Vitamin B 12 and folate deficiency in chronic heart failure

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1 Heart failure and cardiomyopathies Downloaded from on May 11, Published by group.bmj.com ORIGINAL ARTICLE Vitamin B 12 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 Meer 1 Additional material is published online only. To view please visit the journal online ( heartjnl ). 1 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 Published Online First 16 October 2014 To cite: van der Wal HH, Comin-Colet J, Klip IT, et al. Heart 2015;101: ABSTRACT Objective To determine the prevalence, clinical correlates and the effects on outcome of vitamin B 12 and folic acid levels in patients with chronic heart failure (HF). 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 ). Thirteen per cent of the patients had an LVEF >45%. Vitamin B 12 deficiency (serum level <200 pg/ ml), folate deficiency (serum level <4.0 ng/ml) and iron deficiency (serum ferritin level <100 mg/l, or 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 B 12, 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 ( years), 254 subjects died. In multivariable proportional hazard models, vitamin B 12 and folic acid levels were not associated with prognosis. Conclusions Vitamin B 12 and folate deficiency are relatively rare in patients with chronic HF. Since no significant association was observed between mean corpuscular volume and neither vitamin B 12 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 B 12 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 B 12 and folic acid are essential for normal erythrocyte production. Haematinic deficiencies may cause anaemia, although 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 B 12 plays a significant role in DNA synthesis, and deficiencies may lead to inadequate erythropoiesis due to desynchronised maturation of red cell nuclei and cytoplasm. 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 deficiencies in chronic HF is currently unknown. One study from the UK determined the prevalence of haematinic deficiencies in patients with chronic HF. Vitamin B 12 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 B 12 and folic acid in a large, international pooled cohort of patients with HF and studied the prevalence, clinical 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 preserved or reduced LVEF, as described elsewhere. 1 In this subset of 610 patients, clinical follow-up data and serum vitamin B 12 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 transferrin. This value was then multiplied by van der Wal HH, et al. Heart 2015;101: doi: /heartjnl

2 Downloaded from on May 11, Published by group.bmj.com Heart failure and cardiomyopathies Renal function was assessed using the estimated glomerular filtration rate (egfr, ml/min/1.73 m 2 ), calculated from the simplified Modification of Diet in Renal Disease equation. Vitamin B 12, folic acid and N-terminal prohormone brain natriuretic peptide (NT-proBNP) levels were determined using an immunoassay 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 B 12 <200 pg/ml 8 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 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 (haemoglobin, MCV, serum iron, ferritin, transferrin, vitamin B 12 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 percentage 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 B 12, folate, ferritin and TSAT were stratified by MCV levels in tertiles. Changes in serum levels of haematinics were tested using a nonparametric trend test (i.e. extension of Wilcoxon rank-sum test). After baseline analyses, the follow skewed variables were logarithmically transformed for all further analyses to achieve a normal distribution: serum NT-proBNP, erythropoietin, iron, ferritin, vitamin B 12, folic acid, TSAT, hs-crp and egfr. To determine clinical correlates of vitamin B 12 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 B 12 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 B 12 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 B 12 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, 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 significant. 