Randomized supplementation of 4000 IU vitamin D3 daily vs placebo on the prevalence of anemia in advanced heart failure: the EVITA trial

Background Low 25-hydroxyvitamin D (25OHD) levels (< 75 nmol/l) are inversely associated with anemia prevalence. Since anemia and low 25OHD levels are common in patients with heart failure (HF), we aimed to investigate whether vitamin D supplementation can reduce anemia prevalence in advanced HF. Methods EVITA (Effect of Vitamin D on Mortality in Heart Failure) is a randomized, placebo-controlled clinical trial in patients with initial 25OHD levels < 75 nmol/l. Participants received either 4000 IU vitamin D3 daily or a matching placebo for 36 months. A total of 172 patients (vitamin D group: n = 85; placebo group: n = 87) were investigated in this pre-specified secondary data analysis. Hemoglobin (Hb) and other hematological parameters were measured at baseline and study termination. Assessment of between-group differences in anemia prevalence and Hb concentrations was performed at study termination, while adjusting for baseline differences. Results In the vitamin D and placebo group, baseline proportions of patients with anemia (Hb < 12.0 g/dL in females and < 13.0 g/dL in males) were 17.2% and 10.6%, respectively (P = 0.19). At study termination, the proportion of patients with anemia in the vitamin D and placebo groups was 32.2% and 31.8%, respectively (P > 0.99). There was no between-group difference in change in the Hb concentrations (− 0.04 g/dL [95%CI:-0.53 to 0.45 g/dL]; P = 0.87). Results regarding anemia risk and Hb concentrations were similar in the subgroup of patients with chronic kidney disease (vitamin D group: n = 26; placebo group: n = 23). Moreover, results did not differ substantially when data analysis was restricted to patients with deficient baseline 25OHD levels. Conclusions A daily vitamin D supplement of 4000 IU did not reduce anemia prevalence in patients with advanced HF. Data challenge the clinical relevance of vitamin D supplementation to increase Hb levels. Trial registration The study was registered at EudraCT (No. 2010–020793-42) and clinicaltrials.gov (NCT01326650). Electronic supplementary material The online version of this article (doi:10.1186/s12937-017-0270-5) contains supplementary material, which is available to authorized users.

Epidemiological evidence suggests that both aforementioned vitamin D metabolites are inversely associated with Hb levels and anemia prevalence in patients with cardiovascular disease [10][11][12], including patients with HF [2]. There is also evidence from observational studies that anemia is more strongly associated with low circulating 1,25(OH) 2 D levels than with deficient circulating 25OHD levels [2,[11][12][13].
Results of interventional studies with vitamin D on the prevalence of anemia have been inconsistent: Early interventional studies with small numbers of patients and/or no control-groups [14][15][16][17] support the assumption of beneficial vitamin D effects on erythropoiesis and improvement of anemia. However, results of more recent randomized controlled trials (RCTs) are mixed [18][19][20][21]. While two studies showed no effect of vitamin D on Hb levels [19,20], two other studies showed significant decreases in the required dose of erythropoietin (EPO) and erythropoiesis stimulating agents (ESA) in the vitamin D group in patients with chronic kidney disease, respectively [18][19][20][21].
In HF, anemia is an independent risk factor of morbidity [22] and mortality [23] and treatment of anemia can improve patient outcomes [24]. Aim of the present study was to investigate the effect of a daily vitamin D 3 supplementation on the prevalence of anemia and Hb levels in patients with advanced HF and low 25OHD levels.

