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Vitamin B12 and folate deficiency in chronic heart failure

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DOI: 10.1136/heartjnl-2014-306022 · Source: PubMed

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1 (HF).
Department of Cardiology,
University Medical Center
Groningen, University of
Groningen, Groningen,
The Netherlands
2
Heart Failure Program,
Hospital del Mar and
Department of Medicine,
Universitat Autonoma de
Barcelona, Barcelona, Spain
3
Department of Heart Diseases,
Wroclaw Medical University,
Wroclaw, Poland
4
Cardiology Department,
Military Hospital, Wroclaw,
Poland
Correspondence to
Dr Peter van der Meer,
Department of Cardiology,
Thorax Center, University
Medical Center Groningen,
Hanzeplein 1, PO Box 30001,
Groningen 9700 RB,
The Netherlands;
p.van.der.meer@umcg.nl
Received 10 April 2014
Revised 26 August 2014
Accepted 23 September 2014
To cite: van der Wal HH,
Comin-Colet J, Klip IT, et al.
Heart Published Online First:
[please include Day Month
Year] doi:10.1136/heartjnl-
2014-306022
Copyright Article author (or their employer) 2014. Produced by BMJ Publishing Group Ltd (& BCS) under licence.
Heart failure and cardiomyopathies
ORIGINAL ARTICLE
Vitamin B12 and folate deficiency in chronic
heart failure
Haye H van der Wal,1 Josep Comin-Colet,2 Ijsbrand T Klip,1 Cristina Enjuanes,2
Niels Grote Beverborg,1 Adriaan A Voors,1 Waldemar Banasiak,3
Dirk J van Veldhuisen,1 Jordi Bruguera,2 Piotr Ponikowski,3,4 Ewa A Jankowska,3,4
Peter van der Meer1
ABSTRACT
Objective To determine the prevalence, clinical
correlates and the effects on outcome of vitamin B12
and folic acid levels in patients with chronic heart failure
Methods We studied an international pooled cohort
comprising 610 patients with chronic HF. The main
outcome measure was all-cause mortality.
Results Mean age of the patients was 68±12 years
and median serum N-terminal prohormone brain
natriuretic peptide level was 1801 pg/mL (IQR 705–
4335). Thirteen per cent of the patients had an LVEF
>45%. Vitamin B12 deficiency (serum level <200 pg/
mL), folate deficiency (serum level <4.0 ng/mL) and iron
deficiency (serum ferritin level <100 mg/L, or
100–299 mg/L with a transferrin saturation <20%) were
present in 5%, 4% and 58% of the patients,
respectively. No significant correlation between mean
corpuscular volume and vitamin B12, folic acid or ferritin
levels was observed. Lower folate levels were associated
with an impaired health-related quality of life ( p=0.029).
During a median follow-up of 2.10 years (1.31–
3.60 years), 254 subjects died. In multivariable
proportional hazard models, vitamin B12 and folic acid
levels were not associated with prognosis.
Conclusions Vitamin B12 and folate deficiency are
relatively rare in patients with chronic HF. Since no
significant association was observed between mean
corpuscular volume and neither vitamin B12 nor folic
acid levels, this cellular index should be used with
caution in the differential diagnosis of anaemia in
patients with chronic HF. In contrast to iron deficiency,
vitamin B12 and folic acid levels were not related to
prognosis.
INTRODUCTION
Iron deficiency is common in chronic heart failure
(HF) and associated with impaired exercise capacity
and worse clinical outcome, even in the absence of
anaemia.1–3 Several studies have shown beneficial
effects in the treatment of iron deficiency by
administering intravenous iron, resulting in
improved clinical status and quality of life.4–6
Besides iron deficiency, other nutritional deficiencies
may also negatively impact clinical status and outcome
in patients with HF. Both vitamin B12 and folic acid
are essential for normal erythrocyte production.
Haematinic deficiencies may cause anaemia, although
van der Wal HH, et al. Heart 2014;0:1–9. doi:10.1136/heartjnl-2014-306022
haematological parameters (i.e. haemoglobin, mean
corpuscular volume (MCV), haematocrit, red blood
cell distribution width and reticulocyte count) may be
completely normal in these patients.7 Vitamin B12
plays a significant role in DNA synthesis, and deficien-
cies may lead to inadequate erythropoiesis due to
desynchronised maturation of red cell nuclei and cyto-
plasm. This can result in intramedullary haemolysis,
which mimics the characteristics of microangiopathic
haemolytic anaemia.8 Folic acid is also obligatory for
DNA synthesis. Folate deficiency limits erythropoiesis,
which may also lead to megaloblastic anaemia.
To date, studies on haematinic deficiencies in
chronic HF are scarce, and information on clinical
correlates or prognostic consequences of these defi-
ciencies in chronic HF is currently unknown. One
study from the UK determined the prevalence of
haematinic deficiencies in patients with chronic HF.
Vitamin B12 deficiency and folate deficiency were
present in 6% and 8% of the subjects, respectively.9
However, in this relatively small study including
only New York Health Association (NYHA) class
IV patients, clinical outcome data were lacking. In
the present study, we measured vitamin B12 and
folic acid in a large, international pooled cohort of
patients with HF and studied the prevalence, clin-
ical associations and prognosis of these deficiencies.
METHODS
Study population
The study population consisted of 610 patients,
originating from an international pooled cohort of
1506 patients with chronic stable HF with pre-
served or reduced LVEF, as described elsewhere.1
In this subset of 610 patients, clinical follow-up
data and serum vitamin B12 and folic acid levels
were available. Inclusion and exclusion criteria per
study cohort are shown in online supplementary
table S1.
Laboratory measurements
Haematinics were assessed from fresh venous blood.
Blood samples were centrifuged at 3500 rmp for
15 min (4°C) and stored at −70°C afterwards. The
following blood markers reflecting iron metabolism
were assessed using standard methods: serum iron
(μg/dL), ferritin (μg/L) and transferrin (mg/dL). To
calculate the transferrin saturation (TSAT), serum
iron was divided by 25.2 and then divided by trans-
ferrin. This value was then multiplied by 100.10
1
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Heart failure and cardiomyopathies
Renal function was assessed using the estimated glomerular
filtration rate (eGFR, mL/min/1.73 m2), calculated from the sim-
plified Modification of Diet in Renal Disease equation. Vitamin
B12, folic acid and N-terminal prohormone brain natriuretic
peptide (NT-proBNP) levels were determined using an immuno-
assay based on electrochemiluminescence (Elecsys, Roche
Diagnostics, Mannheim, Germany). Serum concentrations of
high-sensitive C-reactive protein (hs-CRP) and other blood
