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Vitamin B12 and Folate Deficiency:
Prevalence, Clinical Correlates and Outcome in
Chronic Heart Failure
Title: Vitamin B12 and folate deficiency: prevalence, clinical correlates and outcome in
chronic Heart Failure
Name student: Haye H. van der Wal
Student number: s2031639
Name faculty supervisor: P. van der Meer, MD, PhD
Name daily supervisor: I.T. Klip, MD
Research institution: University Medical Center Groningen (UMCG), Groningen, The
Netherlands
Research department: Department of Cardiology
2
Abstract (English) INTRODUCTION - Chronic Heart Failure (HF) is a clinical syndrome which is charac-
terized by cardiac dysfunction and/or abnormalities in cardiac structures. Although treat-
ment possibilities have improved over the past decades, chronic HF is still characterized
by a poor clinical status and a five-year survival rate of 50%. Several comorbidities may
have a negative influence on prognosis. Iron deficiency, for example, is common in
chronic HF and associated with impaired exercise capacity and worse clinical outcome.
Other nutritional deficiencies (vitamin B12 and folate deficiency) may also negatively im-
pact functional status and prognosis in patients with chronic HF. Unfortunately, studies
on hematinic deficiencies in chronic HF are scarce, and the clinical correlates of these
deficiencies and their influence on outcome is currently unknown.
METHODS – In an international pooled study cohort comprising 610 patients with stable
chronic HF, we studied the prevalence of vitamin B12 and folate deficiency, their clinical
correlates, and the prognostic value. The main outcome of both deficiencies was all-cause
mortality.
RESULTS – Mean age (± standard deviation) of the patients was 68 ± 12 years, mean left
ventricular ejection fraction was 33 ± 13%, and median serum NT-proBNP level was
1801 pg/mL (interquartile range 705 – 4335 pg/mL). Vitamin B12 deficiency (serum level
<200 pg/mL), folate deficiency (serum level <4.0 ng/mL), and iron deficiency (serum fer-
ritin <100 µg/L, or 100 – 299 µg/L with a transferrin saturation <20%) were present in
5%, 4%, and 58% of all patients, respectively. Serum vitamin B12 levels were positively
associated with higher serum NT-proBNP and ferritin levels (p <0.001), whereas clinical
associates of folic acid included systolic blood pressure, hemoglobin, transferrin satura-
tion, renal function, and atrial fibrillation (all p<0.05). No significant correlation between
mean corpuscular volume (MCV) and serum vitamin B12, folic acid or ferritin levels was
observed. Lower serum folic acid levels were associated with an impaired quality of life
(p = 0.003). During a median follow-up of 2.10 years (interquartile range 1.31 – 3.60
years) 254 patients (42%) died. In multivariate Cox proportional hazard regression mod-
els, serum vitamin B12 and folic acid levels were not significantly associated with all-
cause mortality.
CONCLUSIONS – Vitamin B12 and folate deficiency are relatively rare in patients with
chronic HF, in contrast to iron deficiency. No significant correlation was found between
MCV and serum levels of vitamin B12 and folic acid. MCV may be an unreliable screen-
ing marker to differentiate between possible etiologies of anemia in chronic HF. In con-
trast to iron deficiency, folic acid and vitamin B12 were not related to prognosis.
I
3
Abstract (Dutch) INTRODUCTIE – Chronisch hartfalen is een klinisch syndroom dat gekenmerkt wordt
door dysfunctie van het hart en/of afwijkingen aan de hartspier. Hoewel de behandeling
in de afgelopen decennia is verbeterd, is de prognose van chronisch hartfalen nog steeds
relatief slecht (50% van de patiënten overlijdt binnen vijf jaar). Verschillende comorbidi-
teiten kunnen een negatieve invloed hebben op de prognose van hartfalen. IJzerdeficiën-
tie komt bijvoorbeeld veel voor bij hartfalen en is geassocieerd met een slechter inspan-
ningsvermogen en een slechtere prognose. Andere voedingstekorten zoals vitamine-B12-
en foliumzuurdeficiëntie hebben mogelijk ook een negatieve invloed op het functioneren
en de prognose van patiënten met chronisch hartfalen. Helaas is er nauwelijks onderzoek
gedaan naar deze deficiënties in chronisch hartfalen, evenmin naar de relatie tussen deze
deficiënties en klinische parameters en naar het effect van deze deficiënties op de overle-
ving.
MATERIAAL EN METHODE – In een internationaal studiecohort van 610 patiënten
met stabiel chronisch hartfalen is gekeken naar de prevalentie van vitamine-B12- en foli-
umzuurdeficiëntie, hun klinische parameters, en de prognostische waarde van deze defi-
ciënties. De belangrijkste uitkomstmaat voor beide deficiënties was mortaliteit.
RESULTATEN – De gemiddelde leeftijd van alle patiënten (± standaarddeviatie) was 68
± 12 jaar, de gemiddelde linkerventrikelejectiefractie was 33 ± 13% en de mediane serum
NT-proBNP-spiegel 1801 pg/mL (interkwartielafstand 705 – 4335 pg/mL). Vitamine-
B12-deficiëntie (serumspiegel <200 pg/mL), foliumzuurdeficiëntie (serumspiegel <4,0
ng/mL) en ijzerdeficiëntie (serumspiegel ferritine <100 µg/L of 100 – 299 µg/L met een
transferrinesaturatie <20%) waren aanwezig in respectievelijk 5%, 4% en 58% van alle
patiënten. Serum vitamine-B12-spiegels waren positief geassocieerd met hogere serum
NT-proBNP- en ferritinespiegels (p <0,001). Serum foliumzuurspiegels waren onder an-
dere geassocieerd met systolische bloeddruk, hemoglobine, transferrinesaturatie, nier-
functie en atriumfibrilleren (P <0,05). Er werd geen significante correlatie tussen het
mean corpuscular volume (MCV) en de serum vitamine-B12- en foliumzuurspiegels. Lage
serum foliumzuurspiegels waren geassocieerd met een slechtere kwaliteit van leven (p =
0,003). Na een mediane follow-up van 2.10 jaar (interkwartielafstand 1.31 – 3.60 jaar)
waren 254 patiënten (42%) overleden. In de multivariabele Cox regressieanalyse waren
serum vitamine-B12- en foliumzuurspiegels niet significant geassocieerd met mortaliteit.
CONCLUSIES – Vitamine-B12- en foliumzuurdeficiëntie zijn relatief zeldzaam in patiën-
ten met chronisch hartfalen, in tegenstelling tot ijzerdeficiëntie. Er werd geen significante
correlatie gevonden tussen MCV en serum vitamine-B12- en foliumzuurspiegels. MCV is
derhalve mogelijk een onbetrouwbaar screeningsmiddel om te differentiëren tussen de
verschillende oorzaken van anemie in patiënten met chronisch hartfalen. In tegenstelling
tot ijzerdeficiëntie zijn vitamine-B12- en foliumzuurdeficiëntie niet gerelateerd aan prog-
nose.
