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Abstract

Patients with rheumatoid arthritis have subnormal vitamin B6 status, both quantitatively and functionally. Abnormal vitamin B6 status in rheumatoid arthritis has been associated with spontaneous tumor necrosis factor (TNF)-α production and markers of inflammation, including C-reactive protein and erythrocyte sedimentation rate. Impaired vitamin B6 status could be a result of inflammation, and these patients may have higher demand for vitamin B6. The aim of this study was to determine if daily supplementation with 50 mg of pyridoxine for 30 days can correct the static and/or the functional abnormalities of vitamin B6 status seen in patients with rheumatoid arthritis, and further investigate if pyridoxine supplementation has any effects on the pro-inflammatory cytokine TNF-α or IL-6 production of arthritis. This was a double-blinded, placebo-controlled study involving patients with rheumatoid arthritis with plasma pyridoxal 5'-phosphate below the 25th percentile of the Framingham Heart Cohort Study. Vitamin B6 status was assessed via plasma and erythrocyte pyridoxal 5'-phosphate concentrations, the erythrocyte aspartate aminotransferase activity coefficient (αEAST), net homocysteine increase in response to a methionine load test (ΔtHcy), and 24 h urinary xanthurenic acid (XA) excretion in response to a tryptophan load test. Urinary 4-pyridoxic acid (4-PA) was measured to examine the impact of pyridoxine treatment on vitamin B6 excretion in these patients. Pro-inflammatory cytokine (TNF-α and IL-6) production, C-reactive protein levels and the erythrocyte sedimentation rate before and after supplementation were also examined. Pyridoxine supplementation significantly improved plasma and erythrocyte pyridoxal 5'-phosphate concentrations, erythrocyte αEAST, urinary 4-PA, and XA excretion. These improvements were apparent regardless of baseline B6 levels. Pyridoxine supplementation also showed a trend (p < 0.09) towards a reduction in post-methionine load ΔtHcy. Supplementation did not affect pro-inflammatory cytokine production. Although pyridoxine supplementation did not suppress pro-inflammatory cytokine production in patients with rheumatoid arthritis, the suboptimal vitamin B6 status seen in rheumatoid arthritis can be corrected by 50 mg pyridoxine supplementation for 30 days. Data from the present study suggest that patients with rheumatoid arthritis may have higher requirements for vitamin B6 than those in a normal healthy population.

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.

Study interventions

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.

Results

Indicators of vitamin B6 status before and after treatment are shown in Table . All markers of vitamin B6 status improved significantly in the B6 group after supplementation, except for net increase in total homocysteine concentration, which only showed a modest trend towards improvement. None of the vitamin B6 status parameters showed significant improvements after treatment in the placebo group. Analysis of co-variance further demonstrated that initial levels of plasma pyridoxal 5'-phosphate, ΔtHcy, post-load urinary XA, αEAST (Table ) and CRP and erythrocyte sedimentation rate (ESR) (Table ) in phase 1 were strong predictors of those indicators after treatment in phase 2. After adjusting for the initial levels in (before treatment), the vitamin B6 supplementation significantly improved plasma and erythrocyte pyridoxal 5'-phosphate concentrations, αEAST, post-load XA and 24 h 4-PA excretion. We found a trend for normalization of plasma ΔtHcy in the vitamin B6 treatment versus placebo group (p = 0.086, ANCOVA). In patients who had abnormal ΔtHcy (above 15 μmol/l) before treatment (n = 22/28), the effect of vitamin B6 treatment was significant (p < 0.02). Plasma pyridoxal 5'-phosphate and ΔtHcy levels were related to CRP in patients with rheumatoid arthritis [], thus CRP could be a potential targets for vitamin B6 supplementation. The correlations between CRP and plasma pyridoxal 5'-phosphate and ΔtHcy disappeared in the B6 group after supplementation, whereas the relationships remained in the placebo group after the 30 day treatment (CRP versus plasma pyridoxal 5'-phosphate, r = -0.61, p = 0.02; CRP versus ΔtHcy, r = 0.48, p = 0.098) (n = 14). We found that vitamin B6 supplementation had no effect on inflammatory cytokines, plasma CRP, ESR, or rheumatoid factor levels in these patients (Table ).