markers were assessed using standard methods.
Definitions and study end points
Nutritional deficiencies were defined as follows: serum vitamin
B12<200 pg/mL8 and serum folic acid <4.0 ng/mL.11 Anaemia
was defined as a haemoglobin level <12 g/dL in women and
<13 g/dL in men, in agreement with WHO standards.12 Iron
deficiency was defined as a serum ferritin level <100 mg/L or
serum ferritin 100–299 with a TSAT <20%, reflecting both
absolute and functional iron deficiency. Quality of life was
assessed by means of the Minnesota Living with Heart Failure
Questionnaire (MLHFQ).13 Data regarding demographics and
clinical signs (age at diagnosis, sex, HF aetiology, renal function,
EF, blood pressure and comorbidities), haematinics (haemoglo-
bin, MCV, serum iron, ferritin, transferrin, vitamin B12 and folic
acid) and therapeutics were available for all patients. Follow-up
for surviving patients was censored after 9 years, when <5% of
all patients were at risk. The primary end point of this study
was all-cause mortality.
Statistical analysis
Data are presented as mean±SD when normally distributed, as
median and IQR when non-normally distributed or as percent-
age when categorical. Intergroup differences were tested using
parametric and non-parametric trend tests, when appropriate.
Baseline characteristics stratified by study cohort were compared
using one-way analysis of variance test or Kruskal–Wallis test,
when appropriate. To assess the distribution of haematinics for
different MCV levels, serum levels of vitamin B12, folate, fer-
ritin and TSAT were stratified by MCV levels in tertiles.
Changes in serum levels of haematinics were tested using a non-
parametric trend test (i.e. extension of Wilcoxon rank-sum test).
After baseline analyses, the follow skewed variables were loga-
rithmically transformed for all further analyses to achieve a
normal distribution: serum NT-proBNP, erythropoietin, iron,
ferritin, vitamin B12, folic acid, TSAT, hs-CRP and eGFR.
To determine clinical correlates of vitamin B12 and folic acid,
univariable linear regression models were constructed using age,
sex, body mass index (BMI), LVEF, systolic blood pressure,
haemoglobin, MCV, eGFR, TSAT, serum NT-proBNP, hs-CRP,
ferritin and erythropoietin, comorbidities, NYHA functional
class, HF aetiology, health-related quality of life and treatment
with angiotensin-converting enzyme inhibitors, angiotensin
receptor blockers, aldosterone antagonists, β-blockers, statins,
loop diuretics, antiplatelets and anticoagulants as covariates.
Variables with a significant univariable correlation with either
serum vitamin B12 and/or folic acid levels (i.e. p<0.10) were
entered in the multivariable linear regression models.
Kaplan–Meier curves were constructed to determine the
effect of vitamin B12 and folic acid levels on cumulative survival.
Differences in survival rates were tested using the log-rank
Mantel–Cox test. To determine the prognostic consequences of
vitamin B12 and folic acid, univariable and multivariable Cox
proportional hazard regression models were constructed. In the
multivariable model, adjustment was made for all univariably
significant variables, including age, sex, BMI, LVEF,
2 van der Wal HH, et al. Heart 2014;0:1–9. doi:10.1136/heartjnl-2014-306022
haemoglobin level, eGFR, systolic blood pressure and serum
NT-proBNP, hs-CRP and erythropoietin levels.
A two-sided p value <0.05 was considered statistically signifi-
cant. Data were analysed using STATA V.12.0 (StataCorp LP,
College Station, Texas, USA).
RESULTS
Baseline characteristics
Baseline characteristics for all 610 patients were stratified
according to serum vitamin B12 and folic acid levels in quartiles
(tables 1 and 2). Patients were recruited from Poland, the
Netherlands and Spain. Baseline characteristics for all 610
patients stratified by study cohort are shown in online supple-
mentary table S2. Median serum vitamin B12 and folic acid
levels (+IQR) were 435 (312–600) pg/mL and 9.4 (6.8–12.5)
ng/mL, respectively. Vitamin B12 levels were lowest in the Dutch
subcohort, whereas folic acid levels were lowest in the Polish
subcohort. Vitamin B12 and folate deficiency were present in
5.4% and 4.1% of the patients, respectively. More than half of
the patients were iron deficient (58%). The prevalence of
vitamin B12 and folate deficiency was equal in patients with and
without iron deficiency (5.9% versus 4.7%, p=0.516, and 3.1%
versus 5.5%, p=0.142, respectively). Folate deficiency was more
prevalent in anaemic patients (6.7% versus 2.8% in non-
anaemic patients, p=0.020), whereas vitamin B12 deficiency was
not (6.2% versus 5.0%, p=0.538). None of the patients had
both vitamin B12 and folate deficiency.
Clinical correlates
Univariable and multivariable linear regression models are
shown in tables 3 and 4. Lower serum folic acid levels were
associated with the presence of atrial fibrillation and Polish and
Spanish study cohorts ( p=0.001). Higher concentrations of
serum vitamin B12 were correlated with higher NT-proBNP and
ferritin levels, the presence of atrial fibrillation and the Spanish
study cohort (all p<0.05). Lower serum vitamin B12 levels were
associated with systolic blood pressure ( p=0.008). There was
no significant correlation between haematinics and treatment in
all multivariable models.
Multivariable linear regression models showed that better
quality of life (as assessed by MLHFQ) was significantly corre-
lated to serum folic acid levels ( p<0.029).
Haematinic deficiencies and MCV
Serum levels of haematinics stratified by MCV levels in tertiles
are shown in figure 1. TSAT was significantly lower in patients
with lower MCV at baseline ( p for trend <0.001). A similar
pattern was observed when only anaemic patients were selected
(see online supplementary figure S1). Interestingly, there was no
significant trend in prevalence of vitamin B12 and folate defi-
ciency for different MCV levels.
Haematinics and survival
During a median follow-up of 2.10 years (IQR 1.31–3.60 years),
254 patients (42%) died. Differences in event-free survival strati-
fied by vitamin B12 and folic acid in quartiles are displayed in
figures 2 and 3. Increased mortality rates were seen in patients
with vitamin B12 levels >600 pg/mL versus those with levels
<600 pg/mL (47.7% versus 41.8%; p=0.026). In multivariable
Cox proportional hazard regression models, vitamin B12 lost its
predictive value for all-cause mortality (table 5). Mortality was
similar in patients with higher and lower folic acid levels
(p=0.745), and the absence of an association between folic acid
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Heart failure and cardiomyopathies

