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

References

1. McMurray JJ, Adamopoulos S, Anker SD, Auricchio A, Bohm M, Dickstein K, et al. ESC

guidelines for the diagnosis and treatment of acute and chronic heart failure 2012: The Task Force

for the Diagnosis and Treatment of Acute and Chronic Heart Failure 2012 of the European Socie-

ty of Cardiology. Developed in collaboration with the Heart Failure Association (HFA) of the

ESC. Eur J Heart Fail 2012 Aug;14(8):803-869.

2. Mosterd A, Hoes AW. Clinical epidemiology of heart failure. Heart 2007 Sep;93(9):1137-

1146.

3. Mendez GF, Cowie MR. The epidemiological features of heart failure in developing countries:

a review of the literature. Int J Cardiol 2001 Sep-Oct;80(2-3):213-219.

4. Lam CS, Donal E, Kraigher-Krainer E, Vasan RS. Epidemiology and clinical course of heart

failure with preserved ejection fraction. Eur J Heart Fail 2011 Jan;13(1):18-28.

5. Hogg K, Swedberg K, McMurray J. Heart failure with preserved left ventricular systolic func-

tion; epidemiology, clinical characteristics, and prognosis. J Am Coll Cardiol 2004 Feb

4;43(3):317-327.

6. Meta-analysis Global Group in Chronic Heart Failure (MAGGIC). The survival of patients

with heart failure with preserved or reduced left ventricular ejection fraction: an individual patient

data meta-analysis. Eur Heart J 2012 Jul;33(14):1750-1757.

7. McMurray JJ. Clinical practice. Systolic heart failure. N Engl J Med 2010 Jan 21;362(3):228-

238.

8. Francis GS, Goldsmith SR, Levine TB, Olivari MT, Cohn JN. The neurohumoral axis in con-

gestive heart failure. Ann Intern Med 1984 Sep;101(3):370-377.

9. Davie AP, Francis CM, Caruana L, Sutherland GR, McMurray JJ. Assessing diagnosis in heart

failure: which features are any use? QJM 1997 May;90(5):335-339.

10. Mant J, Doust J, Roalfe A, Barton P, Cowie MR, Glasziou P, et al. Systematic review and

individual patient data meta-analysis of diagnosis of heart failure, with modelling of implications

of different diagnostic strategies in primary care. Health Technol Assess 2009 Jul;13(32):1-207,

iii.

11. Fonseca C. Diagnosis of heart failure in primary care. Heart Fail Rev 2006 Jun;11(2):95-107.

12. Kelder JC, Cramer MJ, van Wijngaarden J, van Tooren R, Mosterd A, Moons KG, et al. The

diagnostic value of physical examination and additional testing in primary care patients with sus-

pected heart failure. Circulation 2011 Dec 20;124(25):2865-2873.

13. Rutten FH, Moons KG, Cramer MJ, Grobbee DE, Zuithoff NP, Lammers JW, et al. Recognis-

ing heart failure in elderly patients with stable chronic obstructive pulmonary disease in primary

care: cross sectional diagnostic study. BMJ 2005 Dec 10;331(7529):1379.

8

28

14. Hawkins NM, Petrie MC, Jhund PS, Chalmers GW, Dunn FG, McMurray JJ. Heart failure

and chronic obstructive pulmonary disease: diagnostic pitfalls and epidemiology. Eur J Heart Fail

2009 Feb;11(2):130-139.

15. Shah AM, Mann DL. In search of new therapeutic targets and strategies for heart failure: re-

cent advances in basic science. Lancet 2011 Aug 20;378(9792):704-712.

16. Ho KK, Anderson KM, Kannel WB, Grossman W, Levy D. Survival after the onset of con-

gestive heart failure in Framingham Heart Study subjects. Circulation 1993 Jul;88(1):107-115.

17. Cowie MR, Wood DA, Coats AJ, Thompson SG, Suresh V, Poole-Wilson PA, et al. Survival

of patients with a new diagnosis of heart failure: a population based study. Heart 2000

May;83(5):505-510.

