 38 with uncomplicated

pregnancies and deliveries. They were taking their habitual diet only and were

suckling their children normally. The sampling was made on admission to the TFC

(Therapeutic Feeding Centre) in order to be sure that none of the therapeutic diet, or

non-habitual foods, were consumed by the mothers before the breast-milk sample was

taken. All mothers who had received supplementary diet at any centre were excluded

from the study. Obesity was recorded in some of the mothers feeding healthy infant

and also in mothers feeding children with kwashiorkor. One kwashiorkor case’s

mother was undernourished.

The infants were all full term healthy singletons appropriate for gestational

age. In conditions of shortage of breast-fed patients, the age criteria were extended

from six to 18 months for all groups. Each child was receiving at least three breast-

feeds par day as well as their habitual supplementary diet. Kwashiorkor was

diagnosed in children who presented with bilateral pitting oedema on the dorsum of

the feet or over the tibia without any other known cause for oedema. Children who

were above -2Z weight-for-height (w/h) and height-for-age (h/a) by NCHS standards,

a mid upper arm circumference (muac) of over 125cm, without any oedema, with

normal vital functions, behaviour and suckling, and with no signs of disease served as

13

the comparison group of normal subjects. Two severely wasted children with w/h of

less then 70% and length over 55cm were selected as marasmic controls. The

characteristic of the patients are given in table 1.

Table 2.1: characteristics of the patients and controls.

Samplecode

Weight inkg

Height incm

MUAC incm

Age inMonths

Oedema Age ofMother

Parity

K1 5.9 67.5 11 ++ 35 4K2 4 62K3 7.5 71 13 12 ++ 32 10K4 5.2 100 11.5 7 ++ 3K5 5.3 64 13.6 11 + 20 5K6 7.8 76.5 13.8 16 ++ 20 2K7 5.6 6.6 11 7 ++S1 8.7 65 16.5 21 1S2 9.6 72 15.5 12 40 9S3 7.5 65 15.5 6 30 5S4 8.3 71 14 9 17 3S5 7.7 6.5 14.5 6 27 8S6 7.8 72 13.5 15 24 2M2 5.3 67.2 17 40 3

2.4. Procedures

A detailed dietary, medical and social history was taken from the mother on

admission to the TFC and the anthropometric measurements of the child.

The whole of the milk from one breast was collected by manual expression

into a plastic cup while the infant was suckling on the other breast to ensure breast-

milk flow. The milk was kept for less than 3-4 hours in shadow (average day

temperature about 29°C) and protected from light with aluminium foil.

The milk volume was recorded. The whole sample was then mixed and

aliquots taken by syringe into six Eppendorf tubes, previously labelled with the

experimental code. Kwashiorkor -K, healthy-S and marasmus-M, followed by the

14

subject number (1 to 7). Solid antioxidant (butylated-hydroxy-toluene, BHT) was

added to the two of the Eppendorf tubes in order to prevent deterioration of the

Vitamin E..

The samples from each patient were kept in a separate plastic sealed bag in the

dark in a home deep freezer. There is no record of the temperature; however, the

contents of the freezer remained solidly frozen throughout the storage period. There

were electricity breaks from time to time but at no stage did the samples thaw during

storage. The samples were brought by air on wet-ice from Kisangani to Kampala

(Uganda) and thence to Aberdeen via London. The total journey took approximately

18 hours during which time the samples defrosted as the cold chain could not be

maintained. However, the samples remained cool. The samples were refrozen on

arrival in Aberdeen. Separate aliquots were defrosted immediately before each assay.

The samples of local food had to be abandoned during the evacuation.

2.5.Ethical considerations

The study was approved by the Ethical Committee of the University Hospital

Kisangani, the Ministry of Health DRC and University of Aberdeen. It was conducted

in accordance with Helsinki Declaration. The volunteers gave their consent.

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2.6. Biochemical methods 2.6.1.Selenium analysis of human milk

Principle of essay: The method uses a perchlorate digestion of samples and the

coupling of selenium to diaminonaphtalene (DAN). The resulting SE/DAN complex

is quantified fluorimetrically.

Chemicals: Concentrated nitric acid (BDH), Perchloric acid 60% (BDH),

concentrated hydrochloric acid (BDH), Hydroxylamine chloride (Sigma), EDTA

(disodium salt) (Sigma), 2,3 diaminonaphtalene (Aldrich), Cyclohexane (BDH),

Ammonia 40% (BDH).

Solutions used:Hydroxylamine/EDTA: 25g hydroxylamine + 9.24g EDTA made up

to 1 litre with distilled water.Cresol red indicator: 0.05g cresol red in 250ml distilled

water containing 1ml of 40% ammonia.DAN: 0.5g diaminonaphtalene dissolved in 1

litre of 0.1M HCl. The DAN was ground with a small volume of the HCl to help it

dissolve more easily. The DAN solution was extracted twice with 60ml of

cyclohexane, which was then discarded. The solution is light sensitive and a

precipitate forms on exposure to direct sunlight. However, it is stable for at least one

month in a foil covered bottle, if stored beneath 60ml of cyclohexane.

Equipment: Fluorimetric spectrophotometer (Excitation:376nm,

Emmission:520nm).Method: Duplicate 1ml milk sample were pre-digested with nitric

acid. The sample was then boiled after the addition of 60% perchloric acid. 10%

hydrochloric acid was then added and used to drive off any excess nitric acid. This

also ensured that any selenium present as selenate is converted to selenite. The

optimal pH of 1.5 to 2.5 for the formation of the diaminonaphtalene-selenium

complex was achieved by adding hydroxylamine/EDTA solution to each digest.

