Study population
Thirty six adults with rheumatoid arthritis were recruited through the Tufts New England Medical Center (NEMC) Rheumatology Clinic as previously described [5]. Written informed consent was obtained from all subjects in accordance with the regulations of the NEMC/Tufts University Human Investigation Review Committee. Briefly, men and women over 18 years old fulfilling the American College of Rheumatology criteria for rheumatoid arthritis were eligible [17]. Patients with pregnancy, oral contraceptive use, anemia (hemoglobin ≤ 10 mg/dl), thrombocytopenia (platelet count ≤ 50,000/ul), abnormal liver transaminase (serum aspartate aminotransferase or alanine aminotransferase ≥ 50 IU/l), renal insufficiency (serum creatinine ≥ 1.5 mg/dl), diabetes, or cancer were excluded. Patients taking supplements containing vitamin B6 were asked to stop for ≥ 1 month before their participation in the study.
Study protocol
This double-blinded, randomized and placebo controlled trial was conducted in the General Clinical Research Center (GCRC) at Tufts-NEMC. Prior to enrollment, blood screening and urinalysis were performed to ensure qualification and to identify individuals with low circulating vitamin B6 for the study. To test the efficacy of vitamin B6 supplementation on those patients with reduced plasma pyridoxal 5'-phosphate, baseline (phase 1) vitamin B6 status was determined using a two day test procedure as follows. Patients taking methotrexate were asked to come at least 24 h after their weekly dose of this drug. On the first day of the evaluation (day 1), each subject arrived in the GCRC at 8 a.m. after having eaten breakfast. Each subject received a standard oral tryptophan load test (5 g powdered L-tryptophan dissolved in chocolate milk; Ajinomoto, Teaneck, NJ, USA) and collected urine for the next 24 h. The urine was kept refrigerated without additives during the collection period. Separate 24 h urine collection was done in the week prior to day 1 for the measurement of baseline XA and 4-pyridoxic acid (4-PA) excretion.
Subjects were asked to fast overnight starting at 8 p.m. on day 1 for the methionine load test next morning. After completion of the 24 h urine collection in the morning of day 2, each subject received a standard methionine load test [18]. Baseline fasting blood was drawn in a tube containing ethylenediaminetetraacetic acid (EDTA) (Becton Dickinson, Franklin Lakes, NJ, USA) for determination of plasma pyridoxal 5'-phosphate, fasting tHcy level, erythrocyte pyridoxal 5'-phosphate concentration, erythrocyte aspartate aminotransferase activity (EAST), and CRP concentrations. Aliquots were also collected for routine hematology and chemistry analyses. Peripheral blood mononuclear cells (PBMC) were collected from heparinized blood and isolated by Ficoll-Hypaque centrifugation, then washed and cultured for 24 h in 96-well flat-bottom plates with ultrafiltered, pyrogen-free RPMI 1640 medium (Sigma, St. Louis, MO, USA) that was supplemented with 100 μg/ml streptomycin and 100 U/ml penicillin, with 1% autologous heat-inactivated pooled serum and 1% L-glutamine. After incubation, plates were then frozen at -80°C until assay.
After collection of fasting blood on day 2, each patient was then given a standard oral methionine load test (100 mg/kg body weight powdered methionine dissolved in orange juice; Ajinomoto, Teaneck, NJ, USA). Blood was drawn 4 h after the methionine load for determination of the post-load tHcy level. Fasting plasma pyridoxal 5'-phosphate levels were determined within 1 week and the level was compared to the 25th percentile of the Framingham Offspring Heart Cohort [19]. Patients with a plasma pyridoxal 5'-phosphate level within the lowest quartile of the appropriate age and gender Framingham population (cycle 6, offspring group) were recruited for the supplementation phase of the study (phase 2). The 25th percentile cutoff for plasma pyridoxal 5'-phosphate in women below 55 years and women at or above 55 years were 33.7 and 37.5 nmol/l, respectively. For men below 55 years and for men at or above 55 years it was 49.7 and 35.6 nmol/l, respectively [19].