Study design and participants
The present investigation is a pre-specified secondary analysis of the EVITA (Effect of Vitamin D on Mortality in Heart Failure) trial. EVITA is a single-center, randomized, placebo-controlled, clinical trial, performed at the Clinic for Thoracic and Cardiovascular Surgery of the Heart-and Diabetes Center North Rhine Westphalia, Bad Oeynhausen, Germany. Between November 2010 and July 2013, 400 patients with HF (332 men and 68 women) were recruited. All patients were ambulatory and regularly seen at our outpatient clinic. Eligible study participants were adults aged ≥ 18 to 79 years with congestive HF, New York Heart Association functional class ≥ II, and circulating 25OHD levels < 75 nmol/L. Participants were randomly allocated to receive 4000 IU (100 μg) cholecalciferol per day as oily drops (Vigantol® Oel, provider: Merck KGaA, Darmstadt, Germany) or a matching placebo (Miglyol Oel, provider: Merck KGaA, Darmstadt, Germany) for 36 months. During the study, participants remained on guideline-recommended medications. None of the study participants received erythropoietin (EPO) or iron preparations. Main study results have already been published elsewhere [25]. Of the 400 patients, 177 completed the study (Fig. 1), whereas 223 patients died, dropped out, or were lost-to follow-up. Finally, data on relevant parameters for this secondary analysis were available in 172 patients.

Outcome measures
In the present analysis of the EVITA trial, we investigated the between-group differences in anemia prevalence at study termination. In addition, we assessed betweengroup differences in Hb concentrations with adjustment for baseline values. We used the World Health Organization's gender-based definition for classifying patients as anemic (< 13 g/dL in men and < 12.0 g/dL in women). . The measuring range for 25OHD lies between 10 and 375 nmol/l. Values < 10 nmol/l were considered 9.9 nmol/l. The limit of 1,25(OH) 2 D quantitation is 12 pmol/L and we considered values below this limit as 11 pmol/L. Creactive protein (CRP), brain natriuretic peptide (BNP), calcium, and creatinine values were analyzed by the Architect Autoanalyzer (Abbott, Wiesbaden, Germany). Estimated glomerular filtration rate (eGFR) was calculated using the Modification of Diet in Renal Disease formula [26]. Hematological parameters such as Hb, hematocrit, erythrocytes, leukocytes, mean corpuscular volume (MCV), mean corpuscular Hb (MCH), mean corpuscular Hb concentration (MCHC), and red blood cell distribution width (RDW) were measured by automated procedures using the Abbott CellDyn 3500 hematology analyzer (Abbott, Wiesbaden, Germany).

Statistics
Categorical data are presented as percentages. Continuous data with a normal distribution are shown as means with standard deviation (SD) and data with a skewed distribution are shown as medians with interquartile range (IQR). Normal data distribution was checked by using the Kolmogorov-Smirnov test and was considered when probability values were > 0.05. We used the chisquared test, unpaired t-test or Mann-Whitney-U-Test for baseline study group comparisons.
The McNemar test was used to assess differences in anemia status within groups. Fisher's exact test was used to assess differences in anemia status between groups. Change from baseline data is shown as mean with 95% confidence interval [CI]. Skewed variables were log(e) transformed before use in parametric statistical analyses. We used ANCOVA with baseline adjustments to test for differences in the outcome variables between the two study groups at follow-up (36 months). Moreover, we used 2-factor repeated-measures ANOVA with time and study group as the between-subject variables for trend analysis in Hb concentrations. We considered P-values < 0.05 as statistically significant. P-values are two-sided. A statistical power calculation for this secondary analysis revealed a probability of 80% that the study will detect a treatment difference at a two-sided 0.05 significance level, if the true difference in Hb levels between treatments is 0.58 mg/dL. This calculation is based on 172 study participants and the assumption that the standard deviation of the Hb concentration is 1.35 mg/dL. The assumption is predicted on observational data [12] in which patients with adequate 25OHD levels had 0.6 g/ dL higher Hb levels than patients with deficient 25OHD levels and their standard deviation in Hb levels was 1.4 g/dL. Statistical analyses were performed using IBM SPSS, version 21 (IBM Corp, Armonk, NY, USA).