16

After addition of 2-3 drops of cresol red indicator, 40% ammonia was added until a

yellow-green colour appeared.

The samples were incubated at 50oC with the diaminonaphtalene solution. Then

"Analar" cyclohexane was added to extract the diamoninaphtalene/SE complex. The

cyclohexane was removed and the fluorescence of the diamoninaphtalene/Se complex

measured. The measurements were compared with the standard curve derived from

sodium selenite solution.

2.6.2.Glutathione Peroxidase Assay

Reaction Mix: 5mg NADPH, 46mg Reduced Glutathione, 3ml Distilled Water, 24ml

Phosphate Buffer (pH 7.6), 1ml Na Azide (0.1125M), 20 Units Glutathione

Reductase, water bath 25° C.

To measure the glutathione peroxidase, the milk was centrifuged at 2000xg for 10

minutes, the clear supernatant was then used for the assay. To a 1ml cuvette, 0.915ml

of the reaction mix and 0.05ml of the milk supranatant was added and the reaction

started by the addition of 0.035ml of 0.022M hydrogen peroxide. The rate of change

was followed at 340nm. A blank rate was measured first by substituting distilled water

for the supernatants.

Calculation of results: A unit of glutathione peroxidase is defined as that which

oxidises 1µmole of NADPH/minute. The molar extinction coefficient of NADPH is

6220 (the conversion factor for the assay, when the volume of milk used is 50µl, is

3.2154).

17

2.6.3. HPLC determination of carotenoids, tocopherols and retinol in human

milk

Principle of assay: The HPLC method allows the simultaneous measurement of

retinol, six carotenoids, α-tocopherol and γ-tocopherol in human milk using

fluorescence and visible detection.

Solutions required: Methanol containing 10g Butylated-hydroxytoluene (BHT)/l;

Hexane containing 500mg BHT/l; DEA – (20% Dioxan, 20%Ethanol, 60%

Acetonitrile); 1% Ammonium Acetate in water. Mobile phase: 67.4% Acetonitrile,

22% Tetrahydrofuran, 6.8% Methanol/BHT, 3.8% Ammonium acetate 1%.

Equipment : Waters 470 Scanning Fluorescence Detector, 486 Tuneable Absorbance

Detector, 600E System Controller, 712 WISP, Jones Chromatography Column Chiller

Model 7955. The system was run using Millennium v 2.1 Software.

Methodology: The milk (200µl) was prepared by adding 200µl water and 400µl

ethanol and vortexing for 10s. Echinone (100µl) was added as an internal standard

which does not co-elute with the other peaks. The carotenoids and vitamin E were

extracted from the sample by addition of 700 µl of hexane/BHT. The sample was

shaken for ten min on the “vortex genie”, centrifuged for 5 min and then 600µl of the

clear fraction taken. The hexane layer, which contains the vitamins of interest, was

evaporated on the speed-vac for 9min. The dried residue was dissolved in 200µl of

DEA before application to the HPLC column.

Chromatography: Beckman ultrasphere ODS 5µm 25cm x 4.6mm I.D. in a column

oven set at 29°C. Flow rate 1.05ml min –1 and the injection volume 150µl. The

18

runtime is 30 minutes. Wavelengths are changed during the run to the appropriate

wavelength for determination of each species.

2.6.4. Total Phenol Compound Concentrations in Human Milk

Principle of assay: The reaction between phenolic compounds , Folin Reagent and

Na2CO3 results in blue colour. The absorbance is read at 765nm and the sample total

phenolic concentration is read from the standard line of the known concentration using

gallic acid. The results are expressed in Gallic Acid Equivalents/ml (GAE µg/ml).

Solutions required: methanol/(1g/lBHT); methanol /perchloric acid (60%) /water

(8:1:1 v/v); diethylether; 10% Folin Reagent; 7.5% Na2CO3; Sep-Pak Alumina B

Cartridges for solid phase extraction.

Methodology: To hydrolysed and separated the proteins and fats from the milk,

duplicate 1ml sample were vortex mixed with 3ml methanol/BHT for 5 min. at 4°C

and centrifuged at 3500rpm for 15 min at 4°C. The supernatant which contains the

phenolic compounds was applied to the alumina cartridge where an alumina phenolic

complex was formed. Contaminating compounds were washed off with methanol and

diethylether washes. Finally, the phenolic compounds were eluted from the alumina-

phenolic complex by applying methanol/perchloric acid/water solution to the

cartridge.

The phenolic content of the elutant was measured adding by 10%Folin Reagent and

after 8.5 minutes adding 7.5% Na2CO3 for 1hour in proportions 1:5:4 (v/v). The

solution was then filtered through Millex-GV 0.22µm filter unit and the colour read at

19

765nm. Working standards of Gallic acid were prepared from 0.1g/100ml methanol

stock standard at four known concentrations and taken through the same extraction.

2.6.5.Assesment of Antioxidant capacity of Human Milk by Electron Spin

Resonance (ESR) Spectroscopy

Principle of assay: The overall ability of human milk to donate electrons to the

synthetic free radical species, potassium nitrosodisulphonate (Fremy’s Salt) is

assessed using electron spin resonance (ESR) spectroscopy. The ESR spectrum of

known free radical concentrations is measured as a standard and the signal intensity

obtained by double integration. The test concentration was calculated by comparison

with the control reaction.

Solutions required: Fremy’s Salt Radical 50µM in distilled water.

Equipment: Bruker ECS 106 spectrometer; frequency – 9.5GHz; microwave power-

2mW; modulation amplitude-0.01mT.

