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A method for enzymatic determination of riboflavin was developed by enzymatic reduction of 2,6-dichlorophenolindophenol using a vitamin B2-aldehyde-forming enzyme prepared from Schizophyllum commune. The enzymatic method enabled us to determine riboflavin in a mixture containing riboflavin, FMN and FAD without separation of riboflavin. Moreover, the total amount of flavins in the mixture was shown to be determined by the enzyme in the presence of phosphatase.
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Vitamin B2 and flavin cofactors are transported tightly bound to immunoglobulin in human serum. We reasoned that anti-mitochondrial flavoprotein autoantibodies (alpha Fp-AB) present in the serum of patients with myocarditis and cardiomyopathy of unknown aetiology may form immunoglobulin aggregates with these serum proteins. However, immunodiffusion and Western blot assays demonstrated that the flavin-carrying proteins were not recognized by alpha Fp-AB. Apparently the flavin moiety in the native protein conformation was inaccessible to alpha Fp-AB. This conclusion was supported by the absence of an immunoreaction between the riboflavin-binding protein from egg white and alpha FP-AB. Intravenous application of vitamin B2 to rabbits immunized with 6-hydroxy-D-nicotine oxidase, a bacterial protein carrying covalently attached FAD, did not neutralize alpha Fp-AB which had been raised in the serum of the animals. FAD-carrying peptides generated from 6-hydroxy-D-nicotine oxidase by trypsin and chymotrypsin treatment were not recognized by the alpha Fp-AB, but those generated by endopeptidase Lys were. This demonstrates that the epitope recognized by alpha Fp-AB comprises, besides the flavin moiety, protein secondary structure elements.
To determine the vitamin B2 supply, 62 athletes in various types of sports and 16 non-athletes were examined, using a weighed food record (7-day), alpha-EGR determination, microbiological blood, and HPLC urinary measurements. If the guidelines of 1.5 or 1.7 mg/day vitamin B2 or 0.6 mg/4,184 kJ (GRDA) are defined for adequate status, all sports groups (M +/- SEM) are above this level. The vitamin B2 blood concentrations show a range (mean) from 303-372 nmol/liter, which indicates that more than 90% of the athletes are above the reference value of 327 nmol/liter (mean). There is a correlation of r = 0.49 between the vitamin B2 intake (mean over 7 days) and the vitamin B2 blood level. Determination of vitamin B2 blood concentration is characterized by good precision (C.V. +/- 3.2%) and recovery (95.7%). The enzyme activation measurement (alpha-EGR) shows that all of the athletes are below the alpha-EGR reference value of < 1.50 (good supply). No significant correlations could be determined between alpha-EGR and vitamin B2 intake, blood concentrations, and urinary excretion. The measurement also shows high precision (C.V. 1.7%). The Vitamin B2 excretion in urine (mumol/g Cr.) was higher than the reference values for non-athletes in more than 90% of the athletes. There is a correlation of r = 0.89 between the vitamin B2 intake on the last day (Day 7) of the food record and the riboflavin excretion, and between vitamin B2 in blood and urinary excretion of r = 0.52. The C.V. of vitamin B2 urinary measurement using HPLC is 2.5%, with recovery of 98.2%. Combined measurement of vitamin B2 intake, blood concentration, and urine excretion appears to be a suitable procedure for determining the vitamin B2 status. Most of the performance athletes were sufficiently supplied with vitamin B2, compared to the reference values for non-athletes.
Assimilability of vitamin B2 from animal and plant foodstuffs has been studied in rats with plasma levels of free riboflavin and liver total riboflavin content as indices of B2 status. Studies have evaluated assimilability of vitamin B2 from diets containing buckwheat, oatmeal, dry milk, beef meat and liver. Assimilability of vitamin B2 from buckwheat and oatmeal was 40-70%, from milk-80%, from meat liver about 90-100% of vitamin determined by chemical methods. The authors make a conclusion that control of vitamin B2 bioavailability from different products is necessary for calculation of vitamin content in rations from the tables of vitamin content in foodstuffs.
AIMS: Development of an agar-diffusion assay to measure vitamin B2 in biological samples and application of the method to determine the amount of vitamin B2 secreted by bacteria. METHODS AND RESULTS: A riboflavin-auxotrophic mutant of Bacillus cereus was generated by mini-Tn10 insertion in the ribD gene. ribD mutant sensitivity to exogenous vitamin B2 was investigated by turbidimetric and agar-diffusion assays. In turbidimetric assays, the B. cereus mutant displayed a similar level of sensitivity to vitamin B2 to that of Lactobacillus casei ATCC 7469, the reference organism used for microbiological vitamin B2 quantification. However, only the ribD mutant could be used as an indicator organism in agar-diffusion assays. A total of eight probiotic strains, from five different probiotic formulations, were analysed by the ribD mutant-based assay on agar plates in order to determine their ability to secrete vitamin B2 during growth. CONCLUSION: The agar diffusion method with the ribD mutant of B. cereus is highly reproducible, sensitive, rapid, inexpensive, and can be applied to measure the amount of vitamin B2 in different samples. SIGNIFICANCE AND IMPACT OF THE STUDY: The method developed in this study appears to be a good candidate for the screening of vitamin B2 secretion by bacteria growing on solid media.