Table 1 Baseline characteristics, stratified by vitamin B12 levels in quartiles

Total cohort <313 pg/mL 313–435 pg/mL 436–600 pg/mL >600 pg/mL p Value
Variables (n=610) (n=153) (n=153) (n=153) (n=151) (trend)

Demographics and clinical signs

Age (years) 68±12 68±12 68±11 67±12 69±12 0.388
Men 71 72 71 73 70 0.882
BMI (kg/m2) 27.7±5.3 27.7±5.3 28.0±5.0 27.9±5.4 27.2±5.4 0.285
BMI <18 1.1 1.3 0 1.3 2.0 0.389
Ischaemic aetiology 55 59 58 58 46 0.027
eGFR (mL/min/1.73 m2) 67.4 (50.3–99.0) 67.7 (50.3–90.5) 71.6 (53.4–117.1) 65.5 (53.8–94.7) 62.2 (44.3–88.2) 0.152
eGFR <60 39 38 34 41 45 0.116
LVEF (%) 33±13 33±13 36±13 32±12 32±14 0.027
LVEF >45% 13 12 17 10 13 0.699
SBP (mm Hg) 122±22 124±21 125±24 122±23 117±19 0.001
MLHFQ 45 (26–61) 45 (23–62) 43 (27–59) 43 (28–61) 47 (25–62) 0.494
Comorbidities
Diabetes mellitus 40 36 36 43 43 0.108
Atrial fibrillation 30 24 29 27 38 0.013
Hypertension 49 44 56 51 45 0.941
Haematinics
Hb (g/dL) 13.4±1.8 13.0±1.7 13.3±1.8 13.7±1.9 13.3±1.9 0.071
MCV (fL) 89.8±5.8 89.8±5.5 89.5±5.9 90.0±5.7 89.7±6.1 0.849
Anaemia* (%) 34 36 34 31 37 0.998
EPO (IU/L) 18.0 (11.3–29.0) 18.1 (13.2–26.2) 17.2 (11.4–27.4) 17.0 (11.2–30.3) 17.9 (10.1–36.2) 0.733
Iron (μg/dL) 67.0 (42.0–91.0) 63.0 (38.0–93.3) 67.0 (44.7–90.0) 71.0 (52.0–89.0) 63.0 (37.0–91.0) 0.992
Ferritin (μg/L) 148 (78–273) 119 (56–239) 137 (78–233) 154 (85–286) 172 (94–340) <0.001
TSAT (%) 19 (12–26) 17 (11–26) 19 (13–26) 19 (14–25) 18 (10–26) 0.738
Iron deficiency† 58 64 61 56 52 0.016
Vitamin B12 (pg/mL) 435 (312–600) 250 (205–280) 377 (343–405) 503 (471–539) 784 (676–948) N/A
Vitamin B12 deficiency‡ 5.4 21.6 0 0 0 N/A
Folic acid (ng/mL) 9.4 (6.8–12.5) 9.4 (7.0–11.9) 8.5 (6.5–11.1) 9.9 (7.2–13.1) 9.7 (6.9–13.6) 0.059
Folate deficiency§ 4.1 3.3 6.5 3.3 3.3 0.664
Cardiac biomarkers
NT-proBNP (pg/mL) 1801 (705–4335) 1556 (498–3926) 1562 (726–3598) 1695 (650–3999) 2987 (956–5463) 0.001
hs-CRP (mg/L)¶ 4.0 (1.5–10.0) 4.0 (1.4–9.7) 3.2 (1.3–7.8) 3.9 (2.0–9.4) 6.0 (2.0–17.0) 0.049
Treatment
ACE inhibitor and/or ARB 90 94 88 92 88 0.186
β-Blocker 87 90 86 92 82 0.160
Aldosterone antagonist 51 50 47 54 52 0.425
Statins 55 54 55 59 50 0.670
Loop diuretics 86 85 85 84 89 0.319
Antiplatelet and/or 85 82 86 88 84 0.478
anticoagulant
Values are given as means±SD, medians (IQR) or proportions (%).
*Anaemia is defined as Hb <12 g/dL in women and <13 g/dL in men.
†Iron deficiency is defined as serum ferritin <100 μg/L or 100–299 μg/L with a transferrin saturation <20%.
‡Vitamin B12 deficiency is defined as serum vitamin B12 level <200 pg/mL.
§Folate deficiency is defined as serum folic acid level <4.0 ng/mL.
¶Serum hs-CRP levels were determined in 385 patients.
BMI, body mass index; eGFR, estimated glomerular filtration rate; EPO, erythropoietin; Hb, haemoglobin; hs-CRP, high-sensitive C-reactive protein; 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; ARB, angiotensin receptor blockers.