Measurements: An aliquot (3ml) of 20-fold diluted milk was placed in a test tube with

an equal volume of Fremy’s radical solution and spectrum was obtained after 5min.

The signal intensity was compared to that of the control, which used water instead of

milk.

2.6.7.Statistical methods

The data were entered into an excel spread sheet and imported into SPSS. The means

and standard deviations calculated. For the purposes of analyses ANOVA analysis

was first performed using the three groups of children. If this analysis was not

significant then the children with marasmus were combined with those for the normal

subjects, and a separate analysis was done to compare the kwashiorkor and normal

samples. Bivariate comparisons were with Student’s unpaired T-test (two tailed).

20

Correlation analysis between variables was by Pearson’s least squares linear

regression. A probability of less than 0.05 was accepted as significant.

3. Results

3.1.Selenium and Glutathione Peroxidase (GSH-Px)

The results of the selenium and GPX concentrations in the milk samples are given in

table 3.1. The mean Se concentration in the milk between the kwashiorkor did not

differ significantly from the healthy and marasmic controls.

Table 3.1: Selenium concentration in µg/l and GSH-Px U/ml in breast-milk fromkwashiorkor and control subjects

sample samplesize

mean extr. Rangemin/max

Std.Error Mean

Std.Dev

Seleniumµµµµg/l /ml k h m

772

8.648.098.10

4.6 - 15.45.0- 10.75.9 - 10.4

1.370.682.27

3.621.803.20

Glutathione Peroxidase k U/ml h M

672

0.0220.0260.022

0.001-0.070.002-0.060.018-0.025

0.010.0090.004

0.030.020.05

k* kwashiorkor h* healthy m* marasmus* the GSH-Px value for one of the kwashiorkor sample excluded

The reference value for Se concentration of mature human milk is 14-21 µg/l with an

extreme range between 8-38µg/l("Nutrition during Lactation" p.116) The Se concentration

is below the lower boundary of the reference value (14 µg/l) in 15 of 16 studied cases.

Nine subjects of 16 are also lower than the lowest extreme reference value of 8µg/l.

This is a significant difference , shown in (graph 3.1) There was no difference

21

between the proportion of kwashiorkor samples and the controls 4/7 and 5/9 which

were below the extreme limit.

Graph 3.1 Reference Selenium concentration in mature human milk and measured Se concentrations in the milk of the study-population

There was no difference in mean GSH-Px activity between the any of the

grouped samples (p > 0.05).

There was a significant correlation (p<0.01) between GSH-Px activity and Se

concentration (r =0.78), after exclusion of one of the kwashiorkor samples which had

an aberrant value. The excluded sample had a very high activity of peroxidase, 0.24

U/ml, despite a low Se level within the range seen for the other samples: this result

was excluded on the basis that the sample was likely to have been contaminated and

the peroxidase activity due to bacteria or inflammatory cells. The relationship is

shown in Figure 3.1.

Figure 3.1: the relationship between Se and GPX in breast milk samples from mothers with children who have kwashiorkor (K), marasmus (M) or are healthy (S)

0

5

10

15

20

25

norm al norm al D R .C on go

Int N o rm alK w ash iorko rH ealthyM aras m us

R2 = 0.6882

R2 = 0.8337

-0.02-0.01

00.010.020.030.040.050.060.070.08

0 5 10 15 20

Se ng/ml

GH

S-P

x U

/ml

Kwashhealthymarasmus

22

3.2. αααα -Tocopherol & γγγγ-Tocopherol

Table 3.2 shows the alpha and gamma tocopherol results. There were no

significant differences. However, the mean values for the mothers of children with

kwashiorkor were lower than for the healthy controls. This almost reached

significance for the gamma-tocopherol values.

Table 3.2: Estimates of the concentrations of α -Tocopherol & γ -Tocopherol in breast-milk from 7kwashiorkor and 9 control subjects compared by one-way ANOVA with p>0.05 .

samplesample size

Mean(mg/L)

extr.rangemin/max

Std.Er.Mean

SD p

αααα-Tocopherol k h m

772

2.423.052.88

0 - 4.120.61 - 8.562.48 - 3.29

0.470.970.40

1.242.580.57

0.83

γγγγ-Tocopherol

k h m

772

0.160.280.08

0.05 - 0.490.16 - 0.450.4 - 0.12

0.060.040.04

0.160.120.05

0.16

k- kwashiorkor h-healthy m- marasmus

The reference Vitamin E concentrations and these of the DRC samples are

shown in Graph 3 .2 The mean reference value for Vitamin E in mature breast-milk is

2.3 +1 mg/L ("Nutrition during Lactation" p.116) and α-Tocopherol is 83% of total

vitamin E content (Kobayashi 1975). 87% of the observed mean concentrations are

distributed in the reference range of 2.3+ 2SD.

23

Graph 3.2 α-Tocopherol concentrations

The equality of the means was examined by ANOVA that showed no

significant difference between the concentration of the case subjects and controls.

There was a strong positive correlation between α-tocopherol concentration

and the total phenolic content of the milk (r= 0.99, p<0.01) . Vitamin E concentrations

did not show significant correlation with the total antioxidant capacity of the milk

examined separately to the kwashiorkor mothers and controls. When this correlation

was observed one of the healthy subject S6 was extrapolated as outlier; that mother

worked in the TFC and was consuming of the therapeutic balanced food. The (Figure

3.2) shows strong linear correlation between the total antioxidant estimate of the

milk with ESR and α -Tocopherol in the healthy sample when the outlier was

included in the test (r =0.9), (p<0.001); no correlation is observed within the milk

0

0.5

1

1.5

2

2.5

3

3.5

normal DR.Congo

Int NormalKwashiorkorHealthyMarasmus

24

from kwashiorkor patients. This example also verify Vitamins' E antioxidant

potential.

