B2 Vitamers Research Articles

Effects of a Folic Acid 5 Mg Daily Intervention on Markers of Vitamin B Status and Kynurenine Pathway

BackgroundIn Brazil, most folic acid (FA) containing supplements contain 5 mg FA. This dose is usually prescribed for patients with chronic hemolytic anemia and for women planning pregnancy. The effects of high FA doses on vitamin B1, B2 and B6 status including the kynurenine pathway are still unclear.AimsTo assess the effects of a daily intervention with 5 mg FA in healthy subjects on vitamins B1, B2 and B6 serum marker status and serum concentration ok kynurenines.Material and MethodsThirty health subjects participated in a 5 mg/day FA intervention. Blood samples were obtained at baseline (D0) and after 45 (D45) and 90 days (D90) of intervention. We assessed serum concentrations of vitamin B6 (pyridoxal-5'-phosphate (PLP), pyridoxal (PL) and 4-pyridoxic acid (PA)), vitamin B2 (riboflavin and flavin mononucleotide (FMN)) and vitamin B1 vitamers (thiamine and thiamine monophosphate (TMP)), as well as tryptophan, kynurenine and several metabolites of tryptophan catabolism in the kynurenine pathway (3-hydroxykynurenine (HK), kynurenic acid (KA), anthranilic acid (AA), xanthurenic acid (XA), 3-hydroxyanthranilic acid (HAA), picolinic acid (Pic) and quinolinic acid (QA)) using LC-MS/MS. PA ratio (PAr) was calculated by PA/(PLP + PL). All variables were normalized by Box-Cox transformation. Comparisons were performed by repeated-measures ANOVA.ResultsConcentrations of all vitamin B6 vitamers (PLP, PL and PA), as well as PAr, were similar at baseline and D45 and D90 of FA intervention. No differences were found among serum vitamin B2 vitamers (riboflavin and FMN) after FA intervention. However, a trend for a decrease in serum TMP concentrations was observed at D90 (P = 0.054). No changes in serum tryptophan and all serum kynurenine pathway metabolites were observed after intervention with FA compared with baseline, except for AA, which was higher at D45 and D90 than baseline (P < 0.001).ConclusionOur data suggest that daily use of 5 mg FA by healthy subjects may interfere with vitamin B1 status markers. In addition, serum AA increased with FA intervention, which suggest that the use of high dose FA supplements might affect the kynurenine pathway.Financing: FAPESP (2016/18854-4) and CNPq (401586/2014-6) DisclosuresNo relevant conflicts of interest to declare.

Rapid determination of the various native forms of vitamin B6 and B2 in cow’s milk using ultra-high performance liquid chromatography

As the formation of pyridoxal phosphate, the active cofactor of vitamin B6, is dependent on riboflavin 5-phosphate, we propose a fast and simple ultra-high performance liquid chromatography method for the simultaneous determination of the native B6 vitamers pyridoxal, pyridoxine, pyridoxamine, their mono phosphorus esters and 4-pyridoxic acid as well as vitamin B2 as riboflavin and its phosphorus ester riboflavin 5-phosphate in milk. Separation was achieved under 6.0min by reversed-phase and pH gradient elution. Sample preparation was optimized regarding various acids and pH levels. Changes in those parameters led to significant deviations of sample matrix breakdown efficiency. The optimized method was then validated regarding specificity, accuracy, precision, linearity, range, detection and quantification limits. As the method performed satisfactory, is was used to study commercial liquid cow’s milk (n=31), regarding effects of the employed preservation technique (pasteurization, extended shelf-life, ultra-high temperature) on the composition and content of B6 and B2 vitamers. In cow’s milk, vitamin B6 mostly consists of pyridoxal and its phosphate ester, with pyridoxal phosphate being the bulk component. The catabolite of the vitamin B6 metabolism, 4-pyridoxic acid was present in significant amounts in all studied samples, with up to 2.69μmolL−1. Vitamin B2 was present as riboflavin and its phosphate ester up to 12.86μmolL−1.