levels and mortality remained present in multivariable Cox pro-
portional hazard regression models.
DISCUSSION
In an international pooled cohort comprising 610 patients with
chronic HF, we demonstrate that vitamin B12 and folate defi-
ciency are relatively rare (4% and 5%, respectively). Second,
there is no significant correlation between MCV and vitamin B12
and folic acid levels. Finally, vitamin B12 and folic acid levels
were not independently associated with mortality.
Prevalence and definition of haematinic deficiencies
Unfortunately, there are no clear definitions of vitamin B12 and
folate deficiency, leading to different thresholds for the pres-
ence of these deficiencies, and consequently to differences in
prevalence. 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,8 serum
vitamin B12 levels <200 pg/mL are suggestive for clinical defi-
ciency (sensitivity and specificity 65%–100% and 50%–60%,
respectively). With serum levels >300 pg/mL, vitamin B12

van der Wal HH, et al. Heart 2014;0:1–9. doi:10.1136/heartjnl-2014-306022 3

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Heart failure and cardiomyopathies

Table 2 Baseline characteristics, stratified by folic acid levels in quartiles

Total cohort <6.9 ng/mL 6.9–9.4 ng/mL 9.4–12.5 ng/mL >12.5 ng/mL p Value
Variables (n=610) (n=153) (n=153) (n=153) (n=151) (trend)

Demographics and clinical signs

Age (years) 68±12 66±12 68±11 67±12 69±12 0.102
Men 71 73 74 71 69 0.388
BMI (kg/m2) 27.7±5.3 28.3±5.5 28.0±5.5 27.7±4.8 26.7±5.2 0.009
BMI <18 1.1 0 1.3 0 3.4 0.025
Ischaemic aetiology 55 55 55 51 61 0.407
eGFR (mL/min/1.73 m2) 67.4 (50.3–99.0) 91.6 (60.7–141.68) 75.9 (58.3–120.1) 62.8 (47.9–82.1) 56.3 (42.6–69.7) <0.001
eGFR <60 39 24 28 45 61 <0.001
LVEF (%) 33±13 36±15 34±14 34±13 30±11 <0.001
LVEF >45% 13 22 14 13 3 <0.001
SBP (mm Hg) 122±22 119±22 125±21 124±23 120±20 0.703
MLHFQ 45 (26–61) 45 (29–63) 46 (26–64) 44 (26–60) 39 (24–57) 0.019
Comorbidities
Diabetes mellitus 40 42 44 39 33 0.085
Atrial fibrillation 30 35 26 31 26 0.158
Hypertension 49 56 62 49 29 <0.001
Haematinics
Hb (g/dL) 13.4±1.8 13.1±2.0 13.3±1.9 13.5±1.7 13.5±1.7 0.022
MCV (fL) 89.8±5.8 89.7±6.3 89.3±5.9 90.4±5.5 89.8±5.4 0.691
Anaemia* (%) 34 46 34 30 27 0.001
EPO (IU/L) 18.0 (11.3–29.0) 18.0 (12.0–30.3) 18.1 (10.3–33.0) 18.0 (12.0–29.0) 16.2 (11.0–27.7) 0.337
Iron (μg/dL) 67.0 (42.0–91.0) 71.0 (45.0–97.0) 70.4 (47.2–95.5) 62.5 (36.0–83.0) 64.8 (36.3–86.0) 0.001
Ferritin (μg/L) 148 (78–273) 167 (78–308) 128 (69–214) 148 (75–292) 154 (84–267) 0.705
TSAT (%) 19 (12–26) 19 (13–28) 20 (13–26) 18 (10–25) 18 (10–24) 0.002
Iron deficiency† 58 59 63 59 61 0.065
Vitamin B12 (pg/mL) 435 (312–600) 428 (318–568) 404 (307–596) 430 (305–547) 485 (326–663) 0.189
Vitamin B12 deficiency‡ 5.4 3.3 8.6 6.3 3.4 0.850
Folic acid (ng/mL) 9.4 (6.8–12.5) 5.4 (4.4–6.2) 8.0 (7.4–8.5) 10.7 (10.0–11.5) 15.5 (13.8–19.2) N/A
Folate deficiency§ 4.1 16.3 0 0 0 N/A
Cardiac biomarkers
NT-proBNP (pg/mL) 1801 (705–4335) 1371 (621–3446) 1746 (580–4680) 1945 (735–4313) 2097 (864–4905) 0.015
hs-CRP (mg/L)¶ 4.0 (1.5–10.0) 4.5 (1.4–7.9) 4.6 (1.5–9.0) 4.0 (1.5–12.2) 4.1 (1.5–10.0) 0.948
Treatment
ACE inhibitor and/or ARB 90 89 86 92 94 0.050
β-Blocker 87 88 91 87 83 0.094
Aldosterone antagonist 51 51 53 47 52 0.801
Statins 55 61 57 50 50 0.027
Loop diuretics 86 85 84 85 90 0.169
Antiplatelet and/or 85 85 84 85 85 0.953
anticoagulant
Values are given as means±SD, medians (IQR) or proportions (%).
*Anaemia is defined as Hb <12 g/dL in women and <13 g/dL in men.
†Iron deficiency is defined as serum ferritin <100 μg/L or 100–299 μg/L with a transferrin saturation <20%.
‡Vitamin B12 deficiency is defined as serum vitamin B12 level <200 pg/mL.
§Folate deficiency is defined as serum folic acid level <4.0 ng/mL.
¶Serum hs-CRP levels were determined in 385 patients.
BMI, body mass index; eGFR, estimated glomerular filtration rate; EPO, erythropoietin; Hb, haemoglobin; hs-CRP, high-sensitive C-reactive protein; MCV, mean corpuscular volume;
MLHFQ, Minnesota Living with Heart Failure Questionnaire; NT-proBNP, N-terminal prohormone brain natriuretic peptide; SBP, systolic blood pressure; TSAT, transferrin saturation; ARB,
angiotensin receptor blockers.

