Characterization of a new flavin metabolite from human urine.

A new flavin metabolite comprising approximately 5% of the total flavin of human urine was isolated and characterized using absorption and fluorescence spectra, oxidation-reduction and hydrolysis data, and ninhydrin reactions. The flavin is a derivative associated with a peptide residue in ester linkage from an amino acid carboxyl to the ribityl chain of riboflavin, probably at the 5'-terminus.

Fungal riboflavin 5'-hydroxymethyl dehydrogenase catalyzes formation of both the aldehyde (riboflavinal) and the acid (riboflavinoic acid).

The purified enzyme from Schizophyllum commune that readily catalyzes the oxidation of the 5'-hydroxymethyl function of riboflavin (vitamin B2) with a redox dye and O2 to form the 5'-aldehyde can more slowly further oxidize the 5'-aldehyde to the 5'-acid. Hence, the formation of these so-called 'schizoflavins' can be accounted for by the action of one enzyme.

Spectroscopic studies of complexes between pyridoxamine (pyridoxine)-5'-phosphate oxidase and pyridoxyl 5'-phosphate compounds differing at position 4'.

The absorbance spectrum of pyridoxamine (pyridoxine)-5'-phosphate oxidase (EC 1.4.3.5) is altered upon the binding of pyridoxal 5'-phosphate or analogs with different substituents at position 4'. The absorbance difference spectra are similar for complexes of the oxidase and pyridoxal 5'-phosphate, 4'-desoxypyridoxine 5'-phosphate, and 4-ethynyl-4-deformylpyridoxal 5'-phosphate; hence, these perturb the flavoprotein absorbance by similar interactions primarily involving the pyridoxyl 5'-phosphate moiety, and not specifically the 4-formyl group or other relatively small and uncharged functions at this position. A different type of spectral perturbation is caused by analogs with larger substituents at position 4' (i.e. 4'-methoxypyridoxine 5'-phosphate, 4-methyl-vinyl-4-deformylpyridoxal 5'-phosphate, and pyridoxal 5'-phosphate oxime and hydrazone). These analogs impose bulky groups in a region of the active site that critically influences the environment of the flavin, and, thus, may reflect positioning of this portion of the substrates close to the flavin ring, as is required for their redox interaction.

Flavin catabolites: identification and quantitation in human urine.

Riboflavin is the primary flavin excreted in human urine but significant amounts of 7 alpha-hydroxyriboflavin and lesser amounts of 8 alpha-hydroxyriboflavin are present and reflect tissue microsomal oxidations. A newly found flavin catabolite of an 8 alpha-sulfonyl type may reflect intake and/or turnover of such thioether-linked flavin as occurs in monoamine oxidase. Additionally, lesser amounts of 10-hydroxyethylflavin (indicative of intestinal microbial action on the vitamin) and traces of lumiflavin (arising from photodecomposition) constitute part of the remaining flavin, which acutely reflects level of intake.

Flavin composition of human milk.

The identity and quantity of greater than 95% of the flavins present in human milk were assessed by acid-phenol extractions followed by high-performance liquid chromatography. Flavin adenine dinucleotide (FAD) and riboflavin were the predominant flavins, followed by 10-(2'-hydroxyethyl)-flavin. In addition, traces of 7 alpha- and 8 alpha-hydroxyriboflavins (7-hydroxymethylriboflavin and 8-hydroxymethylriboflavin, respectively) were detected. The flavin content of human milk samples in this study was higher than contents reported in earlier studies where no correction for the internal fluorescence quenching of FAD was made. This finding may have implications for dietary recommendations concerning both lactating women and infants. In practical terms, the types and amounts of flavins in human milk are very similar to those recently reported for cow milk.

Pharmacokinetics of orally and intravenously administered riboflavin in healthy humans.

The pharmacokinetics and utilization (flavocoenzyme synthesis) of orally and intravenously administered riboflavin in healthy humans were assessed. After the determination of circadian rhythms of riboflavin concentrations in blood plasma and urine of four males and five females (control period), each of these subjects received three different oral riboflavin doses (20, 40, and 60 mg) and one intravenous bolus injection of riboflavin (11.6 mg). Vitamins were administered in a randomized, cross-over design with 2 wk between each administration. Blood plasma and urine specimens were collected repeatedly over a period of 48 h after each administration. Concentrations of flavocoenzymes and riboflavin were analyzed in blood plasma; riboflavin was assayed in urine. During the control period, a small circadian variation was observed: plasma concentrations and urinary excretion of riboflavin were low during the afternoon (P < 0.05). Pharmacokinetics were calculated using a two-compartment open model. The maximal amount of riboflavin that can be absorbed from a single dose was 27 mg per adult. Half-life of absorption was 1.1 h. First-order rate constants describing distribution and elimination of riboflavin were significantly higher after intravenous than after oral administration (P < 0.01). Release of flavocoenzymes into plasma was low compared with the increase of riboflavin concentrations. 7 alpha-Hydroxyriboflavin was identified in plasma. Clearance data indicated that urinary excretion of riboflavin contributes to one-half of the overall removal of riboflavin from plasma. No sex differences were observed for any of the pharmacokinetic variables (P > 0.05).