II
4
Table of contents
List of abbreviations ........................................................................................................... 6
Introduction ........................................................................................................................ 7
1.1 Chronic heart failure ...........................................................................................................7 1.1.1 Definition ......................................................................................................................................7 1.1.2 Epidemiology ................................................................................................................................7 1.1.3 Etiology .........................................................................................................................................7 1.1.4 Pathophysiology ............................................................................................................................7 1.1.5 Clinical presentation .....................................................................................................................8 1.1.6 Diagnostics ....................................................................................................................................8 1.1.7 Treatment ......................................................................................................................................8
1.2 Hematinics in chronic heart failure ....................................................................................8 1.2.2 Vitamin B12 ...................................................................................................................................9 1.2.3 Folic acid .......................................................................................................................................9 1.2.4 Vitamin B12 and folic acid in chronic heart failure ..................................................................... 10
Objectives .......................................................................................................................... 11
Materials and Methods .................................................................................................... 12
3.1 Study population ................................................................................................................12
3.2 Study procedures ................................................................................................................12 3.2.1 Baseline characteristics ............................................................................................................... 12 3.2.2 Laboratory measurements ........................................................................................................... 12 3.2.3 Definitions................................................................................................................................... 13
3.3 Statistical analysis ..............................................................................................................13 3.3.1 Descriptive statistics ................................................................................................................... 13 3.3.2 Linear regression models ............................................................................................................ 14 3.3.3 Survival analysis ......................................................................................................................... 14
Results ............................................................................................................................... 15
4.1 Baseline characteristics ......................................................................................................15
4.2 Hematinics ..........................................................................................................................15
4.3 Clinical correlates of vitamin B12, folic acid, and MCV ..................................................15
4.4 Hematinic deficiencies and mean corpuscular volume ...................................................18
4.5 Hematinics and survival ....................................................................................................19 4.5.1 Kaplan-Meier analysis ................................................................................................................ 19 4.5.2 Cox proportional hazard regression analysis............................................................................... 20
Discussion ......................................................................................................................... 21
5.1 Prevalence and definition of hematinic deficiencies ........................................................21 5.1.1 Vitamin B12 ................................................................................................................................. 21 5.1.2 Folic acid ..................................................................................................................................... 22
5.2 Vitamin B12, folic acid, and mean corpuscular volume ...................................................22
5.3 Hematinics and quality of life ...........................................................................................22
5.4 Hematinics and survival ....................................................................................................23
5.5 Study limitations.................................................................................................................23
III
5
Conclusion ........................................................................................................................ 24 Acknowledgements .............................................................................................................................. 24
Appendices ........................................................................................................................ 25
References ........................................................................................................................ 27
6
List of abbreviations eGFR estimated glomerular filtration rate
HF heart failure
hs-CRP high-sensitivity C-reactive protein
LVEF left ventricular ejection fraction
MCV mean corpuscular volume
MLHFQ Minnesota Living with Heart Failure Questionnaire
NT-proBNP N-terminal prohormone of brain natriuretic peptide
NYHA New York Health Association
TSAT transferrin saturation
New York Heart Association (NYHA) functional classification of heart failure1
Class I No limitation of physical activity. No symptoms of heart failure (e.g. dysp-
nea, fatigue, angina, or palpitations) during normal physical activity
Class II Slight limitation of physical activity. No symptoms of heart failure at rest,
but symptoms during normal physical activity
Class III Marked limitation of physical activity. No symptoms of heart failure at
rest, but symptoms during slight physical activity
Class IV Symptoms of heart failure, even at rest. Symptoms worsening during phys-
ical activity
IV
7
Introduction
1.1 Chronic heart failure
1.1.1 Definition
Traditionally, heart failure is defined as an impaired cardiac function, which leads to in-
sufficient perfusion of body tissue. In other words: the heart is not able to meet the meta-
bolic requirements of the tissues. Heart failure can be considered as a clinical syndrome,
characterized by cardiac dysfunction and/or abnormalities in cardiac structures. This will
lead to typical symptoms and signs of heart failure. The current definition emphasizes the
clinical consequences of heart failure.1
The clinical syndrome of chronic HF is divided into two different types, based on
the left ventricular ejection fraction (LVEF). In general, when LVEF is ≤45%, patients
have HF with reduced Ejection Fraction (HF-rEF). Patients with HF-pEF are character-
ized by a LVEF >45%. This threshold for LVEF is slightly arbitrary, as there is currently
no real consensus on the exact definition of HF-rEF and HF-pEF.
1.1.2 Epidemiology
The prevalence of chronic HF in western countries is approximately 1-2% .2 Chronic HF
is relatively rare in people younger than 50 years, but the prevalence increases gradually
with age (>10% in people aged 70 and older 2). The incidence of HF is generally 5-10 per
1000 persons per year. For developing countries, data on prevalence and incidence are
lacking.3
There are epidemiological differences between HF-rEF and HF-pEF.4,5
Most pa-
tients with HF-pEF are relatively old and more frequently female. HF-rEF is character-
ized by a slight worse prognosis, mainly due to the relatively high prevalence of comor-
bidities.6
1.1.3 Etiology
Chronic HF has many different causes, with no clear classification. Main causes of chron-
ic HF are ischemic heart disease (35-40%), dilated cardiomyopathy (30-34%), and hyper-
tension (15-20%). In western countries, most cases of chronic HF are caused by ischemic
heart disease. The major cause of HF-rEF is coronary artery disease, whereas HF-pEF is
characterized by the presence of many comorbidities like atrial fibrillation, obesity, hy-
pertension, and diabetes.4,5
1.1.4 Pathophysiology
The pathophysiological mechanisms of HF-rEF are best understood, in contrast to HF-
pEF. Most cases of HF-rEF are characterized by myocardial damage (e.g. myocardial in-
farction or overload), followed by pathological remodeling. This will lead to an impaired
left ventricular function, by means of dilation of the left ventricle and/or impaired con-
tractility.7 This process of remodeling and deterioration of left ventricular function is pro-
gressive, due to additional damage to the cardiac muscle and the activation of neurohu-
moral systems (i.e. rennin-angiotensin-aldosterone system and the sympathetic nervous
system).8
1
8
1.1.5 Clinical presentation
Most symptoms and signs of chronic HF are non-specific, and therefore, the discrimina-
tion between chronic HF and other diseases can be challenging. The most typical symp-
toms of chronic HF are dyspnea (in rest and/or on exertion), orthopnea (dyspnea while
lying flat), fatigue, leg swelling, and reduced exercise tolerance. Major signs of chronic
HF include elevated central venous pressure, heart murmurs and/or third heart sound on
auscultation, hepatojugular reflux (sometimes leading to hepatomegaly), and peripheral
edema and/or pulmonary crepitations (especially in decompensated patients).9-12
The in-
terpretation of the symptoms of chronic HF is especially challenging in older patients and
when pulmonary comorbidities are present.13,14
1.1.6 Diagnostics
According to European Society of Cardiology (ESC) guidelines, the diagnosis of heart
failure can be made based on the presence of symptoms and/or signs of heart failure (at
rest or during physical activity) and objective evidence of heart failure (e.g. by means of
echocardiography). Both criteria are obligatory for the diagnosis of chronic HF. The di-
agnosis of chronic HF can be supported by a response to treatment directed towards HF.
The diagnosis of HF-pEF is more challenging than the diagnose of HF-rEF, as HF-pEF
can be considered as a diagnosis of exclusion.
1.1.7 Treatment
The treatment of chronic HF is mainly based on the deactivation of the renin-angiotensin-
aldosterone system and the sympathetic nervous system.15
Main treatment goals are re-
lieving symptoms, preventing and/or shortening of hospitalization, and improving prog-
nosis. Almost all patients with chronic HF are treated pharmacologically with an angio-
tensin-converting enzyme inhibitor (or angiotensin receptor blocker), and/or a beta block-
er, and/or a mineralcorticoid receptor antagonist. In cases of congestion (peripheral
and/or pulmonary edema, elevated central venous pressure), patients can be treated with
diuretics. Medication is up-titrated to doses which relieve symptoms and signs effective-
ly, as long as there are no unacceptable side-effects. In some cases, implantation of de-
vices (e.g. pacemakers, implantable cardioverter-defibrillators) is needed (mainly when
arrhythmias are present).