Figure 3.2: the relation between ESR signal strength and α -Tocopherol

3.3. Carotenoids and Retinol

The carotenoids in the milk samples are shown in table 3. The

carotenoids/retinol high ratio, suggests that the Vitamin A in the examined population

is mostly from vegetable origin. The total carotenoid content of the milk 0.168 µg/ml

from the kwashiorkor mother’s milk is 14 times higher then the retinol content in the

same samples (0.012µg/ml). The same ratio for the healthy samples is 33.5

(0.268/0.008µg/ml). No retinol was found in the milk of the marasmic sample and

their carotenoid content is 0.22µg/ml.

R2 = 0.9218

R2 = 0.4015

-15

-10

-5

0

5

10

15

20

25

30

0 1 2 3 4 5 6 7 8 9

alpha tocopherol

% F

rem

y ra

dica

l red

uced

marasmushealthykwash

25

Table 3.3: the carotenoids in breast milk samples

sample samplesize

meanµµµµg/ml

extr. rangemin/max

Std.ErrorMean

Std.Dev sig2-tail

ββββ-carotene khm

772 total

0.100.100.090.1

0.06- 0.170.05- 0.140.08- 0.110.05-0.1

0.0140.0130.0130.008

0.040.030.020.03

0.90

αααα-carotene khm

772total

0.050.050.060.05

0.02- 0.080.03- 0.080.06- 0.070.02-0.08

0.0070.0070.0040.004

0.020.020.000.02

0.57

Lycopen khm

772total

0.0010.0030.0020.002

0.000- 0.0070.000- 0.0090.000- 0.0030.00- 0.009

0.0000.0010.0020.0007

0.0030.0040.0020.003

0.64

ββββ-Crypt khm

772total

0.0050.0070.0020.005

0.001- 0.0140.002- 0.0170.001- 0.0030.001-0.017

0.0020.0020.0010.001

0.0050.0050.0010.005

0.46

LUT/ZEA khm

772total

0.120.110.060.1

0.08- 0.210.03- 0.320.03- 0.160.03-0.32

0.020.040.040.02

0.060.100.060.08

0.59

Retinol khm

772total

0.0120.0080.0000.008

0.00- 0.20.00- 0.20.00- 0.00.00-0.024

0.0090.0060.0000.002

0.0030.0020.0000.008

0.18

k- kwashiorkor h-healthy m- marasmus

The carotenoids/retinol high ratio, suggests that the Vitamin A in the

examined population is mostly from vegetable origin. The total carotenoid content of

the milk 0.168 µg/ml from the kwashiorkor mother’s milk is 14 times higher then the

retinol content in the same samples (0.012µg/ml). The same ratio for the healthy

samples is 33.5 (0.268/0.008µg/ml). No retinol was found in the milk of the

marasmic sample and their carotenoid content is 0.22µg/ml.

26

There is no significant difference in the means between the samples and

controls examined by ANOVA.

The carotenoids with the highest concentration (counted all 16 samples) are

β carotene 0.1µg/ml, lutein/zeaxantine 0.1µg/ml followed by α carotene 0.05µg/ml.

Lycopene and β Cryptoxantin concentrations are much lower at 0.002µg/ml and

0.005µg/ml respectively. Lycopen was below the limit of detection in 10 of the 16

subjects.

In 6 subjects there was no detectable retinol (two subjects from each sample).

There were positive correlations between α and β carotene (r=0.82), and between

lycopene (0 in 10 cases) and lutein/zeaxantin (r=0.61) β-cryptoxanthine was

positively associated with both the total phenolic compounds in milk (r=0.68) and

lutein/zeaxantin (r=0.61). The correlations between the different fractions of the

carotenoids are result of their appearing in set.

There was a weak correlation between the total antioxidant capacity of the

milk and both β-cryptoxantine (r = 0.55; p<0.05) and α- carotene (r = 0.59; p<0.05)

which is due to the high levels of these nutrients only in the S6 subject. However, the

correlation is a suggestion about the antioxidant characteristics of β-cryptoxantine

and lutein/zeaxantin.

The Vitamin A concentration of mature human milk, as retinol equivalents is

0.670+200 µg/ml (Nutrition During Lactation, Table 6-1). The carotene concentration

varies from 0 to 320µg /L. (Butte and Calloway,1981; Chappell at all., 1986).

Graphs: 3.3 and 3.4 are showing the difference between the normal Vitamin A

(retinol and carotenoids) concentrations and the measured concentrations in the

Congolese population. Vitamin A is remarkably lower than the references.

27

Graph 3.3 Mean retinol concentrations of mature milk - international and studied values

Graph 3.4 Mean carotenoid concentrations of mature milk - international and studied values

3.4.Total Phenolic compound of the milk

The total penolic compounds in the milk samples is given in table 4. Although

the values were lower in the milks from the kwashiorkor mothers, the difference did

0100200300400500600700800900

normal normal DR.Congo

Int Normalkwashhealthymaras

0

50

100150200

250

300350

normal normal DR.Congo

Kwashhealthymarasmnormal

28

not reach significance. There does not seem to be a reference value for the total

phenolic concentration in human milk.