Abstract

YfiBNR is a recently identified bis-(3’-5’)-cyclic dimeric GMP (c-di-GMP) signaling system in opportunistic pathogens. It is a key regulator of biofilm formation, which is correlated with prolonged persistence of infection and antibiotic drug resistance. In response to cell stress, YfiB in the outer membrane can sequester the periplasmic protein YfiR, releasing its inhibition of YfiN on the inner membrane and thus provoking the diguanylate cyclase activity of YfiN to induce c-di-GMP production. However, the detailed regulatory mechanism remains elusive. Here, we report the crystal structures of YfiB alone and of an active mutant YfiBL43P complexed with YfiR with 2:2 stoichiometry. Structural analyses revealed that in contrast to the compact conformation of the dimeric YfiB alone, YfiBL43P adopts a stretched conformation allowing activated YfiB to penetrate the peptidoglycan (PG) layer and access YfiR. YfiBL43P shows a more compact PG-binding pocket and much higher PG binding affinity than wild-type YfiB, suggesting a tight correlation between PG binding and YfiB activation. In addition, our crystallographic analyses revealed that YfiR binds Vitamin B6 (VB6) or L-Trp at a YfiB-binding site and that both VB6 and L-Trp are able to reduce YfiBL43P-induced biofilm formation. Based on the structural and biochemical data, we propose an updated regulatory model of the YfiBNR system.

Abstract

The term vitamin B6 is a designation for the vitamers pyridoxal, pyridoxamine, pyridoxine and the respective phosphate esters pyridoxal-5′-phosphate (PLP), pyridoxamine-5′-phosphate and pyridoxine-5′-phosphate. Animals and humans are unable to synthesise vitamin B6. These organisms have to take up vitamin B6 with their diet. Therefore, vitamin B6 is of commercial interest as a food additive and for applications in the pharmaceutical industry. As yet, two naturally occurring routes for de novo synthesis of PLP are known. Both routes have been genetically engineered to obtain bacteria overproducing vitamin B6. Still, major genetic engineering efforts using the existing pathways are required for developing fermentation processes that could outcompete the chemical synthesis of vitamin B6. Recent suppressor screens using mutants of the Gram-negative and Gram-positive model bacteria Escherichia coli and Bacillus subtilis, respectively, carrying mutations in the native pathways or heterologous genes uncovered novel routes for PLP biosynthesis. These pathways consist of promiscuous enzymes and enzymes that are already involved in vitamin B6 biosynthesis. Thus, E. coli and B. subtilis contain multiple promiscuous enzymes causing a so-called underground metabolism allowing the bacteria to bypass disrupted vitamin B6 biosynthetic pathways. The suppressor screens also show the genomic plasticity of the bacteria to suppress a genetic lesion. We discuss the potential of the serendipitous pathways to serve as a starting point for the development of bacteria overproducing vitamin B6.

Abstract

The term vitamin B6 is a designation for the vitamers pyridoxal, pyridoxamine, pyridoxine and the respective phosphate esters pyridoxal-5′-phosphate (PLP), pyridoxamine-5′-phosphate and pyridoxine-5′-phosphate. Animals and humans are unable to synthesise vitamin B6. These organisms have to take up vitamin B6 with their diet. Therefore, vitamin B6 is of commercial interest as a food additive and for applications in the pharmaceutical industry. As yet, two naturally occurring routes for de novo synthesis of PLP are known. Both routes have been genetically engineered to obtain bacteria overproducing vitamin B6. Still, major genetic engineering efforts using the existing pathways are required for developing fermentation processes that could outcompete the chemical synthesis of vitamin B6. Recent suppressor screens using mutants of the Gram-negative and Gram-positive model bacteria Escherichia coli and Bacillus subtilis, respectively, carrying mutations in the native pathways or heterologous genes uncovered novel routes for PLP biosynthesis. These pathways consist of promiscuous enzymes and enzymes that are already involved in vitamin B6 biosynthesis. Thus, E. coli and B. subtilis contain multiple promiscuous enzymes causing a so-called underground metabolism allowing the bacteria to bypass disrupted vitamin B6 biosynthetic pathways. The suppressor screens also show the genomic plasticity of the bacteria to suppress a genetic lesion. We discuss the potential of the serendipitous pathways to serve as a starting point for the development of bacteria overproducing vitamin B6.

Introduction

Since animals and humans are unable to synthesise vitamin B6, these organisms have to take up vitamin B6 with their diet (Kraemer et al. ). Therefore, vitamin B6 is of commercial interest as a food additive and for applications in the pharmaceutical industry (Acevedo-Rocha et al. ). PN hypochloride, the commercial form of vitamin B6, is synthesised from PN and added usually in combination with other vitamins to food products like bakery products, cereals, baby nutrition, multivitamin juices and other beverages (Domke et al. ; Eggersdorfer et al. ). Moreover, PN is added to the food that is used for intensive animal farming to improve the yield (Johnson et al. ; Verbeek ; Eggersdorfer et al. ). Until today, the B6 vitamers are fully chemically synthesised using expensive and toxic chemicals (Eggersdorfer et al. ). Therefore, there is considerable interest in the biotech industry to develop a sustainable fermentation process for the production of vitamin B6.