1.2 Hematinics in chronic heart failure
Despite optimal treatment regimens, as described earlier, chronic HF is still characterized
by a poor clinical status and very high mortality rates with a five-year survival of approx-
imately 50%.16-20
Several mechanisms may underlie the progression of chronic HF. Im-
portant comorbidities regarding disease progression are anemia and an impaired iron sta-
tus (i.e. absolute or functional iron deficiency).21-23
Iron deficiency is common in chronic
HF, affecting roughly 50% of the patients, and it is associated with impaired exercise ca-
pacity and worse clinical outcome, even in the absence of anemia.24-26
Patients with
chronic HF are more prone to develop iron deficiency, due to depleted iron stores (abso-
lute iron deficiency)27
or reduced iron availability caused by chronic inflammation (func-
tional iron deficiency), which is relatively common in chronic HF.28-30
Several studies
have shown beneficial effects in treatment of iron deficiency by administering intrave-
nous iron, resulting in an improved clinical status and quality of life.31-33
9
Besides iron deficiency, the role of other hematinic deficiencies in chronic HF is current-
ly unknown and data are scarce. Both vitamin B12 and folic acid are essential for normal
erythrocyte production and deficiencies of these vitamins may lead to anemia. However,
hematological parameters (i.e. hemoglobin, mean corpuscular volume (MCV), hemato-
crit, red blood cell distribution width, and reticulocyte count) may be completely normal
in patients with these deficiencies.34
1.2.2 Vitamin B12
Vitamin B12 is a water-soluble vitamin, which plays a significant role in the erythropoie-
sis (i.e. production of red blood cells in the bone marrow) and normal functioning of the
central and peripheral nervous system. Vitamin B12 affects DNA synthesis in virtually all
human body cells. Just like folic acid, vitamin B12 cannot be synthesized by the human
body. The only natural sources of vitamin B12 are animal-derived food products (meat,
dairy products, fish). The minimal recommended daily intake of vitamin B12 is 6-9 µg.35
Dietary vitamin B12 binds to gastric intrinsic factor in the duodenum and is absorbed in
the ileum by means of specialized receptors in the intestinal wall. The total body store of
vitamin B12 comprises 2-5 mg; most vitamin B12 is stored in the liver. As a consequence,
the development of vitamin B12 deficiency may take a few years.35
In general, vitamin B12 deficiency may develop in cases of inadequate dietary in-
take (e.g. strict vegetarians), decreased or absent production of intrinsic factor (e.g. after
gastrectomy, or in pernicious anemia, which is caused by an autoimmune reaction against
gastric parietal cells and autoantibodies against intrinsic factor34
), ileal disorders (e.g.
Crohn’s disease, or significant resection), or as side effect of certain drugs (e.g. metfor-
min36
, proton pump inhibitors37
).
Apart from the typical neurological changes which are characteristic for severe
vitamin B12 deficiency (e.g. progressive symmetrical neuropathy, especially in the legs),
this deficiency also affects erythropoiesis, especially because of the rapid turnover of
erythrocytes. Vitamin B12 deficiency can lead to macrocytic anemia, due to desynchro-
nized maturation of red cell nuclei and cytoplasm. This leads to intramedullary hemolysis
of the erythroid precursors, which mimics the characteristics of microangiopathic hemo-
lytic anemia.38
1.2.3 Folic acid
Folic acid is a water-soluble vitamin, which is mainly present in animal-derived food
products and leaf vegetables. Average daily folic acid requirements are 200-400 µg. The
uptake of folic acid is not dependent on the presence of intrinsic factor, hence the occur-
rence of folate deficiency is mainly dependent on an inadequate intake of folic acid. In
contrast to vitamin B12, body stores of folic acid are limited (5-10 mg) with respect to dai-
ly requirements. Therefore, folate deficiency may develop within months of inadequate
dietary intake.39
Main causes of folate deficiency are malnutrition (e.g. due to overcooking food,
which destroys folic acid, in the elderly40
, and when alcohol abuse is present39
), malab-
sorption, and the use of certain drugs (e.g. folic acid antagonists, like methotrexate41
).
Folic acid has an obligatory role in DNA synthesis, erythropoiesis and the for-
mation of hemoglobin (together with vitamin B12). Folate deficiency leads to a delay in
DNA synthesis, and this limits erythropoiesis (and the maturation of other hematopoietic
10
cell lines). The hematological consequences of folate deficiency are analogous to the ef-
fects of vitamin B12 deficiency: megaloblastic anemia.
The clinical presentation of folate deficiency differs from the characteristics of
vitamin B12 deficiency. Most symptoms are due to the megaloblastic anemia caused by
folate deficiency; neurological symptoms as seen in vitamin B12 deficiency are very rare
and are only present in severe and prolonged folate deficiency42,43
.
1.2.4 Vitamin B12 and folic acid in chronic heart failure
To date, studies on hematinics deficiencies in patients with chronic HF are scarce, and
clinical correlates and the predictive value of these deficiencies on clinical outcome in
chronic HF is currently unknown. One study from the UK focused on the prevalence of
hematinic deficiencies in chronic HF patients. Vitamin B12 and folate deficiency were
present in 6% and 8% of the subjects, respectively.44
Cutoff values for vitamin B12 and
folate deficiency were serum vitamin B12 <180 ng/L and red cell folate <147 μg/L. How-
ever, in this relatively small study including only NYHA class IV patients, clinical corre-
lates and outcome data were lacking.
11
Objectives In this study, we measured serum vitamin B12 and folic acid in a broad and representative
range of chronic HF patients and studied the prevalence and clinical consequences of
these deficiencies. Finally, the prognostic value of serum vitamin B12 and folic acid levels
were assessed.
2
12
Materials and Methods
3.1 Study population
The study population consisted of 610 patients, originating from an international pooled
study cohort of 1,506 patients with stable chronic HF with preserved or reduced LVEF.
Reduced ejection fraction was defined as an LVEF ≤45%, in agreement with the guide-
lines of the European Society of Cardiology.1 For the present study, serum vitamin B12
and folic acid levels were measured in 610 randomly selected patients. Inclusion and ex-
clusion criteria of each study cohort (Poland, Spain, and The Netherlands) are displayed
in Supplementary Table 1.
The present study comprises three study cohorts from Poland, Spain, and The
Netherlands. All patients in this study were suffering from stable chronic HF. From the
Spanish cohort, 256 patients with preserved or reduced ejection fraction were included.45
The Polish cohort comprised 152 patients with stable chronic HF-rEF.25,46
Finally, we
included 202 patients with stable chronic HF-rEF or HF-pEF.47
All study cohorts were
analogous with respect to demographics and clinical signs, laboratory values, treatment,
and follow-up.
The study protocols were assessed and approved by the medical ethical committee
of each institution, and all patients gave written informed consent for participation. The
study was conducted in accordance with the Declaration of Helsinki.
3.2 Study procedures
3.2.1 Baseline characteristics
For all patients in this study, the following information was available: demographics and
clinical signs, including age at diagnosis, sex, etiology of heart failure (ischemic or oth-
er), renal function (eGFR), ejection fraction (LVEF, assessed by means of echocardiog-
raphy or radionuclide ventriculography), blood pressure, and co-morbidities (i.e. atrial
fibrillation, hypertension, and diabetes); hematinics, including hemoglobin, MCV, serum
iron, ferritin, transferrin, vitamin B12, and folic acid); (cardiac) biomarkers, including se-
rum NT-proBNP and hs-CRP; treatment with angiotensin-converting enzyme (ACE) in-
hibitor and/or angiotensin receptor blocker (ARB), beta blocker, aldosterone antagonist,
statins, loop diuretics, and antiplatelet and/or anticoagulant.
3.2.2 Laboratory measurements
Laboratory tests (including hematinics) were performed using venous blood samples.
Blood samples were centrifuged at 3,500 rpm for 15 minutes (4 °C) and were stored at
-70 °C afterwards. Routine blood analysis consisted of hemoglobin, hematocrit, sodium,
potassium, creatinine, eGFR, ureum, AST, ALT, GGT, alkalic phosphatase, bilirubin (to-
tal and direct), LDH, albumin, triglycerides, cholesterol, HDL, LDL, glucose, HbA1c,
NT-proBNP, leukocyte count, trombocyte count, and CRP. The following blood markers
reflecting hematinics (vitamin B12, folic acid, and iron metabolism) were assessed using
standard methods: serum concentrations of vitamin B12 (pg/mL), folic acid (ng/mL), iron
(μg/dL), ferritin (μg/L), and transferrin (mg/dL). To calculate the transferrin saturation
3
13
(TSAT), which is a measure of absolute iron deficiency, serum iron was divided by 25.2
and then divided by serum transferrin. This value was then multiplied by 100.48
Renal function was assessed using the estimated glomerular filtration rate (eGFR,
mL/min/1.73 m2). This was calculated using the simplified Modification of Diet in Renal
Disease (MDRD) equation.49
Serum NT-proBNP levels were determined using an immu-
noassay based on electrochemoluminescence (Elecsys, Roche Diagnostics, Basel,
Switserland). Serum concentrations of hs-CRP and other blood markers were assessed
using standard methods. Unfortunately, serum hs-CRP levels were not available in the
Spanish cohort (only serum CRP levels were measured ).