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 B 12 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 supplementary table S2. Median serum vitamin B 12 and folic acid levels (+IQR) were 435 ( ) pg/ml and 9.4 ( ) ng/ml, respectively. Vitamin B 12 levels were lowest in the Dutch subcohort, whereas folic acid levels were lowest in the Polish subcohort. Vitamin B 12 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 B 12 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 nonanaemic patients, p=0.020), whereas vitamin B 12 deficiency was not (6.2% versus 5.0%, p=0.538). None of the patients had both vitamin B 12 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 B 12 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 B 12 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 correlated 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 B 12 and folate deficiency for different MCV levels. Haematinics and survival During a median follow-up of 2.10 years (IQR years), 254 patients (42%) died. Differences in event-free survival stratified by vitamin B 12 and folic acid in quartiles are displayed in figures 2 and 3. Increased mortality rates were seen in patients with vitamin B 12 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 B 12 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 van der Wal HH, et al. Heart 2015;101: doi: /heartjnl

3 Heart failure and cardiomyopathies Downloaded from on May 11, Published by group.bmj.com Table 1 Baseline characteristics, stratified by vitamin B 12 levels in quartiles Variables Total cohort (n=610) <313 pg/ml pg/ml pg/ml >600 pg/ml (n=151) p Value (trend) Demographics and clinical signs Age (years) 68±12 68±12 68±11 67±12 69± Men BMI (kg/m 2 ) 27.7± ± ± ± ± BMI < Ischaemic aetiology egfr (ml/min/1.73 m 2 ) 67.4 ( ) 67.7 ( ) 71.6 ( ) 65.5 ( ) 62.2 ( ) egfr < LVEF (%) 33±13 33±13 36±13 32±12 32± LVEF >45% SBP (mm Hg) 122±22 124±21 125±24 122±23 117± MLHFQ 45 (26 61) 45 (23 62) 43 (27 59) 43 (28 61) 47 (25 62) Comorbidities Diabetes mellitus Atrial fibrillation Hypertension Haematinics Hb (g/dl) 13.4± ± ± ± ± MCV (fl) 89.8± ± ± ± ± Anaemia* (%) EPO (IU/L) 18.0 ( ) 18.1 ( ) 17.2 ( ) 17.0 ( ) 17.9 ( ) Iron (μg/dl) 67.0 ( ) 63.0 ( ) 67.0 ( ) 71.0 ( ) 63.0 ( ) 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) Iron deficiency Vitamin B 12 (pg/ml) 435 ( ) 250 ( ) 377 ( ) 503 ( ) 784 ( ) N/A Vitamin B 12 deficiency N/A Folic acid (ng/ml) 9.4 ( ) 9.4 ( ) 8.5 ( ) 9.9 ( ) 9.7 ( ) Folate deficiency Cardiac biomarkers NT-proBNP (pg/ml) 1801 ( ) 1556 ( ) 1562 ( ) 1695 ( ) 2987 ( ) hs-crp (mg/l) 4.0 ( ) 4.0 ( ) 3.2 ( ) 3.9 ( ) 6.0 ( ) Treatment ACE inhibitor and/or ARB β-blocker Aldosterone antagonist Statins Loop diuretics Antiplatelet and/or 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 μg/l with a transferrin saturation <20%. Vitamin B 12 deficiency is defined as serum vitamin B 12 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 proportional hazard regression models. DISCUSSION In an international pooled cohort comprising 610 patients with chronic HF, we demonstrate that vitamin B 12 and folate deficiency are relatively rare (4% and 5%, respectively). Second, there is no significant correlation between MCV and vitamin B 12 and folic acid levels. Finally, vitamin B 12 and folic acid levels were not independently associated with mortality. Prevalence and definition of haematinic deficiencies Unfortunately, there are no clear definitions of vitamin B 12 and folate deficiency, leading to different thresholds for the presence of these deficiencies, and consequently to differences in prevalence. Traditionally, serum vitamin B 12 levels have been considered as the gold standard for the detection of clinical vitamin B 12 deficiency. According to Stabler et al, 8 serum vitamin B 12 levels <200 pg/ml are suggestive for clinical deficiency (sensitivity and specificity 65% 100% and 50% 60%, respectively). With serum levels >300 pg/ml, vitamin B van der Wal HH, et al. Heart 2015;101: doi: /heartjnl

4 Downloaded from on May 11, Published by group.bmj.com Heart failure and cardiomyopathies Table 2 Baseline characteristics, stratified by folic acid levels in quartiles Variables Total cohort (n=610) <6.9 ng/ml ng/ml ng/ml >12.5 ng/ml (n=151) p Value (trend) Demographics and clinical signs Age (years) 68±12 66±12 68±11 67±12 69± Men BMI (kg/m 2 ) 27.7± ± ± ± ± BMI < Ischaemic aetiology egfr (ml/min/1.73 m 2 ) 67.4 ( ) 91.6 ( ) 75.9 ( ) 62.8 ( ) 56.3 ( ) <0.001 egfr < <0.001 LVEF (%) 33±13 36±15 34±14 34±13 30±11 <0.001 LVEF >45% <0.001 SBP (mm Hg) 122±22 119±22 125±21 124±23 120± MLHFQ 45 (26 61) 45 (29 63) 46 (26 64) 44 (26 60) 39 (24 57) Comorbidities Diabetes mellitus Atrial fibrillation Hypertension <0.001 Haematinics Hb (g/dl) 13.4± ± ± ± ± MCV (fl) 89.8± ± ± ± ± Anaemia* (%) EPO (IU/L) 18.0 ( ) 18.0 ( ) 18.1 ( ) 18.0 ( ) 16.2 ( ) Iron (μg/dl) 67.0 ( ) 71.0 ( ) 70.4 ( ) 62.5 ( ) 64.8 ( ) Ferritin (μg/l) 148 (78 273) 167 (78 308) 128 (69 214) 148 (75 292) 154 (84 267) TSAT (%) 19 (12 26) 19 (13 28) 20 (13 26) 18 (10 25) 