Abstract
By surveying compounds having isoalloxazine derived from flavins on a high performance liquid chromatogram with fluorescence detection, two new flavin derivatives were found in human urine. These two compounds were purified by partition chromatography on a cellulose column and by paper chromatography with several solvent systems, and their structures were determined to be 7 alpha-hydroxyriboflavin and 8 alpha-hydroxyriboflavin. The relative distributions, measured by high performance liquid chromatography, of 7 alpha- and 8 alpha-hydroxyriboflavin, riboflavin, and hydroxyethylflavin and its derivative were calculated to be 31.1, 5.0, 25.6, 4.9, and 21.9%, respectively, to total flavins in normal human urine obtained in early morning. The excretion of 7 alpha- and 8 alpha-hydroxyriboflavin in human urine indicates the occurrence of a metabolic pathway of the isoalloxazine ring of flavin at its 7 alpha and 8 alpha positions.
Highlights
Rived from flavinson a high performance liquid chro- FAD was released fromD-amino acid oxidase purified fromhog matogram with fluorescence detection, two new flavinkidney [14] by heating in boiling water for 20 s and purified on a dcoonemraivpcaoetulilnvudelsoswsewecreoerleufmopununraidfniineddhubmbyypaanppuaerrrintciethi.ToronhmecsaehttorwgoormaapthoygrapSmRahceiecbyptoohhrfaodlddainevxgoinftyGoSl-gu2tlh5zuuecckoomisliueedmttehanowl..daLso(u1fmp5Fr)fe.alpalHlavyraiendndrdowxaPaycseecttoephrrryidenlpifgnalagr(ve1ditn6ot)ahwfcercaosomrsdmyrininebgttohhtfeolosdaivzthieonedf
Nuclear magnetic resonance spectra were obtained with a JEOLJNM-FX 906 nuclear magnetic resonance spectrometer.Absorption and fluorescence spectra weremeasured in a Beckman DU-8 spectrophotometerand a Shimadzu RF-502 spectrofluorophotometer, respectively
The elution time for each compound depended on the meth- FIG. 1
Summary
Materials-Riboflavin and FMN were purchased from Nakarai Chemical Ltd., Kyoto and purified by paper chromatography using 1-. Solvent systems for column chromatography and paper chromatography were prepared in v/v ratio and referred to asfollows: SI,1butanol/glacial acetic acid/water (15:3:7);S2: 1-butanol/glacial acetic acid/water (4:1:5, upper phase); S:, 1-butanol/glacial acetic acid/. This article must be hereby Analytical Procedure-HPLC was carried out with a Shimadzu marked “advertisement” in accordance with 18 U.S.C. Section 1734 LC-4A system. Shimadzu RF-500LCA spectrofluorophotometer at 530 nm with excitation at 440 nm. The column temperature and theflow rate were fixed at 40 “C and 1.0 ml/min, respectively. A was recorded and converted to the amount from the standard curve of each flavin witha Shimadzu Chromatopacmodel C-R2AX record-. Nuclear magnetic resonance spectra were obtained with a JEOLJNM-FX 906 nuclear magnetic resonance spectrometer.Absorption and fluorescence spectra weremeasured in a Beckman DU-8 spectrophotometerand a Shimadzu RF-502 spectrofluorophotometer, respectively
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