MCV levels for the Dutch cohort were initially not available, but were extracted
from the electronic patients files in the Deventer Hospital (Deventer, The Netherlands).
3.2.3 Definitions
Hematinic deficiencies (vitamin B12 and folate deficiency) were defined as follows: se-
rum vitamin B12 <200 pg/mL35,38,50
, serum folic acid <4.0 ng/mL.39,50
For iron deficiency,
the following definition was used: serum ferritin <100 μg/L or serum ferritin 100-299
μg/L with a TSAT <20%.25,31,32,46,51-55
This definition reflects both absolute (i.e. depleted
iron stores in bone marrow, liver, and spleen, which is characterized by a low serum ferri-
tin) and functional (i.e. inadequate availability of iron for erythropoiesis, in combination
with normal or even increased iron stores, which is characterized by low serum iron lev-
els and/or a low TSAT) iron deficiency.21
Anemia was defined as a hemoglobin <12 g/dL
for women and <13 g/dL for men, in agreement with WHO standards.56
Finally, quality
of life was assessed by means of the Minnesota Living with Heart Failure Question-
naire.57
This questionnaire was developed to assess the quality of life specifically for pa-
tients with chronic HF, as it focusses especially to symptoms and signs which are charac-
teristic for chronic HF (i.e. dyspnea, orthopnea, angina, fatigue, and palpitations). A
higher MLHFQ score signifies a worse quality of life.
3.3 Statistical analysis
3.3.1 Descriptive statistics
Data are presented as means and standard deviation when normally distributed, as medi-
ans and interquartile range when non-normally distributed, or as frequencies and percent-
ages for categorical variables. Intergroup differences between baseline variables were
tested using parametric (i.e. linear contrast analysis) and nonparametric (i.e. extension of
Wilcoxon rank-sum test58
) trend tests, the one-way analysis of variance test, or Kruskal-
Wallis test, when appropriate.
To assess the prevalence of hematinic deficiencies for different MCV levels, the
prevalences of vitamin B12, folate, and iron deficiency were stratified by MCV levels (in
tertiles). This was done for all patients and also for anemic patients separately, as we ex-
pect that the prevalence of different hematinic deficiencies is more pronounced in anemic
patients. Changes in prevalence of hematinic deficiencies were tested using one-way
analysis of variance trend test (linear contrast analysis).
All variables were visually tested for normality by means of Q-Q plots and histo-
grams. In case of doubt, normality was tested using the Kolmogorov-Smirnov test.
14
Skewed variables (e.g. serum NT-proBNP, vitamin B12, folic acid, hs-CRP, and eGFR)
were logarithmically transformed to achieve a normal distribution in order to perform fur-
ther analyses. We used a natural logarithm transformation (binary logarithm, natural loga-
rithm to the base two), as this transformation has a simple interpretation (i.e. the change
in a per doubling in b).
3.3.2 Linear regression models
To determine the clinical correlates of vitamin B12, folic acid, and MCV, multivariate lin-
ear regression models were constructed. First, univariate regression analyses were per-
formed with all baseline variables. Variables with a significant univariate association
with vitamin B12, folic acid, and/or MCV (i.e. p <0.10) were entered in multivariate linear
regression models. The final models consisted of all univariate correlated variables, in-
cluding demographics, laboratory values, comorbidities, and medication.
It was not possible to perform logistic regression analyses (vitamin B12 and folate
deficiency present, yes versus no), as the prevalences of these deficiencies were too low.
3.3.3 Survival analysis
Kaplan Meier curves were constructed to determine the effect of serum vitamin B12 and
folic acid levels on all-cause mortality. This was done by dividing serum vitamin B12 and
folic acid levels into quartiles. Differences in survival rates between quartiles were tested
using the logrank Mantel-Cox test.
To determine the predictive value of vitamin B12 and folic acid levels for all-cause
mortality, univariate and multivariate Cox proportional hazard regression models were
constructed. In consecutive multivariate models, adjustment was made for age, sex, BMI,
LVEF, hemoglobin, eGFR, systolic blood pressure, serum levels of NT-proBNP, hs-CRP
and erythropoietin, and finally for all univariate significant variables. The proportional
hazard assumption was tested using Schoenfeld residuals in each model. The proportion-
ality hazard assumption was proven to hold for both Cox proportional hazard regression
models (folic acid χ2 20.37, p = 0.435, vitamin B12 χ
2 19.22, p = 0.508). For statistical
reasons, follow-up for surviving patients was censored after 9 years, when <5% of all pa-
tients was at risk.
A two-sided p-value <0.05 was considered statistically significant. All analyses were per-
formed using STATA version 12.0 (StataCorp LP, College Station, Texas).
15
Results
4.1 Baseline characteristics
Baseline characteristics (demographics, clinical signs, laboratory measurements, and
treatment) for all 610 patients are shown in Table 1. To determine the effect of disease
severity on these parameters, baseline characteristics were also stratified according to
NYHA functional classification. Baseline characteristics for all 610 patients stratified by
study cohort (Holland, Poland, and Spain) are shown in Supplementary Table 2.
Mean age (± SD) of all patients was 68 ± 12 years, 71% were male, and more than
half of all patients were NYHA functional class III (n = 351, 58%). Patients with a higher
NYHA functional class were older, had a lower systolic blood pressure, LVEF, and quali-
ty of life, had a higher prevalence of anemia and renal failure (i.e. eGFR <60
mL/min/1.73 m2), had lower serum iron levels, and higher serum NT-proBNP levels (all
p for trend <0.001).
4.2 Hematinics
Median serum vitamin B12 and folic acid levels (+ interquartile range) for all patients
were 435 (312 – 600) pg/mL and 9.4 (6.8 – 12.5) ng/mL, respectively. Vitamin B12 and
folate deficiency were present in 5.4% and 4.1% of all patients, respectively. More than
half of all patients were suffering from iron deficiency (58%). The prevalence of vitamin
B12 and folate deficiency was equal in patients with iron deficiency compared to patients
without iron deficiency (5.9% versus 4.7%, p = 0.516, and 3.1% versus 5.5%, p = 0.142,
respectively). As expected, folate deficiency was significantly more prevalent in anemic
patients (6.7% versus 2.8% in non-anemic patients, p = 0.020). However, this was not the
case for vitamin B12 deficiency (6.2% versus 5.0%, p = 0.538). Vitamin B12 and folate
deficiency were not more prevalent in patients with higher NYHA functional class (p for
trend 0.693 and 0.735, respectively). None of the patients were suffering from both vita-
min B12 and folate deficiency.
4.3 Clinical correlates of vitamin B12, folic acid, and MCV
Univariate and multivariate linear regression models are shown in Table 2, 3, and 4. In
multivariate linear regression models, higher serum folic acid levels were observed in pa-
tients with higer hemoglobin levels (p = 0.005). Higher LVEF, eGFR, TSAT, and the
presence of atrial fibrillation were associated with lower serum folic acid levels (all p
<0.05). Higher serum vitamin B12 levels were correlated to higher NT-proBNP and ferri-
tin levels, and the presence of atrial fibrillation (all p<0.05). Lower serum vitamin B12
levels were associated with systolic blood pressure (p = 0.005). Finally, a lower MCV
was observed in younger patients with diabetes (both p <0.001). Only hemoglobin and
TSAT were positively correlated to MCV (both p <0.05). There was no significant corre-
lation between hematinics and treatment (angiotensin-converting enzyme inhibitors, an-
gio-
4
16
Table 1. Baseline characteristics.