deficiency is unlikely (i.e. probability <5%).14 Exceptionally
low serum vitamin B12 levels (i.e. <100 pg/mL) are often asso-
ciated with overt deficiency, but such levels are hardly observed
(n=3 in the present study). Additionally, 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.11 15–17 New assays are currently in
development (e.g. holotranscobalamin), but these assays need
to be validated clinically before they can be used in clinical
healthcare.18
4 van der Wal HH, et al. Heart 2014;0:1–9. doi:10.1136/heartjnl-2014-306022
Measurement of methylmalonic acid and total homocysteine
was introduced several decades ago to diagnose vitamin B12
deficiency, in addition to serum vitamin B12 levels. However,
both serum methylmalonic acid and homocysteine may be ele-
vated in renal failure, making methylmalonic acid and homo-
cysteine 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 dysfunc-
tion (i.e. eGFR <60 mL/min/1.73 m2).19–21 In the current study,
renal dysfunction is present in 39% of all patients. Therefore,
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Heart failure and cardiomyopathies

Table 3 Clinical variables associated with vitamin B12 in chronic heart failure
Univariable regression Multivariable regression
Variable coefficient (95% CI) p Value t Value coefficient (95% CI) p Value t Value

Age, per 5 years 0.015 (−0.011 to 0.040) 0.249 1.15

Sex, female vs male −0.054 (−0.188 to 0.080) 0.429 −0.79
BMI, per 1 kg/m2 −0.004 (−0.016 to 0.008) 0.507 −0.66
LVEF, per 1% −0.003 (−0.008 to 0.001) 0.173 −1.36
SBP, per 1 mm Hg −0.005 (−0.008 to −0.002) <0.001 −3.60 −0.004 (−0.007 to −0.001) 0.008 −2.66
MCV, per 1 fL −0.001 (−0.011 to 0.010) 0.892 −0.14
Hb, per 1 g/dL 0.021 (−0.013 to 0.054) 0.226 0.21
eGFR, per doubling −0.051 (−0.126 to 0.023) 0.176 −1.36
NT-proBNP, per doubling 0.063 (.032 to 0.095) <0.001 3.97 0.063 (0.029 to 0.097) <0.001 3.67
hs-CRP*, per doubling 0.033 (−0.010 to 0.076) 0.133 1.51
EPO, per doubling 0.015 (−0.043 to 0.073) 0.615 0.50
Ferritin, per doubling 0.104 (.059 to 0.148) <0.001 4.57 0.097 (0.051 to 0.143) <0.001 4.14
TSAT, per doubling 0.023 (−0.032 to 0.077) 0.418 0.81
Folic acid, per doubling 0.035 (−0.048 to 0.118) 0.403 0.84
Atrial fibrillation, yes vs no 0.185 (0.053 to 0.317) 0.006 2.75 0.145 (0.012 to 0.278) 0.033 2.14
Diabetes, yes vs no 0.072 (−0.052 to 0.196) 0.253 1.14
Ischaemic aetiology, yes vs no −0.154 (−0.275 to −0.033) 0.013 −2.49 −0.086 (−0.212 to 0.039) 0.178 −1.35
NYHA functional class, III/IV vs I/II −0.016 (−0.142 to 0.111) 0.808 −0.24
MLHFQ, per point 0.001 (−0.001 to 0.004) 0.298 1.04
Study cohort (vs Holland)
Poland 0.161 (0.001 to 0.320) 0.048 1.98 0.121 (−0.044 to 0.285) 0.150 1.44
Spain 0.241 (0.101 to 0.381) 0.001 3.38 0.295 (0.151 to 0.439) <0.001 4.02
Treatment
ACE inhibitor and/or ARB, yes vs no −0.177 (−0.382 to 0.028) 0.090 −1.70 −0.012 (−0.224 to 0.199) 0.908 −0.12
Aldosterone antagonist, yes vs no 0.042 (−0.079 to 0.163) 0.495 0.68
β-Blocker, yes vs No −0.140 (−0.322 to 0.043) 0.133 −1.50
Statins, yes vs no −0.061 (−0.183 to 0.061) 0.324 −0.99
Loop diuretic, yes vs no 0.093 (−0.081 to 0.267) 0.296 1.05
Antiplatelet and/or anticoagulant, yes vs no 0.014 (−0.155 to 0.182) 0.874 0.16
*Serum hs-CRP levels were determined in 385 patients.
BMI, body mass index; eGFR, estimated glomerular filtration rate; EPO, erythropoietin; hs-CRP, high-sensitive C-reactive protein; MCV, mean corpuscular volume; MLHFQ, Minnesota
Living with Heart Failure Questionnaire; NYHA, New York Health Association; SBP, systolic blood pressure; TSAT, transferrin saturation; NT-proBNP, N-terminal prohormone brain
natriuretic peptide; Hb, haemoglobin; ARB, angiotensin receptor blockers.