18 (10 24) Iron deficiency Vitamin B 12 (pg/ml) 435 ( ) 428 ( ) 404 ( ) 430 ( ) 485 ( ) Vitamin B 12 deficiency Folic acid (ng/ml) 9.4 ( ) 5.4 ( ) 8.0 ( ) 10.7 ( ) 15.5 ( ) N/A Folate deficiency N/A Cardiac biomarkers NT-proBNP (pg/ml) 1801 ( ) 1371 ( ) 1746 ( ) 1945 ( ) 2097 ( ) hs-crp (mg/l) 4.0 ( ) 4.5 ( ) 4.6 ( ) 4.0 ( ) 4.1 ( ) Treatment ACE inhibitor and/or ARB β-blocker Aldosterone antagonist Statins Loop diuretics Antiplatelet and/or 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 μg/l with a transferrin saturation <20%. Vitamin B 12 deficiency is defined as serum vitamin B 12 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 B 12 levels (i.e. <100 pg/ml) are often associated 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 B 12 levels can be as high as 50% due to suboptimal assays and highly variable test results 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 Measurement of methylmalonic acid and total homocysteine was introduced several decades ago to diagnose vitamin B 12 deficiency, in addition to serum vitamin B 12 levels. However, both serum methylmalonic acid and homocysteine may be elevated in renal failure, making methylmalonic acid and homocysteine unreliable screening markers for vitamin B 12 deficiency in patients with chronic HF as up to 60% of patients with chronic HF are suffering from moderate to severe renal dysfunction (i.e. egfr <60 ml/min/1.73 m 2 ) In the current study, renal dysfunction is present in 39% of all patients. Therefore, van der Wal HH, et al. Heart 2015;101: doi: /heartjnl

5 Heart failure and cardiomyopathies Downloaded from on May 11, Published by group.bmj.com Table 3 Variable Clinical variables associated with vitamin B 12 in chronic heart failure Univariable regression coefficient (95% CI) p Value t Value Multivariable regression coefficient (95% CI) p Value t Value Age, per 5 years ( to 0.040) Sex, female vs male ( to 0.080) BMI, per 1 kg/m ( to 0.008) LVEF, per 1% ( to 0.001) SBP, per 1 mm Hg ( to 0.002) < ( to 0.001) MCV, per 1 fl ( to 0.010) Hb, per 1 g/dl ( to 0.054) egfr, per doubling ( to 0.023) NT-proBNP, per doubling (.032 to 0.095) < (0.029 to 0.097) < hs-crp*, per doubling ( to 0.076) EPO, per doubling ( to 0.073) Ferritin, per doubling (.059 to 0.148) < (0.051 to 0.143) < TSAT, per doubling ( to 0.077) Folic acid, per doubling ( to 0.118) Atrial fibrillation, yes vs no (0.053 to 0.317) (0.012 to 0.278) Diabetes, yes vs no ( to 0.196) Ischaemic aetiology, yes vs no ( to 0.033) ( to 0.039) NYHA functional class, III/IV vs I/II ( to 0.111) MLHFQ, per point ( to 0.004) Study cohort (vs Holland) Poland (0.001 to 0.320) ( to 0.285) Spain (0.101 to 0.381) (0.151 to 0.439) < Treatment ACE inhibitor and/or ARB, yes vs no ( to 0.028) ( to 0.199) Aldosterone antagonist, yes vs no ( to 0.163) β-blocker, yes vs No ( to 0.043) Statins, yes vs no ( to 0.061) Loop diuretic, yes vs no ( to 0.267) Antiplatelet and/or anticoagulant, yes vs no ( to 0.182) *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 B 12 deficiency in our study. In short, there are currently no reliable screening markers for vitamin B 12 deficiency in patients with chronic HF apart from serum vitamin B 12 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 al 22 stated that serum folic acid measurements may be superior 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 measurement. 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 measured to confirm the diagnosis. The current study shows that serum folic acid levels are correlated 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, and therefore, the prevalence of folate deficiency may be underestimated 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 B 12 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 al 23 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 B 12 levels >600 pg/ml have higher mortality risks compared with patients with lower levels. Vitamin B 12 lost its predictive value on all-cause mortality 306 van der Wal HH, et al. Heart 2015;101: doi: /heartjnl