Table 3.4: the total phenolic compounds in breast-milk samples

Sample sample size mean extr. Rangemin/max

Std.ErrorMean

SD P

µµµµg GAE/mlmilk

khm

772

1.291.661.49

0.023-2.1150.385- 4.5041.263- 1.704

0.240.500.22

0.641.330.31

0.79

k- kwashiorkor h-healthy m- marasmus

Figure 3.4: Total phenolic content versus total antioxidant capacity of milk

Antioxidant features of the phenolics are suggested by the positive linear

correlation at ( 0.01 sig) which was observed among the phenolic contents of the milk

of healthy mothers and the capacity of the milk to reduce radicals (r = 0.9);shown by

R2 = 0 .9 2 44

R2 = 0.37 5 2

-10

0

10

20

30

0 1 2 3 4 5

µ g Gallic Acid Eq/ml milk

% F

rem

ys r

adic

als

redu

ced

maras mushealthyk wash

29

Figure 3.4 When S6 excluded, the correlation within the total of 16 subjects is

loosing the significance (r = 0.5; p = 0.064) and also when only healthy are examined.

β-cryptoxanthine and α-tocopherol show also strong but positive correlation

with the phenolic content of the milk (r = 0.678 and r = 0.992) in that order,

significant at 0.01 level. These relationships has not being explained yet and may be a

point for further examination.

3.5.Total Antioxidant Capacity of the milk

Table5. shows the number of radicals which are reduced by 1L milk. One-

Way ANOVA with p>0.05 showed no significant difference between the mean

antioxidant capacity of the three groups.

Table 3.5: The total antioxidant capacity of milk-samples.

Samp samplesize

mean extr. range min/max Std.ErrMean

Std.Dev p

no of radicalsreduced by1L undilutedmilk

ksm

672total

2.6E+196.1E+184.5E+191.9E+19

-5.9E+18 9.2E+19-4.0E+19 1.6E+20-1.1E+19 1.0E+20-4.0E+19 1.6E+20

1.7E+192.6E+195.6E+192.5E+19

4.1E+196.9E+197.9E+195.7E+19

0.69

k- kwashiorkor h-healthy m- marasmus

Indeed, many of the samples appeared to have no anti-oxidant capacity at all,

compare to water whereas some had antioxidant potential. The individual data are

shown in Figure 3.5. Five of 15 examined subjects show antioxidant potential of

which three were from mothers of children with kwashiorkor. Within the control

30

groups, two subjects show AO potential, between who one is marasmic and the

healthy one is the outlier -S6.

Figure 3.5 . The total antioxidant capacity from each milk sample.

There are no control values for the total antioxidant capacity of human milk to be

compared. These results show that the milks in general do not comprise antioxidant

potential which means that not only the fat soluble antioxidants but also the low-

molecular weight - water soluble compounds and vitamin C, uric acid and sulphydryls

are missing.

-5E +19

0

5E +19

1E +20

1.5E +20

2E +20

m2 m3 s1 s2 s3 s4 s5 s6 s7 k1 k2 k3 k4 k5 k7

subjects

radi

cals

trap

ped

31

4. Discussion

There is evidence that the oedematous malnutrition is due to the free radical

damage to the tissues, not to the protein deficiency. If this is so then an adequate and

balanced intake of antioxidants should be protective. Many of the compounds, such

as vitamins E, C, and the carotenoids, coming mainly from fresh fruits and green

vegetables are strong antioxidants. The family of flavonoids contained in beverages,

mostly in green tea and red wine, also have antioxidant capacity.

Glutathione peroxidase is one of the selenoproteins dependent of enough Se in

the diet. This enzyme removes peroxides, mainly fatty acid hydroperoxides, formed

in the tissues through free radical action, by reduction to their corresponding hydroxy

acids (ROH). This reduction prevents the decomposition of the peroxide (ROOH) to

form alkoxy radicals that initiate further peroxidation. However, the most important

antioxidant reaction catalysed by this Se containing enzyme is probably reduction, and

hence safe disposal, of hydrogen peroxide. (L. Packer & J. Fucks,1992)

Young children and infants are at great risk of developing deficiency due to

their rapid growth.

Se is ingested by humans and animals primarily in the form of

selenomethionine and selenocysteine present in cereal proteins. International

estimated levels showed very different levels of Se in the breast milk; it varies

geographically, for example the levels in the US are 14-750µg/L, in New Zealand 13-

1870µg/L, and in Finland 7-1950 µg/L. There are endemic areas in DRC which are

low in Se (Vanderpas, 1994). This reflects to the low Se level in the soil, plants and

32

animals. We have measured the concentration of Se in the milk of breastfeeding

mothers in DRC to determine whether the levels of antioxidants, especially Se and

vitamin E, are low in the milk of mothers whose children develop kwashiorkor whilst

they are receiving breast milk, and have compared the values with healthy children.

We have found the Se level in the breast milk (8µg/l) to be lower than any

previously reported in the literature – the mean level is as low as the bottom of the

range found in countries that are generally regarded to be selenium deficient (New

Zealand and Finland), and in which there are national programs of selenium dressing

to farmland and crops to increase the selenium levels throughout the food chain. In

these metropolitan countries (New Zealand and Finland) the population generally have

access not only to a wide variety of foods but also to imported foods grown where

there is a higher selenium content of the foods. In these countries, before there was

agricultural use of selenium, there have been major outbreaks of clinical selenium

deficiency in many animal species, mainly livestock and poultry. Even in the USA

losses from selenium deficiency cost farmers many billions of dollars (for example,

turkey X-disease) before selenium supplements were permitted. In DRC the

population have a very restricted diet, almost exclusively of vegetable produce, all of

which is grown locally within a few miles of its consumption. This is similar to the

situation in grazing animals who necessarily consume vegetation grown locally.