Identification of biotin sulfone, bisnorbiotin methyl ketone, and tetranorbiotin-l-sulfoxide in human urine.

In previous studies using the HPLC and avidin-binding assay, five unidentified avidin-binding substances were observed in human urine. The present study investigated the identity of these substances. Urine was collected before and after intravenous administration of 18.5 mumol biotin to healthy adults. Unknown substances 1 and 3 were initially identified as biotin sulfone and bisnorbiotin methyl ketone, respectively, by coelution with authentic standards on HPLC. Identities were confirmed by thin-layer chromatography and by derivatization with p-dimethyl-aminocinnamaldehyde. As expected for biotin metabolites, the urinary excretion of biotin sulfone and bisnorbiotin methyl ketone increased with biotin administration. The urinary excretion of biotin sulfone increased 21-fold from 0.2 nmol/h before to 4.2 nmol/h after administration; the excretion of bisnorbiotin methyl ketone increased 130-fold from 0.4 to 51.8 nmol/h. At presumed steady state in free-living subjects (n = 6), biotin sulfone and bisnorbiotin methyl ketone accounted for 3.6% and 7.9% of total biotin excretion, respectively. Traces of tetranorbiotin-l-sulfoxide were also identified by using thin-layer chromatography and derivatization with p-dimethylaminocinnamaldehyde. However, tetranorbiotin-l-sulfoxide was not detectable in urine by the HPLC and avidin-binding assay because this metabolite has weak avidin-binding affinity. We conclude that biotin sulfone and bisnorbiotin methyl ketone are present in measurable quantities in human urine; their quantitation should allow more accurate studies on human biotin metabolism and turnover.

Factors affecting riboflavin requirements of oral contraceptive users and nonusers.

Riboflavin depletion has been identified in women on oral contraceptives (OC) but change in riboflavin nutriture has not been consistently demonstrated in all OC user groups studied. Discrepant findings in reports have been attributed to differences of pill formulation or riboflavin intake. Aims of this study were to compare the riboflavin requirements of healthy OC users and nonusers on diets prepared in a metabolic unit. A single daily menu and meal pattern was used. The basic diet providing riboflavin at a level of 0.6 mg/1000 kcal was used in the period of acclimation and period 1. In periods 2 and 3, the riboflavin content of the diet was increased to 0.8 and 1.0 mg/1000 kcal, respectively. The riboflavin status of subjects was monitored by erythrocyte glutathione reductase assay and urinary riboflavin excretion. Eight women on OC and 10 nonusers participated. Erythrocyte glutathione reductase assay values and urinary riboflavin excretion showed intersubject and interperiod differences but no significant group differences (OC versus non-OC) in erythrocyte glutathione reductase values or in urinary riboflavin per g creatinine. It was concluded that when dietary intake is controlled, OC do not significantly influence riboflavin status. Riboflavin needs were related to energy requirements of the subjects.

Urinary riboflavin and its metabolites: effects of riboflavin supplementation in healthy residents of rural Georgia (USA).

The following study was designed to provide a comprehensive assessment of urinary riboflavin and flavin metabolites before and after ingestion of a riboflavin load in normal human volunteers. With respect to riboflavin, the total flavin excretion, the flavin-to-creatinine ratio, and the response of the flavin catabolites to the supplement indicate a well-nourished population. Differences in the effects of supplementation on the specific flavin catabolites across sex and age groups are included in detail for the first time. The feasibility of the use of flavin catabolites for determination of riboflavin status should be tested further in individuals with clinical signs of ariboflavinosis.

Isolation of volatile sulfur-containing microbial catabolites of biotin.