Variables Total cohort
(n = 610)
NYHA I
(n = 54)
NYHA II
(n = 167)
NYHA III
(n = 351)
NYHA IV
(n = 38)
p-value
(trend)
Demographics and clinical signs
Age (years) 68 ± 12 58 ± 14 65 ± 11 70 ± 11 72 ± 12 <.001
Men 71 89 69 70 74 .121
BMI (kg/m2)
- Cachexia§
27.7 ± 5.3
1.1
28.3 ± 4.9
0
29.3 ± 5.5
0
27.0 ± 5.1
1.7
26.0 ± 5.1
2.6
.007
.152
Ischemic etiology 55 44 50 58 71 .006
eGFR (mL/min/1.73 m2)
- eGFR <60
67.4 (50.3 – 99.0)
39
89.4 (72.3 – 142.7)
11
85.4 (67.9 – 141.0)
17
58.7 (45.2 – 75.5)
53
57.7 (43.3 – 86.9)
53
<.001
<.001
LVEF (%) 33 ± 13 35 ± 13 36 ± 15 33 ± 12 29 ± 14 .022
SBP (mmHg) 122 ± 22 130 ± 19 122 ± 23 122 ± 22 111 ± 22 <.001
MLHFQ 45 (26 – 61) 18 (9 – 38) 40 (23 – 55) 47 (30 – 62) 67 (57 – 79) <.001
Comorbidities
- Diabetes mellitus
- Atrial fibrillation
- Hypertension
40
30 49
24
30 54
39
31 61
40
29 40
55
29 71
.002
.909
.317
Hematinics
Hb (g/dL) 13.4 ± 1.8 14.1 ± 1.6 13.6 ± 1.8 13.3 ± 1.8 12.0 ± 1.9 <.001
MCV (fL) 89.8 ± 5.8 89.7 ± 5.5 90.0 ± 5.8 89.6 ± 5.8 90.4 ± 6.1 .611 Anemia* (%) 34 17 29 36 71 <.001
EPO (IU/L) 15.4 (8.0 – 26.2) 10.1 (4.0 – 17.0) 13.2 (2.8 – 23) 16.3 (10.1 – 26.9) 33.0 (14.1 – 67) <.001
Iron (μg/dL) 67.0 (42.0 – 91.0) 89.5 (63.0 – 109.0) 82.0 (61.0 – 103.0) 58.1 (35.2 – 79.9) 42.7 (34.0 – 63.0) <.001
Ferritin (μg/L) 148 (78 – 273) 161 (78 – 275) 155 (81 – 269) 142 (75 – 280) 157 (61 – 263) .657
TSAT (%) 19 (12 – 26) 24 (17 – 29) 23 (17 – 28) 17 (10 – 24) 14 (9 – 20) <.001
Iron deficiency 58 43 52 62 71 .002 Vitamin B12 (pg/mL) 435 (312 – 600) 439 (327 – 554) 431 (316 – 602) 428 (306 – 581) 564 (343 – 865) .514
Vitamin B12 deficiency† 5.4 7.4 4.8 5.4 5.3 .693
Folic acid (ng/mL) 9.4 (6.8 – 12.5) 7.3 (5.9 – 9.5) 8.4 (6.1 – 10.5) 10.3 (7.6 – 13.8) 8.2 (6.2 – 11.2) .031 Folate deficiency‡ 4.1 1.9 8.4 2.3 5.3 .735
Cardiac biomarkers
NT-proBNP (pg/mL) 1801 (705 – 4335) 898 (358 – 2515) 1217 (479 – 2984) 2071 (892 – 4687) 6548 (1870 – 12058) <.001
hs-CRP (mg/L)¶ 4.0 (1.5 – 10.0) 1.4 (1.1 – 4.9) 2.2 (1.4 – 5.4) 5.0 (2.0 – 12.7) 13.5 (4.0 – 29.5) .001
Treatment
Ace inhibitor and/or ARB 90 94 90 91 76 .004
Beta blocker 87 94 94 84 79 .007
Aldosterone antagonist 51 30 51 53 55 .013
Statins 55 56 66 47 68 .523
Loop diuretics 86 61 78 93 87 <.001
Antiplatelet and/or anticoagulant 85 76 82 87 84 .192
Values are given as means ± SD, medians (IQR) or proportions (%)
BMI = body mass index; eGFR = estimated glomerular filtration rate; EPO = erythropoietin; Hb = hemoglobin; HF = heart failure; hs-CRP = high-sensitive C-reactive protein; LVEF = left ventricular ejection fraction; MCV = mean corpuscular volume; MLHFQ = Minnesota Living with Heart Failure Questionnaire; NT-proBNP = N-terminal prohormone brain natriuretic peptide; NYHA = New York Health Association; SBP = systol-
ic blood pressure; TSAT = transferrin saturation
* Anemia is defined as Hb <12 g/dL in women and <13 g/dL in men56
† Vitamin B12 deficiency is defined as serum vitamin B12 level <200 pg/mL35,38,50
‡ Folate deficiency is defined as serum folic acid level <4.0 ng/mL39,50
§ Cachexia is defined as BMI <18 kg/m2
¶ Serum hs-CRP levels were determined in 385 patients
17
tensin receptor blockers, aldosterone antagonists, beta blockers, loop diuretics, antiplate-
lets, and anticoagulants) in all multivariate models.
Multivariate linear regression models showed that a worse quality of life (as as-
sessed by MLHFQ) was significantly correlated to lower serum folic acid and MCV lev-
els (all p <0.05).
Table 2. Clinical variables associated with serum vitamin B12 levels in chronic HF. Variable Univariate β
(95% CI)
p-value Multivariate β
(95% CI)
p-value
Age, per 5 year .015 (-.011 ; .040) .249
Sex, female vs. male -.054 (-.188 ; .080) .429
BMI, per 1 kg/m2 -.004 (-.016 ; .008) .507
LVEF, per 1% -.003 (-.008 ; .001) .173
SBP, per 1 mmHg -.005 (-.008 ; .002) <.001 -.004 (-.007 ; -.001) .005
MCV, per 1 fL -.001 (-.011 ; .010) .892
Hb, per 1 g/dL .021 (-.013 ; .054) .226
eGFR, per doubling -.051 (-.126 ; .023) .176
NT-proBNP, per doubling .063 (.032 ; .095) <.001 .049 (.015 ; .083) .004
hs-CRP, per doubling .033 (-.010 ; .076) .133
EPO, per doubling .015 (-.043 ; .073) .615
Ferritin, per doubling .104 (.059 ; .148) <.001 .093 (.047 ; .140) <.001
TSAT, per doubling .023 (-.032 ; .077) .418
Folic acid, per doubling .035 (-.048 ; .118) .403
Atrial fibrillation, yes vs. no .185 (.053 ; .317) .006 .141 (.007 ; .275) .039
Diabetes, yes vs. no .072 (-.052 ; .196) .253
Ischemic etiology, yes vs. no -.154 (-.275 ; -.033) .013
MLHFQ, per point .001 (-.001 ; .004) .298
Treatment
ACEi and/or ARB, yes vs. no -.177 (-.382 ; .028) .090
Aldosterone antag., yes vs. no .042 (-.079 ; .163) .495
Beta blocker, yes vs. no -.140 (-.322 ; .043) .133
Statins, yes vs. no -.061 (-.183 ; .061) .324
Loop diuretic, yes vs. no .093 (-.081 ; .267) .296
Antiplatelet and/or anticoagulant, yes vs. no .014 (-.155 ; .182) .874
Table 3. Clinical variables associated with serum folic acid levels in chronic HF. Variable Univariate β
(95% CI)
p-value Multivariate β
(95% CI)
p-value
Age, per 5 year .022 (-.002 ; .046) .076
Sex, female vs. male -.023 (-.152 ; .106) .724
BMI, per 1 kg/m2 -.012 (-.023 ; -.001) .037
LVEF, per 1% -.009 (-.013 ; -.005) <.001 -.007 (-.012 ; -.002) .005
SBP, per 1 mmHg .001 (-.002 ; .004) .576
MCV, per 1 fL .006 (-.004 ; .016) .251
Hb, per 1 g/dL .040 (.008 ; .072) .014 .051 (.016 ; .087) .005
eGFR, per doubling -.279 (-.347 ; -.211) <.001 -.273 (-.351 ; -.195) <.001
NT-proBNP, per doubling .029 (-.002 ; .059) .064
hs-CRP, per doubling .003 (-.036 ; .042) .881
EPO, per doubling -.033 (-.088 ; .022) .243
Ferritin, per doubling .009 (-.035 ; .052) .699
TSAT, per doubling -.111 (-.162 ; -.059) <.001 -.102 (-.158 ; -.047) <.001
Atrial fibrillation, yes vs. no -.164 (-.291 ; -.037) .011 -.204 (-.329 ; -.079) .001
Diabetes, yes vs. no -.153 (-.272 ; -.035) .011
18
Ischemic etiology, yes vs. no .053 (-.064 ; .170) .376
MLHFQ, per point -.004 (-.006 ; -.001) .007 -.004 (-.006 ; -.001) .003
Treatment
ACEi and/or ARB, yes vs. no -.149 (-.047 ; .346) .137
Aldosterone antag., yes vs. no -.013 (-.129 ; .104) .833
Beta blocker, yes vs. no -.103 (-.278 ; .072) .249