Thus, it could be argued that the clinical expression of illness expected from selenium

deficiency in a country such as DRC is more closely related to veterinary disease, than

to human disease in low selenium, economically rich, areas where large quantities of

imported food are consumed. Thus, the levels of selenium in the breast-milk suggest

that the mothers themselves and all the suckling infants in Kisangani are likely to have

33

dietary selenium deficiency. Thus, the second hypothesis tested, that the levels in the

breast-milk in Kisangani are lower than international reference levels appears to be

correct.

However, we have found no difference between the breast-milk of mothers

whose children are apparently healthy and those that have oedematous or non-

oedematous malnutrition. Thus, the first hypothesis, that the infants who have

kwashiorkor are suckling milk that is lower in selenium than healthy infants is not

correct.

This raises the question of whether the free-radical hypothesis of kwashiorkor

is correct in general and, specifically, whether selenium deficiency is involved in this

disease. Golden’s hypothesis states that there is an imbalance between the production

of radicals and protection from oxidant stress. The idea is that the vulnerable

population has an insufficient intake of antioxidants in general; then whenever an

individual is exposed to a particularly severe “noxa”, such as measles or aflatoxin, that

individual will succumb to the oxidative stress rather than safely dissipating the

radicals. The results of the present study do not show that the children with

kwashiorkor were more vulnerable than the healthy children. On the other hand the

very low levels of selenium do suggest that all the children that I studied, including

the healthy ones are vulnerable to the development of kwashiorkor if they are exposed

to a sufficiently severe stress. Perhaps another way to examine the hypothesis would

be to measure the selenium levels of breast-milk from the indigenous populations in

areas of the world where kwashiorkor is prevalent and where it is uncommon or

unknown.

34

The selenium enzyme GPX was measured in this study and was linearly

correlated to the Se levels. Dodge (1998) examined GPX in human milk from three

different areas in China and found that the activity of the enzyme in mature milk

follows plasma selenium concentration and is a function of the Se intake. In this study

the early milk appeared to have the capacity to maintain GPX levels even when the

mothers Se intake was low. In line with the very low Se concentrations in all the

samples we examined there was not a relationship between the total antioxidant

capacity of the milk and GPX.

We did not find a significant relationship between the Se concentration and

vitamin E. These two antioxidants can compensate for each other. Indeed, small

differences in blood levels of selenium or glutathione peroxidase may not have

significance for human health when the level of vitamin E is adequate

(Packer&Fucks,1992). A combined deficiency is much more severe than a deficiency

of equal magnitude of either one alone. There is frequently a compensatory increase

in the concentration of one when there is a dietary deficiency of the other. Thus, in

the face of selenium deficiency one may have anticipated a compensatory rise in

vitamin E. This did not appear to be the case. Vitamin E was measured in the milk

and the mean concentration is not different than the international reference values (

2.3+ 1)mg/L.

We found strong correlation between α -tocopherol and the total antioxidant

capacity (TAC) of the milks of the healthy sample (r = 0.9), but there was not a

significant correlation within the kwashiorkor sample (r= 0.4). The significant

35

correlation is due to the very high concentration of this vitamin in only one of the

healthy samples (S6), when it is removed from the sample, the relationship is not

significant. This result must thus be viewed with great caution. When TAC of the

milk is considered, the same S6 sample, is only one of the 7 healthy who shows a

high antioxidant capacity. It is noteworthy that this subject, S6, was one of the local

staff of MSF and who was therefore not only already aware of the recommended

balanced diet, but also had access to additional food through her employment.

She is thus atypical of the normal population of Kisangani. However, this one

result, if confirmed, does show what could be achieved if the pregnant women of DRC

were given an adequate and balanced diet, similar to that given to children in

supplementary feeding programs. Thus, subject S6 should not be considered as part

of the normal lactating population of Kisangani from a nutritional point of view and

should be excluded from consideration, in which case the total antioxidant capacity of

all the women’s milk is extremely low and not related to vitamin E.

The total antioxidant capacity of every individual in the sample population

shows either very poor antioxidant activity or not at all. The exception of S6 shows

what a good diet could achieve.

There is a strong correlation between the catechines and total antioxidant

capacity of the milk, which suggests that they are the main antioxidant in the water

soluble phase of the human milk in DRC. Unfortunately, reference values for these

compounds have not been established.

36

The vitamin A concentration of mature human milk (as retinol equivalents) is

670 + 200 µg/L (Nutrition During Lactation, Table 6-1). Ninety-six percent of the

vitamin A content of the human milk is in the form of retinol esters. The concentration

of this vitamin in human milk decreases over the course of lactation from

approximately 2000 to 600µg/L. β- carotene is mostly a marker for the fruit and

vegetable in the diet. The values we measured for all these nutrients are low in

comparison to international standards. This is particularly evident for retinol. The

carotene/retinol ratio shows that nearly all the vitamin A equivalents are derived from

non-animal foods. This raises the question of why the mother had not converted more

of the pro-vitamin A that she was consuming into vitamin A itself, for secretion into

her breast-milk. This conversion is dependent upon zinc status, which was not

measured in the mothers; however, it does raise the possibility that with multiple

deficiency states, it may be unsafe to assume that beta-carotene is converted to

vitamin A with sufficient efficiency. The conversion of pro-vitamin A to retinol in the

young breast fed infant, of a type I nutrient deficient mother, has not been

investigated. However, when mother is chronically deficient in vitamins in her diet

the milk composition is affected and presumably the infant will suffer. The water

soluble vitamins are more responsive to the change of the diet.