Liquid Chromatographic Determination of B2 Vitamers in Human Plasma and Whole Blood

Riboflavin (RF; vitamin B2) participates in redox reactions in 2 coenzyme forms, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) (1). Quantification of these 3 flavins in plasma and erythrocytes is essential for studying vitamin B2 metabolism (1)(2). We describe here an HPLC method for quantifying these flavins in both matrices. Riboflavin and FMN sodium salt were from Sigma, FAD disodium salt and acetonitrile were from Merck, and the internal standard galactoflavin was from Merck & Dohme. All chemicals were analytical grade. For quality control, we used 3 ClinChek serum and whole-blood controls (Recipe), reconstituted and stored at −80 °C. Aliquots of aqueous (9 g/L NaCl) flavin stock solutions (50 μmol/L) were stored at −20 °C in the dark. Stock solution concentrations were verified by measurement of absorbances at 450 nm (3). The HPLC system consisted of a Thermoquest SP 8700 X pump, Rheodyne Model 7125 100-μL loop injector, Jasco FP-920 fluorescence detector (excitation, 456 nm; emission, 512 nm), and Shimadzu Model C-R1B Chromatopac Integrator. A Phenomenex Onyx Monolithic C18 column [50 × 4.6 mm (i.d.)] …

Liquid chromatographic analysis of riboflavin vitamers in foods using fluorescence detection.

The analysis of free riboflavin (RF) and its two coenzymes, flavin mononucleotide (FMN) and flavin-adenine dinucleotide (FAD), is optimized using reversed phase liquid chromatography with fluorescence detection. The stationary phase was amide-based and endcapped with trimethylsilyl, and the isocratic mobile phase consisted of a 10:90 v/v acetonitrile/phosphate buffer (pH 5). Peaks were identified by the retention characteristics and fluorescence spectra. Detection limits were 0.03, 0.05, and 0.24 ng for RF, FMN, and FAD, respectively. The vitamins were extracted using acetonitrile and the phosphate buffer. The procedure was applied to the determination of B2 vitamers in different types of food such as milk and soy-based infant formulas, beer, fruit juices, and honey of different types. Most B2 vitamin appeared as RF, while the coenzymes were present in lower amounts. The method was validated using two certified reference materials, and results within the certified range were obtained.

Riboflavin, Flavin Mononucleotide, and Flavin Adenine Dinucleotide in Human Plasma and Erythrocytes at Baseline and after Low-Dose Riboflavin Supplementation

Vitamin B(2) exists in blood as riboflavin and its cofactors, flavin mononucleotide (FMN) and FAD. The erythrocyte glutathione reductase activation coefficient (EGRAC) has traditionally been used to assess vitamin B(2) status in humans. We investigated the relationships of EGRAC and plasma and erythrocyte concentrations of riboflavin, FMN, and FAD in elderly volunteers and their responses to riboflavin administration. EGRAC and plasma and erythrocyte concentrations of riboflavin, FMN, and FAD were determined in 124 healthy individuals with a mean age of 69 years. The same measurements were made in a subgroup of 46 individuals with EGRAC > or =1.20 who participated in a randomized double-blind 12-week intervention study and received riboflavin (1.6 mg/day; n = 23) or placebo (n = 23). Median plasma concentrations were 10.5 nmol/L for riboflavin, 6.6 nmol/L for FMN, and 74 nmol/L for FAD. In erythrocytes, there were only trace amounts of riboflavin, whereas median FMN and FAD concentrations were 44 and 469 nmol/L, respectively. Erythrocyte FMN and FAD correlated with each other and with EGRAC and plasma riboflavin (P <0.05). All variables except plasma FAD responded significantly to riboflavin supplementation compared with placebo (P < or =0.04). The strongest increases were for riboflavin in plasma (83%) and for FMN in erythrocytes (87%). Concentrations of all B(2) vitamers except plasma FAD are potential indicators of vitamin B(2) status, and plasma riboflavin and erythrocyte FMN may be useful for the assessment of vitamin B(2) status in population studies.

Determination of riboflavin, flavin mononucletide and flavin adenine dinucleotide in biological tissues by capillary zone electrophoresis and laser-induced fluorescence detection.

The separation of riboflavin, flavin mononucleotide and flavin adenine dinucleotide was investigated by capillary zone electrophoresis using laser-induced fluorescence detection. In the systematic approach developed, the differential electrophoretic mobilities were first maximized by adjusting the pH. Increasing the buffer concentration improved the separation at the expense of migration times. A buffer consisting of 50 mM phosphate adjusted to pH 8.5 was found to provide a very efficient and stable electrophoretic system. Responses were linear within the range 0.1-100 micromol L(-1), and the detection limits of B2 vitamers were 0.23 nmol L(-1) or less. The method was successfully applied to a variety of biological tissues from different animals.