AIM: To establish a simple and novel method for the determination of vitamin B2 rapidly in pharmaceutical preparations. METHODS: Vitamin B2 was determined by a chemiluminescence (CL) sensor combined with flow-injection (FI) technology. The analytical reagents involved in the CL reaction, luminol and hexacyanoferrate (III), were both immobilized on an anion-exchange resin column in FI system. The CL signal produced by the reaction between luminol and hexacyanoferrate (III), which were eluted from the column through sodium phosphate injection, decreased in the presence of vitamin B2. RESULTS: The decreased CL intensity was linearly correlated with the vitamin B2 concentration in the range of 0.01-1.0 microgram.mL-1, the detection limit was 4.0 ng.mL-1 vitamin B2 (3 sigma). At a flow rate of 2.0 mL.min-1, the procedure including sampling and washing could be performed in 2 min with a relative standard deviation of less than 3.0%. CONCLUSION: The flow sensor exhibited both good sensitivity and stability. It could be reused more than 450 times and has been applied successfully to the analysis of vitamin B2 in pharmaceutical preparations.
The effect of vitamin B2 on the cellular reduction and cytotoxicity of chromate(VI) was studied using Chinese hamster V-79 cells. Electron spin resonance studies showed that incubation of cells with Na2CrO4 resulted in the formation of both chromium(V) and chromium(III) complex and that cellular pretreatment with riboflavin (Vitamin B2) for 24 h prior to exposure increased the level of chromium(V) complex, but the level of chromium(III) remained unchanged. Analysis of flavin derivatives revealed that pretreatment with vitamin B2 increased free riboflavin without altering flavin adenine dinucleotide and flavin mononucleotide. In addition, the level of the flavoenzyme glutathione reductase, which is capable of reducing chromate, was unaffected by pretreatment with vitamin B2. However, treatment of cells with vitamin B2 and Na2CrO4 augmented the inhibition of glutathione reductase attributable to Na2CrO4 alone. Using a colony-forming assay, pretreatment with vitamin B2 resulted in a decrease of cytotoxicity after exposure to the lethal concentration of chromate (15 microM) but did not affect the cytotoxicity at sublethal concentration of this metal (5-7.5 microM). Alkaline elution studies demonstrated that Na2CrO4 induced alkali-labile sites in the DNA of cells in a concentration-dependent manner (5-15 microM) and pretreatment with vitamin B2 resulted in an increase of these DNA lesions at all concentrations of Na2CrO4. The results, showing that vitamin B2 enhances chromate-induced alkali-labile lesions and chromium inhibition of glutathione reductase, might be due to an increase of chromium(V) species, possibly through its ability to directly reduce chromium(VI). The results also suggest that the extent of DNA lesions induced by chromate may not correlate directly with the cytotoxic effects of this metal.
Inhibitory effects of highly purified vitamin B2 (riboflavin-5'-sodium phosphate, >97%) on the interleukin (IL)-6, macrophage inflammatory protein (MIP)-2 and nitric oxide (NO) in LPS-induced shock mice were evaluated. Vitamin B2 at 20 mg/kg (protective effect on mice mortality induced by LPS), intravenously administered 6 h after LPS injection, significantly decreased the plasma elevated levels of IL-6 and MIP-2 at 9 and 12 h. In addition, vitamin B2 lowered the tissue concentration and the mRNA expression of IL-6 in lung and those of MIP-2 in liver at 9 h. Vitamin B2 also reduced concentration of MIP-2 concentration in lung, and inhibited mRNA expression in kidney, respectively. Vitamin B2 decreased the plasma elevated NO levels in accordance with a reduction in expression of inducible NO synthase (iNOS) both at 21 and 24 h. Accordingly, the reduction in elevated plasma cytokine levels and NO based on the inhibitory effect on local cytokine mRNA expression and iNOS would be responsible for the anti-septic effect of vitamin B2.
The effect of vitamin B2, which is capable of reducing chromium(VI) to chromium(V), on chromosomal aberrations and mutation caused by Na2CrO4 was investigated in Chinese hamster V79 cells. Pretreatment with 200 microM vitamin B2 (riboflavin) for 24 h prior to exposure to Na2CrO4 (2.5-5 microM) resulted in an increase of metal-induced chromosomal aberrations and mutation at the HGPRT locus. These and other previous studies suggest that vitamin B2 enhances the clastogenic and mutagenic action of chromate compounds, through its ability to directly reduce chromium(VI) in cells.