Baseline data
The numbers of patients who died, dropped out or were lost to follow-up in the vitamin D and placebo groups were 39 and 36, 56 and 57, and 17 and 18, respectively. Main causes of drop out were poor health condition (n = 30) and no pleasure to participate (n = 24). Baseline Hb levels were significantly higher in survivors than in nonsurvivors (14.3 ± 1.0 g/dL vs. 13.4 ± 1.2 g/dL; P < 0.001) but were similar in patients who dropped out and did not drop out (14.3 ± 1.1 g/dL vs. 14.2 ± 1.0 g/dL; P = 0.31). Baseline characteristics of the included study participants are shown in Table 1. Initial MCV was significantly lower in the vitamin D group than in the placebo group, while both were within the  Vitamin D effects Table 2 shows the vitamin D effects on biochemical parameters. There was a significant between-group difference in circulating 25OHD levels. The increment was 55.3 nmol/L higher in the vitamin D group than in the placebo group (95% CI: 43.5 to 67.1 nmol/L; P < 0.001).
The corresponding values of circulating 1,25(OH) 2 D were + 17.1 pmol/L (95% CI: 6.2 to 27.9 pmol/L; P = 0.004). Compared with placebo, vitamin D supplementation also resulted in significantly higher in-study plasma calcium levels. Prevalence of anemia increased by 15.0% in the vitamin D group (P = 0.003) and by 21.6% in the placebo group (P < 0.001; Fig. 2). No difference in anemia prevalence was determined between the two study groups at study termination (P > 0.99; Fig. 2). At study termination, causes of anemia in the vitamin D and placebo groups were iron deficiency in 8 and 3 patients, folate or B12 deficiency in 4 and 6 patients, CKD in 10 and 9 patients, inflammation in 2 and 2 patients, and unexplained in 4 and 7 patients, respectively. There was no significant between-group difference in change in the Hb levels ( Table 2). In detail, in-study Hb levels decreased on average by −0.5 g/dL (95% CI: -0.9 to −0.2) g/dL and −0.5 g/dL (95% CI: -0.9 to −0.1) g/dL, in the vitamin D and placebo groups, respectively. The mean between-group difference in change in the Hb levels was −0.04 g/dL (95% CI: -0.53 to 0.45 g/dL; P = 0.87). Fig. 3 shows the trend analysis in hemoglobin concentrations by study group.

Subgroup analyses
In the subgroup of patients with chronic kidney disease (CKD) stage 3 or 4 (vitamin D group: n = 26; placebo group: n = 23) anemia prevalence at baseline was in the vitamin D and placebo groups 23.1% and 21.7%, respectively (P > 0.99). At study termination, anemia prevalence was 50.0% and 47.8%, respectively (P > 0.99). Moreover, vitamin D had no effect on mean change difference in Hb levels (0.0 g/dL [95% CI: -1.0 to 1.0 g/dL]; Additional file 1: Table S1). In addition, results did not differ substantially when data analysis was restricted to patients with baseline 25OHD levels < 30 nmol/L (data not shown).