Analysis of riboflavin and riboflavin cofactor levels in plasma by high-performance liquid chromatography

We describe an assay which determines simultaneously riboflavin (RF), flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) in plasma, using galactoflavin (GF) as an internal standard. The flavins were extracted on a C 18 Sep-Pack cartridge after protein precipitation with 10% trichloroacetic acid, and were analyzed on a C 18 RP-HPLC with 85% phosphate–magnesium acetate buffer (pH 3.4) and 15% acetonitrile. FAD, FMN, GF and RF extraction recoveries were 101.0±5.6, 97.0±6.5, 97.0±2.0 and 95.0±4.1%, and reproducibilities were 5.9, 6.8, 2.1 and 4.3%, respectively. FAD, FMN and RF values in infant and adolescent plasma were in the range 53.5–108.2, 9.0–25.1 and 12.7–53.4 n M, and 36.5–157.20, 7.1–24.6 and 8.2–57.8 n M, respectively. Using GF as an internal standard improved the quantification of these B2 vitamers.

Photokinetic determination of riboflavin and riboflavin 5′-phosphate using flow injection analysis and chemiluminescence detection

A flow injection procedure for the photochemical determination of riboflavin and riboflavin 5′-phosphate has been developed. The method is based on the photo-reduction of both compounds by ethylenediaminetetraacetic acid, the rate of which is a linear function of their concentration at very low levels. The rate of these photochemical reactions was monitored by making use of the chemiluminescent hydrogen peroxide–luminol reaction and determining the hydrogen peroxide reduced by the 1,5-dihydro forms of these flavins, which are generated during the photochemical process. Linear calibration graphs were obtained between 1 × 10–7 and 3 × 10–6 mol l–1. The method has been successfully applied to the determination of B2 vitamers in pharmaceutical preparations, foods and animal tissues.

Determination of B2 vitamers in pharmaceutical preparations, foods and animal tissues by a photokinetic method.

A fast and sensitive photokinetic method for the determination of riboflavin (RF) and riboflavin 5′-phosphate (FMN) is described. It is based on the rate of photoreduction of RF and FMN by EDTA in the absence of oxygen. The rate of photoreduction is a linear function of the concentration of both B2 vitamers at very low concentrations.The rate of these photochemical reactions is monitored polarographically by recording the limiting current of p-benzoquinone, which is reduced by the 1,5-dihydro forms of RF and FMN that are generated in the photochemical reactions. The results obtained by the application of the fixed time, fixed concentration and initial rate kinetic methods have been evaluated. An alternative method for monitoring the rate of the process is by measuring the time necessary for the total reduction of p-benzoquinone. The end-point is detected with two platinum electrodes at an applied voltage of 100 mV. The procedure has been successfully applied to the determination of B2 vitamers in pharmaceuticals, foods and animal tissues.

Determination of B2 vitamers in the body fluid of plankton using high-performance liquid chromatography with fluorescence detection

A sensitive method, using high-performance liquid chromatography (HPLC) with fluorescence detection, has been developed for the separation and determination of B2 vitamers in the body fluid of plankton. B2 vitamers are liberated from the protein moiety in the body fluid by non-hydrolytic extraction with trichloroacetic acid and analysed by the proposed HPLC method. The major B2 vitamer in the body fluid of plankton is riboflavin, although there is also evidence to suggest the presence of endogenous enzymes that can decompose flavin mononucleotide and flavin adenine dinucleotide.

Determination of B2 vitamers in serum of fish using high-performance liquid chromatography with fluorescence detection.

A sensitive method is described for the determination of riboflavin (RF), flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) in serum of different fish using reversed-phase high-performance liquid chromatography (HPLC) with fluorimetric detection. Trichloroacetic acid was used to isolate B2 vitamers from the serum, and an aliquot of this solution was analysed by HPLC with a Zorbax-NH2 column with methanol-0.2 M phosphate buffer (1 + 9) as the mobile phase. The detection limits of RF, FMN and FAD in the serum were 4.89, 9.13 and 73.1 ng ml–1, respectively.