serum methylmalonic acid and homocysteine would definitely
overestimate the prevalence of vitamin B12 deficiency in our
study. In short, there are currently no reliable screening markers
for vitamin B12 deficiency in patients with chronic HF apart
from serum vitamin B12 levels.
Measurement of serum folic acid levels is mainly a reflection
of short-term folic acid status. In the absence of recent fasting,
serum folic acid levels of <4 ng/mL are diagnostic for folate
deficiency.11 Alternatively, erythrocyte folic acid levels can be
used to diagnose folate 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.11 Farrell
et al22 stated that serum folic acid measurements may be super-
ior to erythrocyte folate measurements. As a consequence,
initial screening for folate deficiency can be performed using
serum folic acid levels, which is a relatively inexpensive meas-
urement. More important, serum folic acid measurements are
widely available and easy to perform. When serum folic acid
levels are borderline, erythrocyte folic acid levels can be mea-
sured to confirm the diagnosis.
The current study shows that serum folic acid levels are corre-
lated with renal dysfunction (i.e. serum folic acid levels are
higher when renal dysfunction is present). As stated earlier,
renal dysfunction is a relatively common comorbidity in HF,
van der Wal HH, et al. Heart 2014;0:1–9. doi:10.1136/heartjnl-2014-306022
and therefore, the prevalence of folate deficiency may be under-
estimated in patients with chronic HF when using serum folic
acid levels as a marker for folate deficiency.
Haematinics and quality of life
Our study shows that lower folic acid levels, but not vitamin B12
levels, are related to an impaired health-related quality of life as
assessed by a disease-specific questionnaire. A recent study by
Comin-Colet et al23 shows that iron deficiency is also correlated
to reduced quality of life. In this study comprising 552 patients
with chronic HF, anaemia did not have any significant influence
on quality of life. The importance of quality of life in patients
with chronic HF has been recently stressed by Kraai et al,24
stating that most patients with HF prioritise quality of life above
longevity.
Haematinics and survival
The predictive value of iron deficiency on mortality in patients
with chronic HF has been recently stressed by Klip et al,1
making iron status a strong and independent predictor of
outcome. Our results show that folic acid does not have such
predictive value on mortality. Univariable proportional hazard
analysis shows that patients with vitamin B12 levels >600 pg/mL
have higher mortality risks compared with patients with lower
levels. Vitamin B12 lost its predictive value on all-cause mortality
5
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Heart failure and cardiomyopathies

Table 4 Clinical variables associated with folic acid in chronic heart failure (HF)
Univariable regression coefficient Multivariable regression coefficient
Variable (95% CI) p Value t Value (95% CI) p Value t Value

Age, per 5 years 0.022 (−0.002 to 0.046) 0.076 1.78 0.022 (−0.006 to 0.050) 0.135 1.50
Sex, women vs men −0.023 (−0.152 to 0.106) 0.724 −0.35
BMI, per 1 kg/m2 −0.012 (−0.023 to −0.001) 0.037 −2.08 0.010 (−0.001 to 0.021) 0.086 1.72
LVEF, per 1% −0.009 (−0.013 to −0.005) <0.001 −4.04 −0.004 (−0.009 to 0.001) 0.085 −1.72
SBP, per 1 mm Hg 0.001 (−0.002 to 0.004) 0.576 0.56
MCV, per 1 fL 0.006 (−0.004 to 0.016) 0.251 1.15
Hb, per 1 g/dL 0.040 (0.008 to 0.072) 0.014 2.46 0.002 (−0.034 to 0.037) 0.920 0.10
eGFR, per doubling −0.279 (−0.347 to −0.211) <0.001 −8.06 −0.045 (−0.135 to 0.046) 0.331 −0.97
NT-proBNP, per doubling 0.029 (−0.002 to 0.059) 0.064 1.86 0.003 (−0.033 to 0.038) 0.885 0.14
hs-CRP*, per doubling 0.003 (−0.036 to 0.042) 0.881 0.15
EPO, per doubling −0.033 (−0.088 to 0.022) 0.243 −1.17
Ferritin, per doubling 0.009 (−0.035 to 0.052) 0.699 0.39
TSAT, per doubling −0.111 (−0.162 to −0.059) <0.001 −4.19 −0.012 (−0.067 to 0.043) 0.662 −0.44
Vitamin B12, per doubling 0.033 (−0.044 to 0.109) 0.403 0.84
Atrial fibrillation, yes vs no −0.164 (−0.291 to −0.037) 0.011 −2.54 −0.191 (−0.308 to −0.074) 0.001 −3.21
Diabetes, yes vs no −0.153 (−0.272 to −0.035) 0.011 −2.54 −0.032 (−0.148 to 0.084) 0.591 −0.54
Ischaemic aetiology, yes vs no 0.053 (−0.064 to 0.170) 0.376 0.89
NYHA functional class III/IV vs I/II 0.378 (0.261 to 0.495) <0.001 6.34 0.009 (−0.138 to 0.155) 0.907 0.12
MLHFQ, per point −0.004 (−0.006 to −0.001) 0.007 −2.73 −0.003 (−0.005 to −0.0003) 0.029 −2.18
Study cohort (vs Holland)
Poland −0.602 (−0.734 to −0.470) <0.001 −8.98 −0.520 (−0.702 to −0.338) <0.001 −5.61
Spain −0.878 (−0.993 to −0.763) <0.001 −14.93 −0.794 (−0.983 to −0.606) <0.001 −8.28
Treatment
ACE inhibitor and/or ARB, yes vs −0.149 (−0.047 to 0.346) 0.137 1.49
no
Aldosterone antagonist, yes vs no −0.013 (−0.129 to 0.104) 0.833 −0.21
β-Blocker, yes vs no −0.103 (−0.278 to 0.072) 0.249 −1.15
Statins, yes vs no −0.103 (−0.219 to 0.014) 0.084 −1.73 0.032 (−0.077 to 0.141) 0.563 0.58
Loop diuretic, yes vs no 0.124 (−0.042 to 0.291) 0.144 1.46
Antiplatelet and/or anticoagulant, −0.047 (−0.209 to 0.115) 0.567 −0.57
yes vs no
*Serum hs-CRP levels were determined in 385 patients.
BMI, body mass index; eGFR, estimated glomerular filtration rate; EPO, erythropoietin; hs-CRP, high-sensitive C-reactive protein; MCV, mean corpuscular volume; MLHFQ, Minnesota
Living with Heart Failure Questionnaire; NYHA, New York Health Association; SBP, systolic blood pressure; TSAT, transferrin saturation; ARB, angiotensin receptor blockers; NT-proBNP,
N-terminal prohormone brain natriuretic peptide; Hb, haemoglobin.