18. Bleumink GS, Knetsch AM, Sturkenboom MC, Straus SM, Hofman A, Deckers JW, et al.

Quantifying the heart failure epidemic: prevalence, incidence rate, lifetime risk and prognosis of

heart failure The Rotterdam Study. Eur Heart J 2004 Sep;25(18):1614-1619.

19. Roger VL, Weston SA, Redfield MM, Hellermann-Homan JP, Killian J, Yawn BP, et al.

Trends in heart failure incidence and survival in a community-based population. JAMA 2004 Jul

21;292(3):344-350.

20. Levy D, Kenchaiah S, Larson MG, Benjamin EJ, Kupka MJ, Ho KK, et al. Long-term trends

in the incidence of and survival with heart failure. N Engl J Med 2002 Oct 31;347(18):1397-1402.

21. Okonko DO, Mandal AK, Missouris CG, Poole-Wilson PA. Disordered iron homeostasis in

chronic heart failure: prevalence, predictors, and relation to anemia, exercise capacity, and sur-

vival. J Am Coll Cardiol 2011 Sep 13;58(12):1241-1251.

22. van Deursen VM, Damman K, van der Meer P, Wijkstra PJ, Luijckx GJ, van Beek A, et al.

Co-morbidities in heart failure. Heart Fail Rev 2012 Dec 25.

23. Kleijn L, Belonje AM, Voors AA, De Boer RA, Jaarsma T, Ghosh S, et al. Inflammation and

anaemia in a broad spectrum of patients with heart failure. Heart 2012 Aug;98(16):1237-1241.

24. Klip IT, Comin-Colet J, Voors AA, Ponikowski P, Enjuanes C, Banasiak W, et al. Iron defi-

ciency in chronic heart failure: an international pooled analysis. Am Heart J 2013

Apr;165(4):575-582.e3.

25. Jankowska EA, Rozentryt P, Witkowska A, Nowak J, Hartmann O, Ponikowska B, et al. Iron

deficiency predicts impaired exercise capacity in patients with systolic chronic heart failure. J

Card Fail 2011 Nov;17(11):899-906.

26. van Veldhuisen DJ, Anker SD, Ponikowski P, Macdougall IC. Anemia and iron deficiency in

heart failure: mechanisms and therapeutic approaches. Nat Rev Cardiol 2011 May 31;8(9):485-

493.

27. Alexandrakis MG, Tsirakis G. Anemia in heart failure patients. ISRN Hematol

2012;2012:246915.

29

28. Weiss G. Iron metabolism in the anemia of chronic disease. Biochim Biophys Acta 2009

Jul;1790(7):682-693.

29. Weiss G, Goodnough LT. Anemia of chronic disease. N Engl J Med 2005 Mar

10;352(10):1011-1023.

30. Balla J, Jeney V, Varga Z, Komodi E, Nagy E, Balla G. Iron homeostasis in chronic in-

flammation. Acta Physiol Hung 2007 Mar;94(1-2):95-106.

31. Comin-Colet J, Lainscak M, Dickstein K, Filippatos GS, Johnson P, Luscher TF, et al. The

effect of intravenous ferric carboxymaltose on health-related quality of life in patients with chron-

ic heart failure and iron deficiency: a subanalysis of the FAIR-HF study. Eur Heart J 2013

Jan;34(1):30-38.

32. Anker SD, Comin Colet J, Filippatos G, Willenheimer R, Dickstein K, Drexler H, et al. Ferric

carboxymaltose in patients with heart failure and iron deficiency. N Engl J Med 2009 Dec

17;361(25):2436-2448.

33. Filippatos G, Farmakis D, Colet JC, Dickstein K, Luscher TF, Willenheimer R, et al. Intrave-

nous ferric carboxymaltose in iron-deficient chronic heart failure patients with and without anae-

mia: a subanalysis of the FAIR-HF trial. Eur J Heart Fail 2013 Nov;15(11):1267-1276.