Study interventions
Qualified subjects started taking the study treatment within one week of plasma pyridoxal 5'-phosphate analysis. These subjects were randomly assigned through the NEMC pharmacy to receive either active vitamin B6 (B6 group) or placebo (placebo group) tablets in double-blinded fashion for 30 days. To minimize the potential confounding effect of methotrexate and prednisone treatment on the functional tests, subjects were stratified by prednisone and methotrexate treatment, and then the subjects in each group were randomized to receive either active or placebo treatment. The randomization procedure was under guidance of a statistician and performed by registered pharmacists not directly involved in the present study.
The placebo tablet, made specifically for the study, was identical in appearance as well as ingredients to the active tablet, except that the active tablet (Nutro Laboratories, South Plainfield, NJ, USA) contained 50 mg of pyridoxine hydrochloride and the placebo did not (Tishcon Corp., Westbury, NY, USA). Both tablets contained microcrystalline cellulose, croscarmellose sodium, calcium phosphate, stearic acid, and magnesium stearate, ingredients commonly found in over-the-counter vitamin B6 supplements. Each phase 2 participant was asked to take one assigned tablet daily throughout the 30 day period. To assure compliance with the treatment regimen, each subject was given a personal study calendar with the 30 supplement days highlighted. The subject was asked to record the time of ingestion of each tablet on the calendar. In addition, the study coordinator made phone calls to remind each subject to take the tablets during the 30 day supplement period. The subjects were asked to return the bottle for a tablet count at the end of the 30 day treatment. To test the efficacy of the vitamin B6 supplementation, each subject went through the same 2 day testing procedure described above at the end of the 30 day supplementation period.
Laboratory analyses
Blood hematology and chemistry analyses and urinalysis were performed at the Clinical Laboratory of NEMC, Boston, MA. CRP concentrations were determined by enzyme immunoassay kit (Virgo CRP150 kit, Hemagen, Waltham, MA, USA). Pyridoxal 5'-phosphate concentration was assayed by the tyrosine decarboxylase enzymatic procedure of Camp et al. [20] with a modification of the extraction procedure for plasma and erythrocytes. The modification is described as follows: a 20 μl plasma aliquot was precipitated with 4 volumes of 5% trichloroacetic acid for deproteinization. Erythrocytes were washed with 0.9% saline 3 times and the freshly washed erythrocytes were extracted with an equal volume of 10% (w/v) perchloroacetic acid. After centrifugation, the supernatants were stored at -70°C until the analysis. The erythrocyte pyridoxal 5'-phosphate results were expressed as nmol/l of packed erythrocyte at a hemotocrit of 100%. Fasting and post-methionine load plasma tHcy concentrations [21] and 4-PA [22] were determined by high performance liquid chromatography (HPLC) using a Hitachi L-7100 intelligent pump connected to an L-7400 UV detector (Hitachi, Tokyo, Japan). Baseline and post-tryptophan load urinary XA were measured by a colorimetric method [23]. EAST activity was measured using the Cobas Fara II Centrifugal Analyzer (Roche Dianostic system Inc., Nutley, NJ, USA) [24]. The ratio of pyridoxal 5'-phosphate saturated and unsaturated enzyme is expressed as the activity coefficient αEAST. Plasma TNF-α concentrations and PBMC TNF-α and IL-6 production was assayed with the commercially available quantitative enzyme immunoassays (Quantikine, R&D Systems, Minneapolis, MN, USA). Total PBMC cytokine production was measured in unstimulated cells (spontaneous production).
Statistical analysis
Differences in means between the baseline indices of the active group versus the placebo group were evaluated by Student's t-tests to examine if the randomization was successful. Differences were considered significant if the two-tailed p-value was <0.05. Plasma pyridoxal 5'-phosphate, tHcy, urinary XA, and 4-PA levels were log-transformed to achieve normality. Analysis of covariance (ANCOVA) was used to test the treatment effect of pyridoxine. The model was adjusted for the baseline (phase 1) value. A Pearson's correlation coefficient was calculated to examine the relationship between plasma pyridoxal 5'-phosphate levels and the inflammatory marker CRP before and after the treatment period. All statistical analyses were performed using Systat 10.0 for Windows ™ (SPSS, Chicago, IL, USA).