A soil pseudomonad was grown on biotin as sole source of carbon, nitrogen, and sulfur. A product of thiolane ring degradation, methyl thioacetate, was isolated by Tenax trapping and identified by GC-Mass spectrometry in comparison to the synthetic compound. H2S is another catabolite which was isolated via heavy metal trapping and was quantified in culture supernatant. Studies of acetylation and methylation activities of broken pseudomonad cells indicated that methyl thioacetate may be formed by chemical alterations of H2S as the primary sulfur-containing catabolite of biotin. H2S can also serve as sole sulfur source for the pseudomonad.

Bacterial catabolism of lipoic acid. Isolation and identification of a methyl ketone.

A soil bacterium, Pseudomonas putida LP, can be grown on lipoate as sole source of carbon, sulfur, and energy. In addition to the previously identified catabolites (bisnorlipoate, tetranorlipoate, beta-hydroxybisnorlipoate, lipoate thiolsulfinate, and two bisnorlipoate thiolsulfinates) isolated from cultures of the organism grown on [1,6-14C[lipoate, a methyl ketone (1,2-dithiolane-3-butyl-3'-one) has now been isolated and identified. This catabolite was isolated by solvent extraction and hydrophobic gel filtration and characterized by chromatographic mobilities and spray reactions and by UV, IR, PMR, and mass spectrometries. The methyl ketone presumably arises by decarboxylation of the beta-keto acid formed during the beta-oxidation of lipoate to bisnorlipoate by the microorganism.

The identification and kinetics of 7 alpha-hydroxyriboflavin (7-hydroxymethylriboflavin) in blood plasma from humans following oral administration of riboflavin supplements.

Following the administration of different oral (20, 40, 60 mg) and intravenous (11.6 mg) doses of riboflavin to healthy humans and female patients with liver cirrhosis (oral 40-mg dose), 7 alpha-hydroxyriboflavin (7-hydroxymethylriboflavin) was identified in blood plasma by fluorescence after high-performance liquid and thin-layer chromatographies, and by its absorbance spectrum. The apparent first-order absorption rate constant of 7 alpha-hydroxyriboflavin was 1.2 per hour in healthy subjects. Plasma peak concentrations of 40 nmol/l in males and 20 nmol/l in females (p < 0.01) were achieved within two hours. Peak concentrations and areas under the plasma curves (smaller in females, p < 0.01) of 7 alpha-hydroxyriboflavin were 5 to 16% of those observed for riboflavin. Healthy females showed an approximately 2.5-fold faster disposition of 7 alpha-hydroxyriboflavin from plasma than males (p < 0.01). Correction of peak concentrations and areas under the plasma curves by the rate constants of disposition led to the finding of approximately equal amounts of 7 alpha-hydroxyriboflavin released into plasma by both sexes (p > 0.05). No significant influence of different oral riboflavin doses on 7 alpha-hydroxyriboflavin kinetics was found (p > 0.05). Liver cirrhosis had no significant effect on the amount of 7 alpha-hydroxyriboflavin released into blood plasma (p > 0.05). However, the failure to detect this metabolite following intravenous riboflavin administration indicates a substantial influence of gastrointestinal- or liver-passage.

The metabolism of riboflavin in female patients with liver cirrhosis.

The metabolism of vitamin B2 was studied in five female patients with liver cirrhosis of varying etiology. Following the oral administration of 40 mg (106.3 mumol) riboflavin, plasma concentrations of riboflavin and flavo-coenzymes as well as urinary riboflavin excretion were analyzed over a period of 48 h. Results were compared to data obtained for healthy controls (Zempleni J. et al, Am. J. Clin. Nutr., 1996 [15]). About 18% of the administered vitamin was recovered from patients' urine, indicating an absorption similar to healthy subjects (p > 0.05). The area under the riboflavin plasma concentration vs time curve was 1.2-fold larger among patients than controls, but the difference was not significant (p > 0.05). Riboflavin peak concentrations in plasma (315.6 nmol/l) and times when those concentrations were achieved (3.0 h) were similar to those found for healthy subjects (p > 0.05). Flavocoenzyme peak plasma concentrations were increased 1.4-fold above their baseline levels in cirrhotics which was equal to controls (p > 0.05). 7 alpha-Hydroxyriboflavin was detected in the plasma of patients. Distribution and elimination kinetics of riboflavin were analyzed by using a two-compartment open model; the riboflavin plasma disposition rate constants of the patients (k alpha = 0.7232 h-1; k beta = 0.0627 h-1) were not different from controls (p > 0.05). No differences between both groups were found regarding renal excretion (renal clearance, first-order rate constants for renal excretion; p > 0.05). In conclusion, patients with liver cirrhosis of varying etiology and varying medical treatment did not show alterations of riboflavin turnover.