6 Downloaded from on May 11, Published by group.bmj.com Heart failure and cardiomyopathies Table 4 Variable Clinical variables associated with folic acid in chronic heart failure (HF) Univariable regression coefficient (95% CI) p Value t Value Multivariable regression coefficient (95% CI) p Value t Value Age, per 5 years ( to 0.046) ( to 0.050) Sex, women vs men ( to 0.106) BMI, per 1 kg/m ( to 0.001) ( to 0.021) LVEF, per 1% ( to 0.005) < ( to 0.001) SBP, per 1 mm Hg ( to 0.004) MCV, per 1 fl ( to 0.016) Hb, per 1 g/dl (0.008 to 0.072) ( to 0.037) egfr, per doubling ( to 0.211) < ( to 0.046) NT-proBNP, per doubling ( to 0.059) ( to 0.038) hs-crp*, per doubling ( to 0.042) EPO, per doubling ( to 0.022) Ferritin, per doubling ( to 0.052) TSAT, per doubling ( to 0.059) < ( to 0.043) Vitamin B 12, per doubling ( to 0.109) Atrial fibrillation, yes vs no ( to 0.037) ( to 0.074) Diabetes, yes vs no ( to 0.035) ( to 0.084) Ischaemic aetiology, yes vs no ( to 0.170) NYHA functional class III/IV vs I/II (0.261 to 0.495) < ( to 0.155) MLHFQ, per point ( to 0.001) ( to ) Study cohort (vs Holland) Poland ( to 0.470) < ( to 0.338) < Spain ( to 0.763) < ( to 0.606) < Treatment ACE inhibitor and/or ARB, yes vs ( to 0.346) no Aldosterone antagonist, yes vs no ( to 0.104) β-blocker, yes vs no ( to 0.072) Statins, yes vs no ( to 0.014) ( to 0.141) Loop diuretic, yes vs no ( to 0.291) Antiplatelet and/or anticoagulant, yes vs no ( to 0.115) *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 B 12 levels >600 pg/ml. The univariable effect on mortality is caused by the paradoxical association between serum vitamin B 12 levels and disease severity (serum NT-proBNP levels). This association was observed earlier by Herrmann et al 25 and may be caused by the cardiohepatic syndrome. 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 B 12 analogues, making serum vitamin B 12 measurements less accurate. However, this cardiohepatic syndrome remains only a hypothesis and is an incomplete explanation of the association between serum vitamin B 12 levels and disease severity. Clinical implications Traditionally, MCV is one of the conventional erythrocyte parameters 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 B 12 and folate deficiency since these deficiencies are frequently characterisedbyanincreaseinmcv.inthepresentstudy,noassociation between MCV and neither serum vitamin B 12 nor folic acid levels were observed, although we found a significant association between MCV levels and the presence of iron deficiency. While MCV is a reliable screening marker for haematinic 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 B 12 and/or folate deficiency as the influence of serum vitamin B 12 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 haematinic deficiencies as macrocytosis is frequently absent in vitamin B 12 and folate deficiency. 28 Therefore, MCV should be used van der Wal HH, et al. Heart 2015;101: doi: /heartjnl

7 Heart failure and cardiomyopathies Downloaded from on May 11, Published by group.bmj.com Figure 1 Distribution of serum vitamin B 12, folic acid, ferritin and transferrin saturation levels, stratified by mean corpuscular volume (MCV) levels (in tertiles). with caution when screening for vitamin B 12 or folate deficiency in patients with chronic HF. With respect to prognosis, vitamin B 12 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 currently 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 B 12 and folate deficiency is relatively challenging as a result of lacking gold standard tests for vitamin levels and no clear definitions of vitamin B 12 and folate deficiency. Low serum vitamin B 12 and folic acid levels are not always conclusive for clinical deficiency. However, current screening methods for haematinic deficiencies are validated thoroughly and are mainstream practice in daily healthcare. The cut-offs for haematinic deficiencies used in this study are based on an optimal balance between sensitivity and specificity. Serum vitamin B 12 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 2 Kaplan Meier survival analysis, stratified by vitamin B 12 levels (in quartiles). Figure 3 Kaplan Meier survival analysis, stratified by folic acid levels (in quartiles). 308 van der Wal HH, et al. Heart 2015;101: doi: /heartjnl

8 Downloaded from on May 11, Published by group.bmj.com Heart failure and cardiomyopathies Table 5 Cox proportional hazard regression model for the prediction of mortality of patients with heart failure (HF) Univariable HR (95% CI) Multivariable HR (95% CI) Folic acid (ng/ml) < (0.72 to 1.65) 1.16 (0.52 to 2.56) (0.64 to 1.41) 0.78 (0.37 to 1.63) > (0.60 to 1.33) 0.74 (0.36 to 1.55) Vitamin B 12 (pg/ml) < (0.79 to 1.64) 1.49 (0.96 to 2.32) (0.80 to 1.64) 1.03 (0.66 to 1.60) > (1.16 to 2.31) 0.95 (0.60 to 1.51) The multivariable model is adjusted for age, sex, N-terminal prohormone brain natriuretic peptide (NT-proBNP), presence of anaemia and renal failure (i.e. estimated glomerular filtration rate <60 ml/min/1.73 m 2 ), body mass index, left ventricular ejection fraction, New York Heart Association functional class, history of hypertension, serum levels of erythropoietin and high-sensitive