Discussion
The present work suggests that a daily vitamin D supplement of 4000 IU for 36 months does not reduce the prevalence of anemia in patients with advanced HF. Moreover, vitamin D had no significant effect on Hb levels. Similar results were obtained in the subgroup of patients with initial eGFR values < 60 mL/min/1.73 m 2 and in patients with deficient baseline 25OHD levels.
Obviously, hematological parameters were unaffected by the higher plasma calcium levels and the tendency for a higher risk of hypercalcemia which has been reported in those EVITA participants who were assigned to vitamin D [25]. Our data do not support results of a recent metaanalysis of observational studies [29]. This meta-analysis included seven studies with 5183 participants. Vitamin  Results are presented as means and 95% confidence interval; b probability of group differences in mean change differences between groups D deficiency was associated with an increased incidence of anemia (odds ratio = 2.25, 95% CI = 1.47-3.44). Our data are however in line with results of three other recent RCTs [19,20,30] in subjects with concurrent iron-deficiency anemia, ethnic minorities living in Norway, and hypertensive patients. In these earlier investigations, study duration ranged from 8 to 16 weeks and vitamin D doses were 400 IU and 2800 IU daily or 600,000 IU once intramuscularly. The sample sizes in these earlier studies were 30, 214, and 188 participants, respectively, and the group-differences in change in mean Hb levels were −0.8 g/dL, −0.02 g/dL and 0.04 g/ dL, respectively. Although in another small, placebocontrolled trial [21] 650,000 IU vitamin D (50,000/ weekly over a 4-month period) significantly decreased the required dose of EPO, no correlation between Hb levels and 25OHD concentrations could be shown. Moreover, it remains unclear why in that RCT 20 out of the 64 study subjects were excluded from data analysis. The required ESA dose was also reduced in a small RCT in children with CKD stage 5, who received vitamin D 2 or placebo in conjunction with oral alfacalcidol (0.25 μg/capsule) for 12 weeks [18]. Unfortunately, no data on circulating 1,25(OH) 2 D levels were presented in this study. 1,25(OH) 2 D has been shown to stimulate erythroid progenitor cell proliferation via increased erythropoietin sensitivity [31]. Some earlier observational studies reported that compared with the reference category of circulating 1,25(OH) 2 D levels > 70 pmol/L, the multivariable-adjusted odds ratio for anemia was 2.35 to 4.08 in the categories of circulating 1,25(OH) 2 D levels < 40 pmol/L [2,11,12].
In the present RCT, the mean change difference in circulating 1,25(OH) 2 D was on average + 17.1 pmol/L. The increment is in line with a recent meta-analysis reporting that vitamin D supplements increase circulating 1,25(OH) 2 D on average by + 18.8 pmol/L (95% CI: 9.2 to 28.4 pmol/L) [32], but was probably too small to achieve significant effects on anemia prevalence. It might also be that in the present RCT the average initial 1,25(OH) 2 D level of 81.6 pmol/L was already above the threshold for stimulating erythropoiesis. It is also noteworthy that anemia risk is associated with the severity of HF [4,23,33,34]. Therefore, there may simply be no causal relationship between vitamin D status and anemia in advanced HF. This assumption is further supported by the fact that even in the subgroup of patients with eGFR values of < 60 mL/min/ 1.73 m 2 , where EPO production and circulating 1,25(OH) 2 D levels are reduced, vitamin D treatment had no effect on anemia prevalence and Hb levels. Although vitamin D treatment decreased MCHC values, median values remained in the reference range. We therefore conclude that this vitamin D-associated between-group difference is not of clinical relevance. Our study has several strengths, and a number of limitations. Strengths are the design of an RCT, the study duration of 3 years, and the measurement of the active form of vitamin D, 1,25(OH) 2 D, in addition to circulating 25OHD levels. One limitation is that only surrogate parameters were available on nutritional risk factors that may contribute to the development of anemia such as  iron, folate or vitamin B12 deficiency, whereas data on ferritin concentrations and/or transferrin saturation for the assessment of iron deficiency were missing. Considering that iron therapy is increasingly used in HF patients [35], we have to acknowledge that previous studies suggest a suppressive effect of high-dose vitamin D on hepcidin [36], a protein with profound effects on iron metabolism including inhibition of intestinal iron absorption. However, our findings including a decrease in MCHC with vitamin D treatment strongly argue against a clinically relevant beneficial effect of a moderately high daily vitamin D dose on iron metabolism. Additional limitations are that the study population was restricted to Caucasian ethnicity and that the vast majority of patients were male. Finally, it is noteworthy that nonsurvivors had significantly lower baseline Hb levels than survivors. We therefore cannot definitively exclude the possibility that patients with lower Hb levels might have a different/more response to vitamin D supplementation compared to patients with normal or higher Hb levels.

Conclusions
In summary, the present study found no significant effects of a daily vitamin D supplementation with 4000 IU for 36 months on the prevalence of anemia and Hb values in patients with advanced HF. Our data challenge the clinical relevance of vitamin D supplementation to increase Hb levels.

Additional file
Additional file 1: Table S1. Results of vitamin D treatment on biochemical parameters in patients with chronic heart failure and estimated glomerular filtration rate values < 60 mL/min/1.73 m2. (DOCX 27 kb)