The aim of this experiment was to clarify whether an amino acid supplement could enhance the therapeutic effect of vitamin B2 (riboflavin 5'-sodium phosphate; purity > 97%) in mice with lipopolysaccharide-induced shock. Six hours after injection of a lethal dose of lipopolysaccharide, treatment (6-h i.v. infusion) was commenced. All mice died in the groups treated with saline or aminolevane (an amino acids mixture used to treat hepatopathy); however, the survival rates in the vitamin B2 (10 mg/kg/6 h) and vitamin B2 plus aminolevane groups were 45% (P < 0.05) and 80% (P < 0.05), respectively. Valine (200 mg/kg/6 h) alone had little effect on the survival rate (10%), but the combination of vitamin B2 (10 mg/k/g/6 h) and valine was highly effective (80%, P < 0.05). Clinical trials of vitamin B2 plus amino acids for the treatment of patients with sepsis would appear to be warranted.
The induction of DNA single strand breaks by carcinogenic chromate compounds has been found to be altered by vitamin E and vitamin B2. Pretreatment with vitamin E for 24 h prior to exposure to Na2CrO4 resulted in a decrease of chromate-induced DNA single strand breaks, whereas similar treatment with vitamin B2 enhanced levels of breaks induced by chromate. In contrast, levels of DNA protein crosslinks, the other major lesion induced, were not affected by vitamin E or vitamin B2. The uptake of Na2CrO4 was not affected by pretreatment with these vitamins. The role of vitamins in chromate-induced DNA damages is discussed.
The present study is carried out to explore whether the liver microsomal drug-metabolizing enzymes induced by PCB are associated with the PCB-induced liver lipid peroxide formation in rats. For this purpose, variations of the drug-metabolizing enzyme activities mediated by vitamin B2 deficiency were utilized. The administration of PCB to rats induced the liver microsomal cytochrome P-450 and vitamin B2 deficiency promoted the induction. The cytochrome b5 was also induced by PCB but no further induction by vitamin B2 deficiency was observed. The flavoenzyme, NADPH-cytochrome c reductase, was induced by PCB when the vitamin B2-supplemented PCB diet was fed to rats, but the activity of the enzyme was decreased by vitamin B2 deficiency and PCB further decreased the activity. The liver lipid peroxide levels increased in PCB groups with and without vitamin B2 compared with each PCB-free group, but the lipid peroxide level in vitamin B2-deficient PCB group was significantly lower than in vitamin B2-supplemented PCB group. From these results, the PCB-induced liver lipid peroxide formation was not necessarily related to the variations of the liver microsomal drug-metabolizing enzymes.
Rectal administration of metaside (20 mg per 100 g body weight) as suppositories for 10 days was found to cause vitamin B2 deficiency both in rats on low-vitamin B2 diet and in those on balanced diet. This appeared as decreased vitamin B2 levels in the liver and plasma. The design and application of suppositories containing the mixed preparation of metaside and riboflavin drug (in a dose of 100 micrograms per rat, which corresponds to the recommended daily requirement) completely prevents vitamin B2 deficiency.
It has been estimated that ten-day rectal administration of metazid in the form of suppositories (20 mg per 100 g of body mass) gives rise to vitamin B2 deficiency both in rats with initial vitamin B2 insufficiency and in those adequately supplied. This is confirmed by a decrease in the vitamin B2 content both in the liver and in the blood plasma. Development and use of suppositories containing combined preparation of metazid and riboflavin (in the dose of 100 micrograms per a rat according to the daily recommended allowance) completely prevent metazid-induced vitamin B2 deficiency.
Riboflavin and vitamin B6 status of mothers from a low-income group were assessed by erythrocyte glutathione reductase activation and erythrocyte aspartate aminotransferase activation tests respectively, at different stages of lactation. Levels of these vitamins in milk were also measured. The majority of the women had biochemical evidence of vitamins B2 and B6 deficiency. There was a bizarre reduction in erythrocyte glutathione reductase activation from 6 to 30 d post partum. Levels of riboflavin in milk were in general satisfactory, but vitamin B6 levels were lower than the values reported from developed countries. Clear-cut correlations between the enzymatic indices of vitamin status evaluation and milk levels of the corresponding vitamins were not apparent.
The effects of vitamin E, vitamin B2 and selenite on DNA single strand breaks induced by Na2CrO4 were examined by alkaline elution. Incubation of Chinese hamster V-79 cells with alpha-tocopherol succinate (vitamin E) for 24 h prior to exposure to Na2CrO4 resulted in a decrease of DNA breaks produced by this compound. However, similar pretreatment with riboflavin (vitamin B2) or Na2SeO3 resulted in an enhanced formation of breaks induced by Na2CrO4. Pretreatment with Na2SeO3 resulted in increased levels of glutathione in these cells while levels of glutathione remained the same with vitamin E or vitamin B2. These results suggest that Na2CrO4 induced DNA breaks appear to be mediated by the formation of free radicals and/or cellular reductive metabolism.