34. Pruthi RK, Tefferi A. Pernicious anemia revisited. Mayo Clin Proc 1994 Feb;69(2):144-150.

35. Green R, Kinsella LJ. Current concepts in the diagnosis of cobalamin deficiency. Neurology

1995 Aug;45(8):1435-1440.

36. de Jager J, Kooy A, Lehert P, Wulffele MG, van der Kolk J, Bets D, et al. Long term treat-

ment with metformin in patients with type 2 diabetes and risk of vitamin B-12 deficiency: ran-

domised placebo controlled trial. BMJ 2010 May 20;340:c2181.

37. Lam JR, Schneider JL, Zhao W, Corley DA. Proton pump inhibitor and histamine 2 receptor

antagonist use and vitamin B12 deficiency. JAMA 2013 Dec 11;310(22):2435-2442.

38. Stabler SP. Clinical practice. Vitamin B12 deficiency. N Engl J Med 2013 Jan 10;368(2):149-

160.

39. Antony AC. Megaloblastic anemias. In: Hoffman R, Benz EJ, Shattil SJ, editors. Hematology:

Basic principles and practice. 4th ed. New York: Churchill Livingstone; 2005. p. 519.

40. Clarke R, Grimley Evans J, Schneede J, Nexo E, Bates C, Fletcher A, et al. Vitamin B12 and

folate deficiency in later life. Age Ageing 2004 Jan;33(1):34-41.

41. Bertino JR. Karnofsky memorial lecture. Ode to methotrexate. J Clin Oncol 1993 Jan;11(1):5-

14.

42. Okada A, Koike H, Nakamura T, Watanabe H, Sobue G. Slowly progressive folate-deficiency

myelopathy: Report of a case. J Neurol Sci 2014 Jan 15;336(1-2):273-275.

30

43. Green R, Miller JW. Folate deficiency beyond megaloblastic anemia: hyperhomocysteinemia

and other manifestations of dysfunctional folate status. Semin Hematol 1999 Jan;36(1):47-64.

44. Witte KK, Desilva R, Chattopadhyay S, Ghosh J, Cleland JG, Clark AL. Are hematinic defi-

ciencies the cause of anemia in chronic heart failure? Am Heart J 2004 May;147(5):924-930.

45. Comin-Colet J, Enjuanes C, Gonzalez G, Torrens A, Cladellas M, Merono O, et al. Iron defi-

ciency is a key determinant of health-related quality of life in patients with chronic heart failure

regardless of anaemia status. Eur J Heart Fail 2013 Oct;15(10):1164-1172.

46. Jankowska EA, Rozentryt P, Witkowska A, Nowak J, Hartmann O, Ponikowska B, et al. Iron

deficiency: an ominous sign in patients with systolic chronic heart failure. Eur Heart J 2010

Aug;31(15):1872-1880.

47. Bruggink-Andre de la Porte PW, Lok DJ, van Wijngaarden J, Cornel JH, Pruijsers-Lamers D,

van Veldhuisen DJ, et al. Heart failure programmes in countries with a primary care-based health

care system. Are additional trials necessary? Design of the DEAL-HF study. Eur J Heart Fail

2005 Aug;7(5):910-920.

48. Beilby J, Olynyk J, Ching S, Prins A, Swanson N, Reed W, et al. Transferrin index: an alter-

native method for calculating the iron saturation of transferrin. Clin Chem 1992 Oct;38(10):2078-

2081.

49. Levey AS, Bosch JP, Lewis JB, Greene T, Rogers N, Roth D. A more accurate method to es-

timate glomerular filtration rate from serum creatinine: a new prediction equation. Modification

of Diet in Renal Disease Study Group. Ann Intern Med 1999 Mar 16;130(6):461-470.

50. Selhub J, Jacques PF, Dallal G, Choumenkovitch S, Rogers G. The use of blood concentra-

tions of vitamins and their respective functional indicators to define folate and vitamin B12 status.

Food Nutr Bull 2008 Jun;29(2 Suppl):S67-73.