Statins, yes vs. no -.103 (-.219 ; .014) .084
Loop diuretic, yes vs. no .124 (-.042 ; .291) .144
Antiplatelet and/or anticoagulant, yes vs. no -.047 (-.209 ; .115) .567
Table 4. Clinical variables associated with mean corpuscular volume (MCV) in chronic HF. Variable Univariate β
(95% CI)
p-value Multivariate β
(95% CI)
p-value
Age, per 5 year .020 (.002 ; .040) .047 .445 (.245 ; .646) <.001
Sex, female vs. male .266 (-.769 ; 1.30) .614
BMI, per 1 kg/m2 -.037 (-.125 ; .051) .410
LVEF, per 1% .003 (-.032 ; .039) .846
SBP, per 1 mmHg -.008 (-.030 ; .014) .487
Hb, per 1 g/dL .549 (.293 ; .805) <.001 .336 (.056 ; .616) .019
eGFR, per doubling .191 (-.396 ; .779) .523
Folic acid, per doubling .373 (-.264 ; 1.01) .251
NT-proBNP, per doubling .039 (-.207 ; .286) .755
hs-CRP, per doubling -.244 (-.575 ; .087) .149
EPO, per doubling -.191 (-.648 ; .267) .414
Ferritin, per doubling .447 (.101 ; .793) .011
TSAT, per doubling 1.452 (1.04 ; 1.87) <.001 1.358 (.895 ; 1.82) <.001
Vitamin B12, per doubling -.043 (-.664 ; .578) .892
Atrial fibrillation, yes vs. no -.058 (-1.09 ; .973) .912
Diabetes, yes vs. no -2.25 (-3.19 ; -1.32) <.001 -2.15 (-3.11 ; -1.20) <.001
Ischemic etiology, yes vs. no -1.19 (-2.13 ; -.256) .013
MLHFQ, per point -.038 (-.059 ; -.017) <.001 -.021 (-.041 ; -.001) .037
Treatment
ACEi and/or ARB, yes vs. no -.145 (-1.73 ; 1.44) .858
Aldosterone antag., yes vs. no -.488 (-1.43 ; .449) .307
Beta blocker, yes vs. no .352 (-1.04 ; 1.74) .619
Statins, yes vs. no -.301 (-1.24 ; .640) .530
Loop diuretic, yes vs. no .213 (-1.13 ; 1.56) .756
Antiplatelet and/or anticoagulant, yes vs. no -.873 (-2.16 ; .415) .184
4.4 Hematinic deficiencies and mean corpuscular volume
The prevalence of hematinic deficiencies stratified by MCV levels in tertiles is displayed
in Figure 1 and 2. The prevalence of iron deficiency was significantly higher in patients
with lower MCV levels at baseline (p for trend <0.001). A similar pattern was observed
when only anemic patients (n = 210) were selected (p for trend =0.009). Interestingly,
there was no significant trend in prevalence of vitamin B12 and folate deficiency for dif-
ferent MCV levels.
19
4.5 Hematinics and survival
4.5.1 Kaplan-Meier analysis
During median follow-up of 2.10 years (interquartile range 1.31 – 3.60 years) 254 pa-
tients (42%) died. Differences in event-free nine-year survival stratified by quartiles of
serum vitamin B12 and folic acid levels are displayed in Figure 3 and 4. Increased mortal-
ity rates were observed in patients with serum vitamin B12 levels >600 pg/mL versus
those with levels <600 pg/mL (47.7% versus 41.8% mortality in nine years, p = 0.026).
No differences in mortality rates were observed between higher and lower serum folic
acid levels (p = 0.745).
Figure 1. Figure 2.
Prevalence of hematinic deficiencies in the total study cohort
(n = 610), stratified by MCV in tertiles.
Prevalence of hematinic deficiencies in anemic patients (n = 210),
stratified by MCV in tertiles.
Figure 3. Figure 4.
Kaplan-Meier survival analysis, stratified by vitamin B12
levels in quartiles.
Kaplan-Meier survival analysis, stratified by folic acid
levels in quartiles.
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
<87.4 87.4 - 92.0 >92.0
Fre
qu
ency
(%
)
MCV (fL)
No deficiencies ID
Folate deficiency Vitamin B12 deficiency
ID + folate deficiency ID + vitamin B12 deficiency
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
<87.4 87.4 - 92.0 >92.0
Fre
qu
ency
(%
)
MCV (fL)
No deficiencies ID
Folate deficiency Vitamin B12 deficiency
ID + folate deficiency ID + vitamin B12 deficiency
0.0
00
.25
0.5
00
.75
1.0
0
Cum
ula
tive s
urv
ival (%
)
0 2 4 6 8 10Follow-up time (years)
Vitamin B12 <313 pg/mL Vitamin B12 313-435 pg/mL
Vitamin B12 436-600 pg/mL Vitamin B12 >600 pg/mL
0.0
00
.25
0.5
00
.75
1.0
0
Cum
ula
tive s
urv
ival (%
)
0 2 4 6 8 10Follow-up time (years)
Folic acid <6.9 ng/mL Folic acid 6.9-9.4 ng/mL
Folic acid 9.5-12.5 ng/mL Folic acid >12.5 ng/mL
Logrank P = 0.026 Logrank P = 0.745
20
4.5.2 Cox proportional hazard regression analysis
Univariate and multivariate Cox proportional hazard regression models for serum vitamin
B12 and folic acid levels are presented in Table 5. In the univariate Cox proportional haz-
ard regression model, serum levels of vitamin B12 >600 pg/mL were associated with
higher mortality rates (hazard ratio [HR] 1.64, 95% CI 1.16 – 2.31). In multivariate Cox
regression models, serum vitamin B12 lost its predictive value for all-cause mortality (HR
0.95, 95% CI 0.60 – 1.51). Mortality was similar in patients with higher and lower serum
folic acid levels, and the absence of an association between serum folic acid levels and
mortality remained present in multivariate Cox regression models (HR 0.74, 95% CI 0.36
– 1.55).
Table 5. Cox proportional hazard regression models for the prediction of mortality of chronic
HF patients Univariate HR Multivariate HR
model I Multivariate HR model II
Multivariate HR model III
Folic acid (ng/mL) <6.9 6.9 – 9.4 9.5 – 12.5 >12.5
1.00 1.09 (.72 – 1.65) .95 (.64 – 1.41) .89 (.60 – 1.33)
1.00 1.05 (.69 – 1.59) .92 (.61 – 1.36) .87 (.58 – 1.30)
1.00 .98 (.65 – 1.49) .86 (.57 – 1.29) .76 (.50 – 1.15)
1.00 1.16 (.52 – 2.56) .78 (.37 – 1.63) .74 (.36 – 1.55)
Vitamin B12 (pg/mL) <313 313 – 435 436 – 600 >600
1.00 1.14 (.79 – 1.64) 1.14 (.80 – 1.64) 1.64 (1.16 – 2.31)
1.00 1.17 (.81 – 1.68) 1.17 (.82 – 1.68) 1.60 (1.14 – 2.26)
1.00 1.17 (.81 – 1.69) 1.14 (.79 – 1.63) 1.30 (.92 – 1.84)
1.00 1.49 (.96 – 2.32) 1.03 (.66 – 1.60) .95 (.60 – 1.51)
21
Discussion In this well-described international pooled study cohort comprising a broad and repre-
sentative range of patients with stable chronic HF, we demonstrate that vitamin B12 and
folate deficiency are relatively rare (4% and 5%, respectively). Secondly, there is no sig-
nificant correlation between the prevalence of hematinic deficiencies and disease severity
(NYHA functional class). Thirdly, MCV is not related to serum levels of vitamin B12 and
folic acid. Finally, serum vitamin B12 and folic acid levels are not independently associat-
ed with mortality.