C-reactive protein, ischaemic aetiology of heart failure, treatment with angiotensin-converting-enzyme inhibitor and/or angiotensin receptor blocker, loop diuretics, and study cohort. established. Additional studies with multiple haematinic measurements over time should be conducted. Third, although our study cohort consists of >600 patients, a relative small number has an LVEF of >45% (i.e. heart failure with preserved EF (HFpEF)). Consequently, we should be slightly circumspect regarding our conclusions in patients with HFpEF. Unfortunately, hs-crp levels were not available in the Spanish cohort. However, results for both vitamin B 12 and folic acid remained similar when excluding hs-crp from the multivariable Cox regression model. Chronic HF is characterised by lowgrade inflammatory processes in which hs-crp plays an important role. Additionally, several studies have stressed out the strong prognostic value of hs-crp in patients with HF. 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 B 12 and folate deficiency are relatively rare in patients with chronic HF. Mean corpuscular volume is neither related to vitamin B 12 nor to folic acid levels. Unlike iron deficiency, vitamin B 12 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 B 12 nor folic acid levels, this cellular index should be used with caution in the differential diagnosis of anaemia in patients with chronic HF. In addition, data regarding neither diet nor vitamin supplementation were available. Food products naturally rich in vitamins, vitamin-fortified food products and administration of vitamin supplements might influence serum vitamin levels, which might explain the significant differences in vitamin levels across the subcohorts. Future studies addressing haematinics should be aware of possible international differences in serum haematinic levels. Finally, although this international study comprises patients from several countries in Europe, our results cannot be extrapolated directly to other continents or populations, for example, due to differences in diet across the world. CONCLUSION Vitamin B 12 and folate deficiency are relatively rare in patients with chronic HF. Higher serum folic acid levels are independently correlated to better health-related quality of life as assessed by a disease-specific questionnaire. Whether treatment of folate deficiency has beneficial effects is currently unknown. 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 B 12 and folic acid levels are not associated with mortality. Contributors HHvdW, IJTK and PvdM were involved in designing the study. JC-C and EAJ provided help with the study design. They were assisted by CE, JB, WB and PP. Statistical analysis and writing of the manuscript were performed by HHvdW, 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 by AAV, DJvV, EAJ and JC-C. Funding This study was financially supported by the grant of National Centre of Research (Poland) no. UMO-2012/05/E/NZ5/ Competing interests JCC and ITK received speaker fees from Vifor Pharma. AAV and PvdM received speaker fees and an unrestricted grant from Vifor Pharma. DJvV received board membership fees from Vifor Pharma. PP received consulting fees and related travel expenses from Vifor Pharma. EAJ received speaker and advisory board 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. REFERENCES 1 Klip IT, Comin-Colet J, Voors AA, et al. Iron deficiency in chronic heart failure: an international pooled analysis. Am Heart J 2013;165: e3. 2 Jankowska EA, Rozentryt P, Witkowska A, et al. Iron deficiency predicts impaired exercise capacity in patients with systolic chronic heart failure. J Card Fail 2011;17: van Veldhuisen DJ, Anker SD, Ponikowski P, et al. Anemia and iron deficiency in heart failure: mechanisms and therapeutic approaches. 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10 Downloaded from on May 11, Published by group.bmj.com Vitamin B 12 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 : originally published online October 16, 2014 doi: /heartjnl Updated information and services can be found at: Supplementary Material References alerting service These include: Supplementary material can be found at: DC1.html This article cites 29 articles, 7 of which you can access for free at: Receive free alerts when new articles cite this article. Sign up in the box at the top right corner of the online article. Topic Collections Articles on similar topics can be found in the following collections Drugs: cardiovascular system (8673) Epidemiology (3663) Notes To request permissions go to: To order reprints go to: To subscribe to BMJ go to: