Biotin metabolites

Zempleni J and Mock DM (1999a) Advanced analysis of biotin metabolites in body fluids allows a more accurate measurement of biotin bioavailability and metabolism in hnmans./oMrnaf of Nutrition 129,494S-7S. 

Figure 11.2. Biotin metabolites. Relative molecular masses (Mr) biotin, 244.3 biotin sulfoxide, 260.3 biotin sulfone, 276.3 bisnorbiotin, 212.3 tetranorbiotin, 180.3 and bisnorbiotin sulfoxide, 228.3.

Unidentified biotin metabolites were analyzed and identified in urine from healthy adults by TLC. Three unknown biotin metabolites were identified as... 

Unidentified biotin metabolites were analyzed and identified in urine from healthy adults by TLC. " Three unknown biotin metabolites were identified as biotin sul-fone (Rf values 0.49 and 0.17), bisnorbiotin methyl ketone (Rf values 0.78 and 0.29), and tetranorbiotin-/-sulfoxide (/ f values 0.22 and 0.01) by derivatization with -demethylaminocinnamaldehyde after TLC on microcellulose with 1-butanol-acetic acid-water (4 1 1) and 1-butanol as solvents, respectively." ... 

Mock, D.M., Mock, N.I., and Stratton, S., 1997. Concentrations of biotin metabolites in human milk. Journal of Pediatrics. 131 456-458. 

To allow estimation of human biotin requirements and evaluation of potential deleterious efiects of marginal degrees of biotin deficiency, indicators of biotin status need to be determined and validated. Several explored directions include serum concentrations and urinary excretion rates of biotin and biotin metabolites, activities of the biotin-dependent decarboxylases in peripheral blood mononuclear cells, and urinary excretion rates of 3-hydroxy-isovaleric acid 3-methylcrotonyl glycine and 2-methylcitric acid. 

Excretion rates of biotin and biotin metabolites are early and sensitive indicators of biotin deficiency. A decrease in excretion to the lower limit of normal or even below the limit was observed in normal adults in whom biotin deficiency was experimentally induced. 

Unlike the urinary excretion rates, serum concentrations of biotin and biotin metabolites seem not to reflect accurately the biotin deficiency and are therefore inadequate candidates to track it (Mock 1999). Egg-white feeding studies (Mock et al. 1997) and biotin-free diets in patients under total parenteral nutrition (Velazquez et al. 1990) showed that the serum concentrations of biotin, bisnorbiotin and biotin sulfoxide did not decrease significantly during a period of 20 days, whereas other indicators confirmed the presence of biotin deficiency. 

Zempleni, J., McCormick, D.B., Stratton, S.L., and Mock, D.M., 1996. Lipoic acid (thioctic add) analogs, tryptophan analogs, andurea do not interfere with the assay of biotin and biotin metabolites by high-performance liquid chromatography/avidin-binding assay. The Journal of Nutritional Biochemistry. 1 518 523. 

Little is known about biotin degradation (26), but the main pathway so far identified in micro-organisms involves a P-oxidation of the side chain via bio-tinyl-5 -coenzyme A leading to bisnorbiotin and teranorbiotin (Fig. 6). Interestingly, bisnorbiotin has been identified as the main biotin metabolite in human, pig, and rat urine (27-29). 

Figure 6 Structures of biotin metabolites bisnorbiotin and tetranorbiotin.

The use of radiolabeled biotin or analogs, as tracers or in biosynthetic experiments, provides a way to increase the sensitivity of the detection after HPLC separation. For instance, Birch et al. have used a reversed-phase column (C18 column, isocratic elution, 10% acetonitrile in 50 mM sodium phosphate buffer pH 3.5 containing 20 mM triethylamine) with on-line radioactive detection to monitor the transformation of dethiobiotin to biotin catalyzed by biotin synthase (107). Using reversed-phase HPLC with radiometric flow detection, Mock et al. have detected biotin metabolites (biotin sulfoxides and bisnorbiotin) in pig and rat urine, after injection of the animal with radiolabeled biotin (28,29). 

Mock has described a very sensitive method (the limit of detection is around 5 fmol) for the detection of biotin and its metabolites, involving C18 reversed-phase HPLC separation followed by detection using an avidin-horseradish peroxidase conjugate (92). The principle of this method has already been discussed. This methodology has allowed the identification and quantification of biotin, bisnorbi-otin, biotin sulfoxides, and biotin sulfone in human and animal serum and urine. Furthermore, new biotin metabolites (tetranorbiotin sulfoxide and bisnorbiotin methyl ketone) were detected in human urine (114). This technique should potentiate more accurate studies on human biotin metabolism (see, e.g., 115). 

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