in multivariable proportional hazard analyses, especially in
patients with vitamin B12 levels >600 pg/mL. The univariable
effect on mortality is caused by the paradoxical association
between serum vitamin B12 levels and disease severity (serum
NT-proBNP levels). This association was observed earlier by
Herrmann et al25 and may be caused by the cardiohepatic syn-
drome. Chronic HF may cause blood congestion in the liver,
leading to hepatic cell damage and, consequently, to mild liver
dysfunction.26 Liver function deterioration may lead to the
release of hepatic and metabolically inactive vitamin B12 analo-
gues, making serum vitamin B12 measurements less accurate.
However, this cardiohepatic syndrome remains only a hypoth-
esis and is an incomplete explanation of the association between
serum vitamin B12 levels and disease severity.
Clinical implications
Traditionally, MCV is one of the conventional erythrocyte para-
meters and is used in the differential diagnosis of anaemia in
mainstream clinical practice, prior to measuring haematinics.
The importance of MCV in the differential diagnosis of
anaemia has been recently emphasised by Brugnara and
Mohandas.27 Levels of MCV have always been considered as
one of the hallmarks in the diagnosis of vitamin B12 and
folate deficiency since these deficiencies are frequently charac-
terised by an increase in MCV. In the present study, no associ-
ation between MCV and neither serum vitamin B12 nor folic
acid levels were observed, although we found a significant
association between MCV levels and the presence of iron defi-
ciency. While MCV is a reliable screening marker for haemati-
nic deficiencies in otherwise healthy people, the diagnostic
value of this cellular index seems limited in patients with
chronic HF.
In general, lower MCV levels increase the probability of iron
deficiency but do not effectively rule out vitamin B12 and/or
folate deficiency as the influence of serum vitamin B12 and folic
acid levels on MCV is limited when iron deficiency is present.
Thus, MCV levels in patients with chronic HF seem to be
mainly driven by iron status. Since iron deficiency is a common
comorbidity in patients with chronic HF,1 MCV levels will be
inevitably lower as the result of microcytic anaemia caused by
iron deficiency. The simultaneous presence of more than one
haematinic deficiency might cause MCV levels to be in the
normal range (i.e. normocytic anaemia). Even normal MCV
levels in the absence of iron deficiency do not rule out haemati-
nic deficiencies as macrocytosis is frequently absent in vitamin
B12 and folate deficiency.28 Therefore, MCV should be used

6 van der Wal HH, et al. Heart 2014;0:1–9. doi:10.1136/heartjnl-2014-306022

 Downloaded from http://heart.bmj.com/ on November 12, 2014 - Published by group.bmj.com
Figure 1 Distribution of serum vitamin B12, folic acid, ferritin and transferrin saturation levels, stratified by mean corpuscular volume (MCV) levels
(in tertiles).
with caution when screening for vitamin B12 or folate deficiency
in patients with chronic HF.
With respect to prognosis, vitamin B12 or folate supplementation
does not constitute a therapeutic target in patients with chronic
HF. Whether folate supplementation might be warranted in view
of a possible improvement of health-related quality of life is cur-
rently unknown. For iron deficiency, several studies have shown
beneficial effects of intravenous iron supplementation with respect
to improved clinical status, exercise capacity and quality of life.4–6
Study limitations
The main limitation of this study is that the diagnosis of vitamin
B12 and folate deficiency is relatively challenging as a result of
Figure 2 Kaplan–Meier survival analysis, stratified by vitamin B12
levels (in quartiles).
van der Wal HH, et al. Heart 2014;0:1–9. doi:10.1136/heartjnl-2014-306022
Heart failure and cardiomyopathies
lacking gold standard tests for vitamin levels and no clear defini-
tions 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 haematinic
deficiencies are validated thoroughly and are mainstream prac-
tice in daily healthcare. The cut-offs for haematinic deficiencies
used in this study are based on an optimal balance between sen-
sitivity and specificity. Serum vitamin B12 and folic acid levels
remain the most important screening markers for these vitamin
deficiencies in patients with chronic HF as long as more reliable
markers are lacking.
Second, haematinics were only available at baseline.
Therefore, haematinic changes over time could not be
Figure 3 Kaplan–Meier survival analysis, stratified by folic acid levels
(in quartiles).
7
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Heart failure and cardiomyopathies