The results of this study, emphasise the importance of the type I, type II

classification of nutrients. In particular, measurement of the anthropometry of the

mother (and several of the mothers in this study appeared to be obese) and finding that

they are not underweight, does not inform us about their type I nutrient status. Thus,

the infants of anthropometrically “well nourished” mothers may well develop serious

type I nutrient deficiency (in this study, selenium and vitamin A) without any health

37

worker being aware of the problem. This is compounded by the fact that the clinical

expression of major type I nutrient deficiency is often quite different in the young

infant than at other ages. For example, thiamine deficiency presents as a meningo-

enchaphalitis or aphonia in the infant but as classical beri-beri in older age groups.

The diet of the observed sample is poor in animal food. Meat, eggs, milk are

almost not excluded or consumed for festival times. Cereals, fruits and vegetables are

expensive and not available ordinary food. The main diet is cassava; both the green

leaves and the tuber (usually prepared as flour) are consumed. Banana is also

frequently eaten and is a highly appreciated food. Rice and maize are rarely

consumed and are very expensive. Red palm oil is the main source of fat. The

dietary information shows a very poor or no consumption of fresh vegetables and

fruits. There is a general awareness among the population about the health benefits

from the use of these foods, but they are in short supply and expensive.

The sample was relatively homogeneous in terms of gestational age, maternal

age and birth weight. Unfortunately, we could neither measure the milk volume

accurately nor the breast-milk intake of the infants. It is uncertain how much the

infants consumed, nevertheless, they had at least 3 feeds per day of breast milk and

there was a free flow from each of the mothers. The study had been planned for many

more mother’s to participate. With the need to evacuate the project area after taking

only 16 subjects, the study in under-powered statistically.

The question arises as to the reliability of the results in terms of deterioration

of the samples between collection and analysis, and whether this could account for the

38

very low levels of retinol, carotenoids, TAC and other labile organic constituents. The

samples were quickly frozen after collection and did not de-frost with the exception of

during transport from DRC to Aberdeen. They had a potent antioxidant added to them

shortly after collection. Nevertheless, if there had been a major deterioration in the

samples then the vitamin E levels would perhaps be much lower. The selenium

content, which is not labile, was exceptionally low and related to the GPX activity.

This correlation between Se and GPX indicates that the enzymatic activity had not

deteriorated during storage and transport. Nevertheless, deterioration is a potential

source of error and could account for the very low TAC.

The analytical methods used were in routine use for other projects. The

coefficients of variation of duplicate samples varied from 4.2% for selenium to 0.5%

for the ESR. Unfortunately there do not seem to be comparable data for the TAC or

phenolic compounds in human milk, or published methods for estimation of these

quantities in milk samples. We therefore adapted the methods used in plasma. If the

methods for plasma are not appropriate for breast-milk then these values will be in

error.

This study was small. Nevertheless, the results were relatively homogeneous

(with the exception of S6, for which there is a plausible explanation) with a variance

that is as low or lower than that found in other series. For this reason, I suggest that

these samples are indeed representative of the women of Kissangani. Kissangani

itself lies in the heart of DRC in the middle of the Congo river catchment area,

surrounded by tropical jungle. There is a high rainfall, and the soil is laden with iron

(red), factors which will lower the available selenium content of the soil into the food-

39

chain. It is likely that similar results would be obtained from geographically similar

areas where the population is dependent upon locally grown produce. The results

cannot be extrapolated to other areas of Africa or to areas with different geochemistry,

soil characteristics, staple foods or climate.

The implications of this study are that maternal nutrition does affect the type I

nutrient content of breast milk, and through this mechanism the health of the suckling

infant. Although I found no evidence of a difference in the mother’s breast-milk

composition of the infants that had kwashiorkor or those who were seemingly healthy,

I did find that all the infants were consuming diets that were potentially deficient. I

suggest that if we are to lower infant mortality and improve infant growth and health

then every lactating mother should have an adequate supply of antioxidant and other

type I nutrients in her diet.

Conclusion

The antioxidant status of the breast milk in mothers from DRC is low,

compared to the international standard concentrations and there is no difference

between the kwashiorkor samples and healthy or marasmic controls. Se status in the

milk from the same mothers was particularly low. Vitamin E concentration of the milk

from the three groups was not different and agreed with the reference mean

concentration.

40

references:

Butte NF, CallowayDH at all. Evaluation of lactational Performance of Navajo women. Am J

Clin Nutr. (1981)34:2210-2215.

Dodge RC at all. Glutathione peroxidase activity modulates fatty acid profiles of plasma and

breast-milk in Chinese women. Trac El Med Biol (1998); 12:221-230

Gardner et al.(1998), Electron Spin Resonans Spectroscopic (ESR)Assesment of the

Antioxidant Potential of Teas in Aqueous and Organic Media, J. Sci. Food Agric., 76(2), 257-

262.

Geigy Scientific Tables 1.Composition of the Body Nutrition. Breast Milk. Sigma Chemical

Co. Ltd. Dorset England & Deisenhofen, W.Germany.

Golden MH, Ramdath D. Free Radicals in pathogenesis of kwashiorkor,Proc Nutr Soc (1987);

46:53-68

Golden MH. The nature of nutritional deficiency in relation to grouth failure and poverty.

Acta Pediatr Scand (1991);374:95-110.

Golden MH. Oedematous Malnutrition. Brit Med Bull (1998);54(2):433-444

Golden MH. Severe malnutrition . In: Weatherall DJ, LedingtonJGG, Warrell DA, eds.Oxford

Textbook of Medicine, 3rd ed. Oxford: Oxford University Press, 1996: 1996:1278-1296.

Hess D.et al, Simultaneous Determination of Retinol, Tocopherols, Carotens and Lycopene in

Plasma by Means of High Performance Liquid Chromatography on Reverse Phase, Internat.