51. Bolger AP, Bartlett FR, Penston HS, O'Leary J, Pollock N, Kaprielian R, et al. Intravenous

iron alone for the treatment of anemia in patients with chronic heart failure. J Am Coll Cardiol

2006 Sep 19;48(6):1225-1227.

52. Toblli JE, Lombrana A, Duarte P, Di Gennaro F. Intravenous iron reduces NT-pro-brain na-

triuretic peptide in anemic patients with chronic heart failure and renal insufficiency. J Am Coll

Cardiol 2007 Oct 23;50(17):1657-1665.

53. Okonko DO, Grzeslo A, Witkowski T, Mandal AK, Slater RM, Roughton M, et al. Effect of

intravenous iron sucrose on exercise tolerance in anemic and nonanemic patients with sympto-

matic chronic heart failure and iron deficiency FERRIC-HF: a randomized, controlled, observer-

blinded trial. J Am Coll Cardiol 2008 Jan 15;51(2):103-112.

54. Usmanov RI, Zueva EB, Silverberg DS, Shaked M. Intravenous iron without erythropoietin

for the treatment of iron deficiency anemia in patients with moderate to severe congestive heart

failure and chronic kidney insufficiency. J Nephrol 2008 Mar-Apr;21(2):236-242.

31

55. Parikh A, Natarajan S, Lipsitz SR, Katz SD. Iron deficiency in community-dwelling US

adults with self-reported heart failure in the National Health and Nutrition Examination Survey

III: prevalence and associations with anemia and inflammation. Circ Heart Fail 2011

Sep;4(5):599-606.

56. Nutritional anaemias. Report of a WHO scientific group. World Health Organ Tech Rep Ser

1968;405:5-37.

57. Rector TS, Cohn JN. Assessment of patient outcome with the Minnesota Living with Heart

Failure questionnaire: reliability and validity during a randomized, double-blind, placebo-

controlled trial of pimobendan. Pimobendan Multicenter Research Group. Am Heart J 1992

Oct;124(4):1017-1025.

58. Cuzick J. A Wilcoxon-type test for trend. Stat Med 1985 Jan-Mar;4(1):87-90.

59. Matchar DB, McCrory DC, Millington DS, Feussner JR. Performance of the serum cobalamin

assay for diagnosis of cobalamin deficiency. Am J Med Sci 1994 Nov;308(5):276-283.

60. Lindenbaum J, Savage DG, Stabler SP, Allen RH. Diagnosis of cobalamin deficiency: II. Rel-

ative sensitivities of serum cobalamin, methylmalonic acid, and total homocysteine concentra-

tions. Am J Hematol 1990 Jun;34(2):99-107.

61. Carmel R, Agrawal YP. Failures of cobalamin assays in pernicious anemia. N Engl J Med

2012 Jul 26;367(4):385-386.

62. Carmel R, Brar S, Agrawal A, Penha PD. Failure of assay to identify low cobalamin concen-

trations. Clin Chem 2000 Dec;46(12):2017-2018.

63. Carmel R. How I treat cobalamin (vitamin B12) deficiency. Blood 2008 Sep 15;112(6):2214-

2221.

64. Oberley MJ, Yang DT. Laboratory testing for cobalamin deficiency in megaloblastic anemia.

Am J Hematol 2013 Jun;88(6):522-526.

65. Solomon LR. Cobalamin-responsive disorders in the ambulatory care setting: unreliability of

cobalamin, methylmalonic acid, and homocysteine testing. Blood 2005 Feb 1;105(3):978-85; au-

thor reply 1137.

66. Carmel R. Holotranscobalamin: not ready for prime time. Clin Chem 2012 Mar;58(3):643-5;

author reply 645-6.

67. Savage DG, Lindenbaum J, Stabler SP, Allen RH. Sensitivity of serum methylmalonic acid

and total homocysteine determinations for diagnosing cobalamin and folate deficiencies. Am J

Med 1994 Mar;96(3):239-246.

