Study setting, participants and design
This is a randomized open labelled controlled study that was conducted between January and May 2016 at BioTeSys GmbH, Esslingen am Neckar, Germany. The primary outcome of the study was changes in serum vitamin D after supplementation of two different doses of vitamin D provided as multinutrient supplements [18]. The secondary outcomes were changes in folate concentrations (serum and RBC-folate) and plasma tHcy. The present paper is concerned with the secondary outcome markers.
Participating women were randomized to receive either 400 or 800 µg/day folate as oral multinutrient supplements containing 1:1 of folic acid and (6S)-5-methyltetrahydrofolate-Ca [(6S)-5-CH3-H4folate-Ca]. The randomization was conducted using the software Randist.exe. The supplementation was conducted for 8 weeks starting from the recruitment visit 1 (Study flow chart, Supplemental Fig. 1).
Inclusion criteria were: healthy women, age 18–45 years, and BMI between 17 and 30 kg/m2. Exclusion criteria included taking supplements containing folate or vitamin D during the last 2 months, vascular diseases, history of cancer, diabetes, or contraindication of iodine supplementation. Gastrointestinal disorders that affect folate absorption such as Crohn’s disease, celiac disease, colitis ulcerosa, inflammatory bowel disease, and peptic ulcer were also exclusion criteria. Exclusion criteria and medical history were based on self-reported information, but several laboratory markers were measured to ensure the general health condition (i.e., liver markers, creatinine, and blood count). Since vitamin D was the main outcome of the study, an exclusion criterion was traveling to southern countries during the intervention (see study design under DRKS-ID: DRKS00009770).
Announcements in local newspapers and medias were used to invite volunteers. Interested women (n = 403) were preliminary screened by a structured telephone interview. Women who were likely to meet the inclusion criteria and none of the exclusion criteria (n = 213) were invited to the study centre and all of them attended the screening visit. After the screening visit, 12 women had to be excluded because of at least one of the exclusion criteria. The remaining 201 women were randomized and started the intervention (100 women in the 400 µg/day group and 101 in the 800 µg/day group). One woman in the 400 µg/day group and 2 in the 800 µg/day group did not return for the following visits, 1 woman in the 400 µg/day group and 1 in the 800 µg group did not return for visit 3, thus leaving 98 women in each of the study groups after 8 weeks (Supplemental Fig. 1).
A 3 days food diary protocol including alcohol intake was obtained prior to visit 1 and again 8 weeks later (prior to visit 3). Average intake levels were calculated from both assessment periods.
Participants received study fees as single payment upon completion of the study to enhance motivation and compensate for time and transportation to the study site. The study was conducted according to the ethical principles for medical research involving human subjects stated in Helsinki Declaration. The study protocol was reviewed and approved by the medical ethics commission of the Baden-Württemberg region (approval number: F-2015-102). All participants provided a signed consent to the study.
Supplement compositions, compliance, and safety
Participants were randomized to receive a daily capsule containing 400 µg folate [folic acid and (6S)-5-CH3-H4folate-Ca (1:1)] (Elevit® gynvital) or a tablet containing 800 µg folate [folic acid + (6S)-5-CH3-H4folate-Ca (1:1)] (Femibion® 1). The compositions of the multinutrients preparations are shown in Supplemental Table 1. Compliance with the study supplementation was documented through counting the tablets or capsules at each visit in addition to recording the intake in a study diary. The participants were asked to document any suspected adverse events in the volunteer’s diaries.