5.1 Prevalence and definition of hematinic deficiencies
Unfortunately, there are no clear definitions of vitamin B12 and folate deficiency, leading
to different thresholds for the presence of these deficiencies and consequently to differ-
ences in prevalence.
5.1.1 Vitamin B12
Traditionally, serum vitamin B12 levels have been considered as the gold standard for the
detection of clinical vitamin B12 deficiency. According to Stabler et al., serum vitamin
B12 levels <200 pg/mL are suggestive for clinical deficiency (sensitivity and specificity
65-100% and 50-60%, respectively).38,59
A higher threshold of 350 pg/mL was character-
ized by a sensitivity of 90% to determine clinical vitamin B12 deficiency. Vitamin B12 de-
ficiency is unlikely (i.e. probability <5%) with serum levels >300 pg/mL.60
Exceptionally
low serum levels of vitamin B12 (i.e. <100 pg/mL) are often associated with overt defi-
ciency, but such levels are hardly observed (n = 3 in this study, <1%). Furthermore, the
false negative and false positive value of laboratory assays for serum vitamin B12 levels
can be as high as 50% due to suboptimal assays and highly variable test results.39,61-65
New assays are currently in development (e.g. holotranscobalamin), but these assays need
to be validated clinically before they can be used in clinical and daily health care.66
Measurement of methylmalonic acid (MMA) and total homocysteine (HCY) was
introduced several decades ago to diagnose vitamin B12 deficiency, additionally to serum
vitamin B12 levels. Virtually all patients (>98%) with clinical vitamin B12 deficiency have
elevated serum MMA and HCY, including patients without megaloblastic anemia.38,67
Serum MMA is considered more specific for vitamin B12 deficiency than serum HCY,
since serum HCY can also be elevated in folate deficiency.67,68
However, both serum
MMA and HCY may be elevated when renal failure is present, making MMA and HCY
unreliable screening markers for vitamin B12 deficiency in patients with chronic HF, as
up to 60% of patients with chronic HF are suffering from moderate to severe renal dys-
function (i.e. eGFR <60 mL/min/1.73 m2).
69-74 In the current study, renal dysfunction is
present in 39% of all patients. Serum MMA and HCY would definitely overestimate the
prevalence of vitamin B12 in our study. Our results show that serum vitamin B12 levels are
not dependent on the presence of renal failure. In short, there are currently no reliable
screening markers for vitamin B12 deficiency in patients with chronic HF, apart from se-
rum vitamin B12 levels. Serum vitamin B12 levels give a fair indication of the prevalence
of vitamin B12 deficiency in patients with chronic HF and are still of value in the diagno-
sis of vitamin B12 deficiency in those patients.
5
22
5.1.2 Folic acid
Measurement of serum folic acid levels is mainly a reflection of short-term folic acid sta-
tus.35
In the absence of recent fasting, serum folic acid levels of <4 ng/mL are diagnostic
for folate deficiency.50
Alternatively, red cell folic acid levels can be used to diagnose
folic acid deficiency, as this parameter reflects average folic acid availability over time
and is less subject to daily fluctuations. However, test results are occasionally difficult to
interpret and are therefore not always conclusive.39
Several studies showed that serum
and red cell folic acid levels were of equal value (or even superior to red cell folic acid)
in the diagnosis of folate deficiency.75,76
As a consequence, initial screening for folate
deficiency can be performed using serum folic acid levels, which is a relatively inexpen-
sive measurement. More important, serum folic acid measurements are widely available
and are relatively easy to perform, in contrast to red cell folic acid measurements. When
serum levels of folic acid are borderline, red cell folic acid levels can be measured to con-
firm the diagnosis.
The current study shows that serum folic acid levels are correlated to renal dys-
function (i.e. serum folic acid levels are higher when renal dysfunction is present, due to
reduced renal clearance of folic acid). As stated earlier, renal dysfunction is a relatively
common comorbidity in chronic HF, and, as a consequence, the prevalence of folate defi-
ciency may be underestimated in patients with chronic HF when using serum folic acid
levels as marker for folate deficiency. Nonetheless, serum folic acid remains the best ini-
tial screening marker for folate deficiency in patients with chronic HF.
5.2 Vitamin B12, folic acid, and mean corpuscular volume
Traditionally, mean corpuscular volume (MCV) is one of the standard red cell parame-
ters. Its importance in the differential diagnosis of anemia has recently been emphasized
by Brugnara et al..77
MCV has always been considered as one of the hallmarks in the di-
agnosis of vitamin B12 and folate deficiency, since these deficiencies are frequently char-
acterized by an increase in MCV.
In the current study, no significant correlation between MCV and either serum
vitamin B12 or folic acid levels was observed, although we found a significant association
between MCV and transferrin saturation (used to diagnose iron deficiency). Iron defi-
ciency is significantly more prevalent in patients with lower MCV levels, as expected
(iron deficiency may lead to microcytic anemia). Apparently, MCV is more dependent on
iron status than on vitamin B12 and/or folic acid levels. Although MCV is a reliable
screening marker for hematinic deficiencies in otherwise healthy people, this seems not to
be the case in patients with chronic HF. MCV levels in chronic HF patients are mainly
driven by iron status, and since iron deficiency is a common comorbidity in patients with
chronic HF, MCV levels will be inevitably lower as the result of microcytic anemia
caused by iron deficiency. Therefore, the effect of vitamin B12 or folate deficiency on
MCV levels (i.e. megaloblastic anemia) will be negated.
5.3 Hematinics and quality of life
The present study shows that lower serum folic acid levels, but not serum vitamin B12
levels, are related to an impaired quality of life. A recent study of Comin-Colet et al.
shows that iron deficiency is also correlated to reduced quality of life.45
In this study
comprising 552 patients with chronic HF, anemia did not have any significant influence
23
on quality of life. The importance of quality of life in patients with chronic HF has re-
cently been stressed by Kraai et al., stating that most chronic HF patients prioritize quali-
ty of live above longevity.78
5.4 Hematinics and survival
The predictive value of iron deficiency on mortality in patients with chronic HF has re-
cently been underscored by Klip et al., making iron status a strong and independent pre-
dictor of outcome.24
The present study shows that vitamin B12 and folic acid do not have
such predictive value on mortality in patients with chronic HF. Univariate Cox propor-
tional hazard regression analysis shows that patients with serum vitamin B12 >600 pg/mL
have higher mortality risks compared to patients with lower levels. This effect on mortali-
ty is caused by the paradoxical association between serum vitamin B12 levels and disease
severity (i.e. serum NT-proBNP levels). This association was observed earlier by
Herrmann et al. and may be caused by the cardiohepatic syndrome.79
Chronic HF may
cause blood congestion in the liver, due to a poor hemodynamic status in patients with
chronic HF.80
This can lead to hepatocyte damage and, as a consequence, mild liver dys-
function.81
Deterioration of liver function may lead to the release of hepatic and metabol-
ically inactive vitamin B12 analogues, making serum vitamin B12 measurements less accu-
rate.82,83
However, the cardiohepatic syndrome remains only a hypothesis and is an in-
complete explanation of the association between serum vitamin B12 and disease severity.