J.Vit.Nutr.Res., (1991) 61,232-238.

Kobayashi H, Kanno C, Yamauchi K, Tsugo T. Identification of alfa-, beta-, gama-,and delta-

tocopherols and their contents in human milk. Biochim Biophys Acta1975 20; 380(2):282-90.

Nutrition During Lactation.. Ch" Milk Composition". National Academy Press,

Washington,D.C. 1991

41

Packer L, Fuchs J. Vitamin E in Health and Disease: Vitamin E interactions with Selenium.

J.(1992) New York

Olsen et al. (1975) J. of Assoc. of Analytical Chemists 58(1) 117 - 121. With modifications

suggested by Michael, S and White, C.L. Analytical Chem. (1975) 48 1484 - 1486.

Serafini M. et al, Alcohol-Free Red Wine Enhances Plasma Antioxidant Capacity in Humans,

J.Nutr (1998).,128 1003-1007.

42

Annexe 1

Measurement of the Se and Glutathione Peroxidase

Measurements of the carotenoids and tocopherol

Se.read 1 Se.read 2 Se.ng/ml1 Se.ng/ml2 Se.ng/ml mean U/ml gpx mean

K1 5.8 9.9 4.799 8.839 6.819 0.018K2 10.8 9.4 9.726 12.750 11.238 0.022K3 16.1 17 14.948 15.835 15.392 0.070K4 7.2 7.7 6.178 6.671 6.424 0.005K5 8.6 8.2 7.558 7.164 7.361 -0.001K6 5.6 4.601 4.601 0.243K7 9.1 10.4 8.050 9.331 8.691 0.022S1 7.5 9.4 6.474 8.346 7.410 0.002S2 10.2 9.6 9.134 8.543 8.839 0.031S3 7 5 5.981 4.010 4.996 0.005S4 8.4 9.3 7.361 8.248 7.804 0.005S5 9.1 8.1 8.050 7.065 7.558 0.031S6 12.2 11.3 11.105 10.218 10.662 0.060S7 8.8 12.1 7.755 11.007 9.381 0.047M2 7.5 6.2 6.474 5.193 5.833 0.018M3 13.2 9.7 12.091 8.642 10.366 0.025

b-caroten a-caroten retinol Lycopen b-crypt lut/zea a-tocophe g-tocophe

0.08 0.038 0.024 0 0.004 0.2 0 0.0450.1 0.049 0.016 0 0.001 0.088 2.107 0.055

0.167 0.075 0.018 0.001 0.003 0.08 2.705 0.0810.138 0.05 0.013 0 0.014 0.118 4.122 0.1080.061 0.02 0.011 0.007 0.006 0.167 2.804 0.2360.124 0.063 0 0 0.007 0.209 2.356 0.4930.076 0.025 0 0 0.001 0.081 2.828 0.1110.107 0.055 0.013 0 0.002 0.058 2.313 0.3220.115 0.05 0.011 0 0.004 0.076 3.377 0.2490.144 0.058 0 0 0.002 0.051 1.581 0.1960.085 0.048 0.006 0.005 0.008 0.097 2.505 0.1550.079 0.033 0.016 0.005 0.005 0.114 0.611 0.1590.141 0.084 0 0.009 0.017 0.324 8.558 0.449

0.05 0.031 0.01 0 0.01 0.033 2.408 0.420.107 0.065 0 0.003 0.003 0.105 2.483 0.0430.081 0.057 0 0 0.001 0.027 3.285 0.123

43

Annexe 2

Measurement of the free radicals of the milk by ESR

mean%red of radical No. of radicals reduced by oneA B MEAN sollution litre of undiluted milk

control 0 1.717 1.696 1.6735 0control1 1.641 1.64

m2 1.719 1.691 1.705 -1.882 -1.13351E+19m3 1.426 1.361 1.3935 16.731 1.00756E+20

s1 1.722 1.71 1.716 -2.540 -1.52934E+19s2 1.712 1.708 1.71 -2.181 -1.31343E+19s3 1.706 1.676 1.691 -1.046 -6.29728E+18s4 1.781 1.708 1.7445 -4.243 -2.5549E+19s5 1.896 1.672 1.784 -6.603 -3.97628E+19s6 1.221 1.233 1.227 26.681 1.60671E+20s7 1.746 1.7 1.723 -2.958 -1.78123E+19

k1 1.661 1.678 1.6695 0.239 1.43938E+18k2 1.686 1.692 1.689 -0.926 -5.57759E+18k3 1.53 1.491 1.5105 9.740 5.86547E+19k4 1.407 1.428 1.4175 15.297 9.21202E+19k5 1.697 1.683 1.69 -0.986 -5.93744E+18k7 1.631 1.634 1.6325 2.450 1.47536E+19

44

Annexe 3

Measurements of the phenolic compound of the milk

µgGAE/mlmilk/mean

GAE/milk/1

GAE/milk/2

K1 0.0225 3.174617 2.991695K2 1.081 0.88026 1.340176K3 1.393 1.538777 1.643303K4 2.115 1.016144 1.779188K5 1.52 1.507419 1.413345K6 1.4245 1.204292 1.246103K7 1.4695 1.852356 1.726925S1 1.3175 3.280596 2.883135S2 1.813 2.0957 2.136881S3 0.8885 2.376481 3.012918S4 1.33 4.693984 5.465489S5 0.385 3.174617 3.514328S6 4.5035 6.022964 7.14289S7 1.414 5.709384 3.932434M2 1.263 10.03744 2.720906M3 1.704 4.637988 4.402803