Blood samples and processing
Blood samples were collected at baseline (visit 1), week 4 (visit 2) and week 8 (visit 3). To avoid an acute effect caused by the last supplemental dose on plasma and serum biomarkers, participants were instructed to abstain from the study products 24 h before the blood collection (visits 2 and 3). Blood was collected after ≥10 h fasting into tubes without any anticoagulant and those containing K+EDTA or K+EDTA plus NaF (for plasma glucose). Serum and K+EDTA-whole blood were centrifuged and separated within 30 min of blood collection. For the measurement of whole blood folate, whole blood haemolysates were immediately prepared by diluting whole blood with 0.5% ascorbic acid and incubation for 3 h at ambient temperature. After separation (plasma, serum) or preparation (blood haemolysates), samples were immediately frozen at −70°C until analyses of the biomarkers. Samples from the same participant were measured at the end of the study in the same run. All markers were measured using aliquots that were not thawed before.
Biochemical assays
Concentrations of serum- and whole blood folate were measured using Chemoluminescence immunoassay (IMMULITE®). The concentrations of RBC-folate (nmol/L) were calculated according to the recommendation of the Centers for Disease Control and Prevention by adjusting for haematocrit and serum folate measured in the same individual https://www.cdc.gov/nchs/data/nhanes/nhanes_03_04/l06_c_met_folates-b12.pdf:
$${\text{RBC-folate }} = \, \left( {{\text{whole blood folate }} \times {\text{ dilution factor}}} \right) \, - \, \left[ {{\text{serum folate }} \times \, \left( { 1- {\text{haematocrit}}} \right)} \right] \, \times { 1}00/{\text{haematocrit}}.$$
The concentrations of tHcy were measured in EDTA plasma using commercially available reagents (Chromsystems Instruments & Chemicals GmbH) and a reversed phase-high pressure liquid chromatography connected to a fluorescence detector. The between-day coefficients of variation (CV%) for the folate assay were 8.8% at 3.6 nmol/L and 5.2% at 24.9 nmol/L. The CVs% for the tHcy assay were ≤8.2% at 10.0 µmol/L and 20.6 µmol/L.
Blood count (in K+EDTA whole blood), glucose (in NaF-plasma) and markers of liver and kidney functions (in serum) were measured. Measurements of the routine markers were conducted on the same day of blood collection (visits 1, 2, 3) using automatic analysers (Advia 2120i and Advia 2400).
Study power and statistical analyses
Folate markers (RBC- and serum folate) were secondary outcomes in this trial. The samples size (n = 100 per group) calculation was performed for the primary outcome variable, vitamin D. Based on earlier studies in young women from the same country [14, 19], we predicted a mean [standard deviation (SD)] baseline serum folate to be approximately 18.0 (9.0) nmol/L and that of RBC-folate to be 600 (250) nmol/L. For the priority folate marker, RBC-folate, the differences between the intervention groups were expected to be at least 15% after 4 or 8 weeks [mean difference = 170 (250) nmol/L]. Using different scenarios, we estimated that 100 participants in each group will be sufficient for detecting the differences in RBC-folate between the groups with 0.95 power and α = 0.05.
Data analyses included the 198 women who returned at least for visit 2 (intention-to-treat). For the two subjects stopping the study prior to visit 3, missing data were imputed by LOCF principle. The distributions of the continuous variables were tested using the Shapiro–Wilk test. The following variables showed normal distribution; creatinine, fasting glucose, haemoglobin, and haematocrit, while all folate markers were not normally distributed.
Testing the differences in continuous variables between two independent groups was performed by using unpaired t-test for variables with normal distribution and the Wilcoxon rank sum test for the variables that were not normally distributed. The Chi-square test was used for testing the differences in categorical variables between the independent groups. Comparisons within the group (over time) were tested by Friedman test with Dunn’s post test. Results of the continuous variables are expressed as mean ± SD. The changes of blood markers were calculated as post- minus pre-treatment concentrations. The differences were calculated for the first interval after 4 weeks (visit 2–visit 1) and for the whole duration of 8 weeks (visit 3–visit 1). Spearman test was used to study the correlations between different variables.
All statistical tests were two-sided and p values <0.05 were considered statistically significant and those between 0.05 and 0.10 were considered to indicate a tendency. Data analyses were performed using SPSS 24.0 and Graph Pad Prism Version 5.04.