5.5 Study limitations
The main limitation of this study is that the diagnosis of vitamin B12 and folate deficiency
is relatively challenging as the result of lacking gold standard tests for serum levels and
no clear definitions of vitamin B12 and folate deficiency. Low serum vitamin B12 and folic
acid levels are not always conclusive for clinical deficiency. However, current screening
methods for hematinic deficiencies are validated thoroughly and are mainstream practice
in daily health care. The cut-offs for hematinic deficiencies used in this study are based
on an optimal balance between sensitivity and specificity. Serum vitamin B12 and folic
acid levels remain the most important screening markers for deficiencies of these vita-
mins in patients with chronic HF, as long as more reliable markers are lacking.
Secondly, measurements of hematinics were only available at baseline, so hema-
tinic changes over time could not be established. Additional studies with multiple meas-
urements of hematinics over time should be conducted.
Finally, although this international study comprises patients from several coun-
tries in Europe, our results cannot be extrapolated directly to other continents or popula-
tions, for example due to differences in diet.
24
Conclusion In a large international pooled cohort study, we showed that vitamin B12 and folate defi-
ciency are relatively rare in patients with stable chronic HF. The prevalence of hematinic
deficiencies is independent of NYHA functional class. Lower serum folic acid levels are
independently correlated to worse quality of life. Whether treatment of folic acid defi-
ciency has beneficial effects is currently unknown. Interestingly, there was no significant
correlation between serum vitamin B12 and folic acid levels and MCV levels, making
MCV an unreliable screening marker for hematinic deficiencies in patients with chronic
HF. Finally, serum vitamin B12 and folic acid levels are not associated with mortality.
Acknowledgements
I would like to thank Peter van der Meer and IJsbrand Klip for their great support during
my internship. I am looking forward to conduct my MD/PhD programme during the next
years.
Finally, many thanks to Dirk Lok, Dian Pruijsers, and Joanneke Penninkhof from
the Department of Cardiology of the Deventer Hospital, for the opportunity to extract
some data from the electronic patients files.
6
25
Appendices Supplementary Table 1. Inclusion and exclusion criteria per participating study cohort.
Inclusion criteria Exclusion criteria
Dutch
cohort47
(n = 202)
- NYHA class III-IV
- Stable HF with echocardiographic findings of
reduced left ventricular ejection fraction (LVEF
≤45%) or preserved left ventricular ejection fraction
- Patients able to understand study procedures and
willing to provide informed consent
- Dementia or psychiatric illness
- Residents of a nursing home
- Diseases other than HF with expected survival
<1 year
- Participation in other studies
- Current or planned hospitalisation
- Ongoing kidney replacement therapy
Polish
cohort25,46
(n = 152)
- NYHA class I-IV
- HF duration ≥6 months
- LVEF ≤45% (echocardiographic)
- Stable HF with stable medical therapy for ≥1 month
before study
- Patients able to understand study procedures and
willing to provide informed consent
- Acute coronary syndrome, coronary
revascularisation or any major sugery ≤3 months
before study
- Unplanned hospitalisation due to HF deterioration
- Any acute or chronic diseases with possible
influence on iron metabolism
- Treatment for anaemia and/or iron deficiency at the
time of study and/or ≤12 months before study
Spanish
cohort45
(n = 256)
- NYHA class I-IV
- Stable HF ≥1 month before study
- Reduced (≤45%) or preserved LVEF
- Patients able to understand study procedures and
willing to provide informed consent
- Significant primary valvular disease
- Severe anaemia (hemoglobin <8.5 g/dL)
- Significant pericardial disease
- Hypertrophic or restrictive cardiomyopathy
- Current malignancy
- Current infection
- Clinically significant liver function abnormalities
7
26
Supplementary Table 2. Baseline characteristics, stratified by study cohort Variables Total cohort
(n = 610)
Holland47
(n = 202)
Poland25,46
(n = 152)
Spain45
(n = 256)
p-value
Demographics and clinical signs
Age (years) 68 ± 12 71 ± 10 58 ± 11 71 ± 11 <.001 Men (%) 71 73 91 58 <.001
BMI (kg/m2)
- Cachexia§
27.7 ± 5.3
1.2
26.3 ± 4.7
2.0
27.4 ± 4.7
0.7
29.0 ± 5.7
0.8
<.001
.396
Ischemic etiology (%) 55 62 59 48 .007 eGFR (mL/min/1.73 m2)
- eGFR <60 (%)
67.4 (50.3 – 99.1)
39
53.1 (41.9 – 61.6)
71
70.9 (58.5 – 81.8)
28
110.2 (67.1 – 154.6)
21
<.001
<.001
LVEF (%) 33 ± 13 31 ± 9 28 ± 9 39 ± 16 <.001
SBP (mmHg) 124 ± 22 123 ± 22 118 ± 19 123 ± 23 .085 MLHFQ 45 (26 – 61) 43 (25 – 57) 44 (22 – 66) 46 (29 – 61) .263
Comorbidities (%)
- Diabetes mellitus
- Atrial fibrillation - Hypertension
39 30
49
29 29
26
32 35
38
52 27
73
<.001 .258
<.001
Hematinics
Hb (g/dL) 13.4 ± 1.8 13.6 ± 1.6 14.1 ± 1.6 12.7 ± 1.9 <.001
MCV (fL) 89.7 ± 5.8 90.3 ± 5.6 88.5 ± 4.9 90.1 ± 6.3 .011
Anemia* (%) 34 27 22 48 <.001
EPO (IU/L) 18.0 (11.2 – 29.1) 16.7 (11.4 – 26.7) 0.0 (0.0 – 12.3) 20.0 (12.0 – 33.5) <.001 Iron (μg/dL) 67.0 (42.0 – 91.2) 50.0 (23.5 – 73.2) 91.5 (70.0 – 114.5) 63.0 (44.0 – 86.0) <.001
Ferritin (μg/L) 148 (78 – 273) 140 (75 – 272) 166 (92 – 271) 143 (73 – 274) .205
TSAT (%) 19 (12 – 26) 14 (6 – 22) 23 (15 – 30) 19 (13 – 26) <.001
Iron deficiency 58 65 48 59 .005
Vitamin B12 (pg/mL) 435 (313 – 600) 399 (274 – 539) 447 (322 – 598) 451 (342 – 652) .006
Vitamin B12 deficiency† 5.4 7.9 5.3 3.5 .117 Folic acid (ng/mL) 9.4 (6.8 – 12.4) 12.5 (10.3 – 15.5) 8.8 (6.6 – 11.4) 7.5 (5.5 – 9.4) <.001
Folate deficiency‡ 4.1 0 3.3 7.8 <.001
Cardiac biomarkers
NT-proBNP (pg/mL) 1801 (705 – 4335) 2135 (989 – 4473) 2501 (796 – 4761) 1202 (487 – 3002) <.001
hs-CRP (mg/L)¶ 4.0 (1.5 – 10.0) 5.0 (2.0 – 14.0) 3.2 (1.3 – 7.3) N/A N/A
Treatment
Ace inhibitor and/or ARB 90 95 95 84 <.001
Beta blocker 87 78 97 89 <.001 Aldosterone antagonist 50 50 44 55 .097
Statins 55 40 68 59 <.001
Loop diuretics 86 97 68 88 <.001 Antiplatelet and/or anticoagulant 85 90 80 84 .043
Values are given as means ± SD, medians (IQR) or proportions (%)
BMI = body mass index; eGFR = estimated glomerular filtration rate; EPO = erythropoietin; Hb = hemoglobin; HF = heart failure; hs-CRP = high-sensitive C-reactive protein; LVEF = left ventricular ejection fraction; MCV = mean corpuscular volume; MLHFQ = Minnesota Living with Heart Failure Questionnaire; NT-proBNP = N-terminal prohormone brain natriuretic peptide; NYHA = New York
Health Association; SBP = systolic blood pressure; TSAT = transferrin saturation
* Anemia is defined as Hb <12 g/dL in women and <13 g/dL in men56 † Vitamin B12 deficiency is defined as serum vitamin B12 level <200 pg/mL35,38,50
‡ Folate deficiency is defined as serum folic acid level <4.0 ng/mL39,50
§ Cachexia is defined as BMI <18 kg/m2
¶ Serum hs-CRP levels were determined in 385 patients
27
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