In addition, data regarding neither diet nor vitamin supple-

Table 5 Cox proportional hazard regression model for the
mentation were available. Food products naturally rich in vita-
prediction of mortality of patients with heart failure (HF)
mins, vitamin-fortified food products and administration of
Univariable HR (95% CI) Multivariable HR (95% CI) vitamin supplements might influence serum vitamin levels,
which might explain the significant differences in vitamin levels
Folic acid (ng/mL)
across the subcohorts. Future studies addressing haematinics
<6.9 1.00 1.00
should be aware of possible international differences in serum
6.9–9.4 1.09 (0.72 to 1.65) 1.16 (0.52 to 2.56)
haematinic levels.
9.5–12.5 0.95 (0.64 to 1.41) 0.78 (0.37 to 1.63)
Finally, although this international study comprises patients
>12.5 0.89 (0.60 to 1.33) 0.74 (0.36 to 1.55)
from several countries in Europe, our results cannot be extrapo-
Vitamin B12 (pg/mL)
lated directly to other continents or populations, for example,
<313 1.00 1.00
due to differences in diet across the world.
313–435 1.14 (0.79 to 1.64) 1.49 (0.96 to 2.32)
436–600 1.14 (0.80 to 1.64) 1.03 (0.66 to 1.60)
CONCLUSION
>600 1.64 (1.16 to 2.31) 0.95 (0.60 to 1.51)
Vitamin B12 and folate deficiency are relatively rare in patients
The multivariable model is adjusted for age, sex, N-terminal prohormone brain with chronic HF. Higher serum folic acid levels are independ-
natriuretic peptide (NT-proBNP), presence of anaemia and renal failure (i.e. estimated
glomerular filtration rate <60 ml/min/1.73 m2), body mass index, left ventricular ently correlated to better health-related quality of life as assessed
ejection fraction, New York Heart Association functional class, history of hypertension, by a disease-specific questionnaire. Whether treatment of folate
serum levels of erythropoietin and high-sensitive C-reactive protein, ischaemic
aetiology of heart failure, treatment with angiotensin-converting-enzyme inhibitor
deficiency has beneficial effects is currently unknown.
and/or angiotensin receptor blocker, loop diuretics, and study cohort. Interestingly, MCV levels are not a reliable screening marker for
haematinic deficiencies in patients with chronic HF and should
be used with caution in the differential diagnosis of haematinic
deficiencies in chronic HF. Finally, serum vitamin B12 and folic
established. Additional studies with multiple haematinic mea-
acid levels are not associated with mortality.
surements over time should be conducted.
Third, although our study cohort consists of >600 patients, a Contributors HHvdW, IJTK and PvdM were involved in designing the study. JC-C
relative small number has an LVEF of >45% (i.e. heart failure and EAJ provided help with the study design. They were assisted by CE, JB, WB and
with preserved EF (HFpEF)). Consequently, we should be PP. Statistical analysis and writing of the manuscript were performed by HHvdW,
slightly circumspect regarding our conclusions in patients with IJTK and PvdM. NGB provided assistance with the statistical analyses and revised the
manuscript several times. A final critical revision of the manuscript was performed
HFpEF. by AAV, DJvV, EAJ and JC-C.
Unfortunately, hs-CRP levels were not available in the Spanish
Funding This study was financially supported by the grant of National Centre of
cohort. However, results for both vitamin B12 and folic acid Research (Poland) no. UMO-2012/05/E/NZ5/00590.
remained similar when excluding hs-CRP from the multivariable
Competing interests JCC and ITK received speaker fees from Vifor Pharma. AAV
Cox regression model. Chronic HF is characterised by low- and PvdM received speaker fees and an unrestricted grant from Vifor Pharma. DJvV
grade inflammatory processes in which hs-CRP plays an import- received board membership fees from Vifor Pharma. PP received consulting fees and
ant role. Additionally, several studies have stressed out the related travel expenses from Vifor Pharma. EAJ received speaker and advisory board
strong prognostic value of hs-CRP in patients with HF.29 30 fees, and related travel expenses from Vifor Pharma. Wroclaw Medical University
received an unrestricted grant from Vifor Pharma.
Ethics approval Local ethics committees at each institution.
Provenance and peer review Not commissioned; externally peer reviewed.

Key messages
What is already known on this subject?
Iron deficiency is common in chronic heart failure (HF) and
affects clinical parameters and outcome. However, studies on
haematinic deficiencies other than iron deficiency in chronic HF
are scarce, and clinical correlates or the effects on prognosis are
currently unclear.
What might this study add?
Vitamin B12 and folate deficiency are relatively rare in patients
with chronic HF. Mean corpuscular volume is neither related to
vitamin B12 nor to folic acid levels. Unlike iron deficiency,
vitamin B12 and folic acid levels are not related to prognosis.
Lower folic acid levels are associated with an impaired
health-related quality of life as assessed by a disease-specific
questionnaire.
How might this impact on clinical practice?
Since no significant association was observed between mean
corpuscular volume and neither vitamin B12 nor folic acid levels,
this cellular index should be used with caution in the
differential diagnosis of anaemia in patients with chronic HF.
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9
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Vitamin B12 and folate deficiency in chronic

heart failure
Haye H van der Wal, Josep Comin-Colet, Ijsbrand T Klip, Cristina
Enjuanes, Niels Grote Beverborg, Adriaan A Voors, Waldemar Banasiak,
Dirk J van Veldhuisen, Jordi Bruguera, Piotr Ponikowski, Ewa A
Jankowska and Peter van der Meer

Heart published online October 16, 2014

Updated information and services can be found at:

http://heart.bmj.com/content/early/2014/10/16/heartjnl-2014-306022

Supplementary Supplementary material can be found at:

Material http://heart.bmj.com/content/suppl/2014/10/16/heartjnl-2014-306022.
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References This article cites 29 articles, 7 of which you can access for free at:
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Epidemiology (3268)

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