68. Stabler SP, Marcell PD, Podell ER, Allen RH, Savage DG, Lindenbaum J. Elevation of total

homocysteine in the serum of patients with cobalamin or folate deficiency detected by capillary

gas chromatography-mass spectrometry. J Clin Invest 1988 Feb;81(2):466-474.

32

69. Heywood JT, Fonarow GC, Costanzo MR, Mathur VS, Wigneswaran JR, Wynne J, et al.

High prevalence of renal dysfunction and its impact on outcome in 118,465 patients hospitalized

with acute decompensated heart failure: a report from the ADHERE database. J Card Fail 2007

Aug;13(6):422-430.

70. Adams KF,Jr, Fonarow GC, Emerman CL, LeJemtel TH, Costanzo MR, Abraham WT, et al.

Characteristics and outcomes of patients hospitalized for heart failure in the United States: ra-

tionale, design, and preliminary observations from the first 100,000 cases in the Acute Decom-

pensated Heart Failure National Registry (ADHERE). Am Heart J 2005 Feb;149(2):209-216.

71. Hillege HL, Girbes AR, de Kam PJ, Boomsma F, de Zeeuw D, Charlesworth A, et al. Renal

function, neurohormonal activation, and survival in patients with chronic heart failure. Circula-

tion 2000 Jul 11;102(2):203-210.

72. Ezekowitz J, McAlister FA, Humphries KH, Norris CM, Tonelli M, Ghali WA, et al. The as-

sociation among renal insufficiency, pharmacotherapy, and outcomes in 6,427 patients with heart

failure and coronary artery disease. J Am Coll Cardiol 2004 Oct 19;44(8):1587-1592.

73. Smith GL, Lichtman JH, Bracken MB, Shlipak MG, Phillips CO, DiCapua P, et al. Renal im-

pairment and outcomes in heart failure: systematic review and meta-analysis. J Am Coll Cardiol

2006 May 16;47(10):1987-1996.

74. van der Meer P, van Veldhuisen DJ. Anaemia and renal dysfunction in chronic heart failure.

Heart 2009 Nov;95(21):1808-1812.

75. Galloway M, Rushworth L. Red cell or serum folate? Results from the National Pathology

Alliance benchmarking review. J Clin Pathol 2003 Dec;56(12):924-926.

76. Farrell CJ, Kirsch SH, Herrmann M. Red cell or serum folate: what to do in clinical practice?

Clin Chem Lab Med 2013 Mar 1;51(3):555-569.

77. Brugnara C, Mohandas N. Red cell indices in classification and treatment of anemias: from

M.M. Wintrobes's original 1934 classification to the third millennium. Curr Opin Hematol 2013

May;20(3):222-230.

78. Kraai IH, Vermeulen KM, Luttik ML, Hoekstra T, Jaarsma T, Hillege HL. Preferences of

heart failure patients in daily clinical practice: quality of life or longevity? Eur J Heart Fail 2013

Oct;15(10):1113-1121.

79. Herrmann M, Muller S, Kindermann I, Gunther L, Konig J, Bohm M, et al. Plasma B vita-

mins and their relation to the severity of chronic heart failure. Am J Clin Nutr 2007

Jan;85(1):117-123.

80. van Deursen VM, Damman K, Hillege HL, van Beek AP, van Veldhuisen DJ, Voors AA. Ab-

normal liver function in relation to hemodynamic profile in heart failure patients. J Card Fail

2010 Jan;16(1):84-90.

81. Giallourakis CC, Rosenberg PM, Friedman LS. The liver in heart failure. Clin Liver Dis 2002

Nov;6(4):947-67, viii-ix.

33

82. Kanazawa S, Herbert V. Total corrinoid, cobalamin (vitamin B12), and cobalamin analogue

levels may be normal in serum despite cobalamin in liver depletion in patients with alcoholism.

Lab Invest 1985 Jul;53(1):108-110.

83. Baker H, Leevy CB, DeAngelis B, Frank O, Baker ER. Cobalamin (vitamin B12) and holo-

transcobalamin changes in plasma and liver tissue in alcoholics with liver disease. J Am Coll Nutr

1998 Jun;17(3):235-238.