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Department of Pediatrics, University of Arkansas for Medical Sciences/Arkansas Children's Hospital Research Institute, Little Rock, AR 72202
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONPATHWAYS OF BIOTIN CATABOLISMANALYSIS OF BIOTIN AND...ORIGIN OF BIOTIN METABOLITES...BIOAVAILABILITY OF BIOTIN IN...REFERENCES |
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KEY WORDS: • analysis • bioavailability • biotin • biotin metabolites • humans
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONPATHWAYS OF BIOTIN CATABOLISMANALYSIS OF BIOTIN AND...ORIGIN OF BIOTIN METABOLITES...BIOAVAILABILITY OF BIOTIN IN...REFERENCES |
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, Clevidence et al. 1988
). In these studies, 24–58% of the
biotin dose was recovered in urine, suggesting that biotin
bioavailability is <100%. However, these studies could theoretically
underestimate the bioavailability because of the limitations of assay
procedures used. Biotin was analyzed either by Ochromonas
danica assay (Clevidence et al. 1988
) or by an avidin-binding
assay without metabolite separation (Bitsch et al. 1989
). The O.
danica assay measures biotin specifically, but does not detect
biotin metabolites. Thus, the bioavailability will be underestimated.
Avidin-binding assays are normally calibrated using biotin as a
standard and fail to compensate for the smaller affinities of biotin
metabolites to avidin (Zempleni and Mock 1999a
). Hence, both assays
will underestimate the true excretions or serum concentrations of
biotin plus metabolites in the presence of significant amounts of
biotin metabolites. In this symposium presentation, we describe the
progress that has been made in the past 10 years in the identification
and quantitation of biotin metabolites in mammals and the effect of
improved analysis on the quantitation of biotin turnover in humans. |
PATHWAYS OF BIOTIN CATABOLISM |
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TOPABSTRACTINTRODUCTIONPATHWAYS OF BIOTIN CATABOLISMANALYSIS OF BIOTIN AND...ORIGIN OF BIOTIN METABOLITES...BIOAVAILABILITY OF BIOTIN IN...REFERENCES |
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). In one pathway,
biotin is catabolized by ß-oxidation of its valeric acid side chain.
The repeated cleavage of two-carbon units leads to the formation of
bisnorbiotin, tetranorbiotin and related intermediates known to result
from ß-oxidation (e.g., 
,ß-dehydro–, ß-hydroxy– and
ß-keto–intermediates). Whether the ß-oxidation of biotin takes
place in mitochondria or peroxisomes is uncertain (Lee et al. 1972
,
Wang et al. 1997
).
In another pathway, biotin is catabolized by oxidation of the sulfur in
its heterocyclic ring. Sulfur oxidation leads to the formation of
biotin-l-sulfoxide, biotin-d-sulfoxide and biotin
sulfone. The sulfur oxidation of biotin may occur in the smooth
endoplasmic reticulum by an NADPH-dependent process (Lee et al. 1970
).
Combinations of both pathways of biotin catabolism occur, leading to
the formation of metabolites such as
bisnorbiotin-l-sulfoxide.
Biotin metabolites that originate from ß-oxidation or sulfur
oxidation have also been identified in mammals. In urine from rats and
pigs, biotin metabolites accounted for 47–66% of total biotin (Lee et al. 1972
, Mock et al. 1997
, Wang et al. 1996
). Hence, biotin
metabolites have to be quantitated in studies of human biotin turnover.
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ANALYSIS OF BIOTIN AND METABOLITES BY HPLC/AVIDIN-BINDING ASSAYS |
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TOPABSTRACTINTRODUCTIONPATHWAYS OF BIOTIN CATABOLISMANALYSIS OF BIOTIN AND...ORIGIN OF BIOTIN METABOLITES...BIOAVAILABILITY OF BIOTIN IN...REFERENCES |
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). First,
biotin and its metabolites are separated by HPLC; the eluate is
collected in individual fractions. Next, the fractions are assayed
using a sequential, solid-phase avidin-binding assay. Figure 1
shows a chromatogram from human urine analyzed by the
HPLC/avidin-binding assay. Biotin accounted for only one half of the
total avidin-binding substances; bisnorbiotin,
biotin-d,l-sulfoxide and five unidentified peaks accounted
for the remainder (Mock et al. 1993
). A similar profile was found in
human serum (Mock et al. 1995
).
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).
Lipoates and urea do not bind to avidin, whereas tryptophan shows some
binding but elutes discretely from biotin and its metabolites on HPLC.
We conclude that the unidentified peaks in our assay system are biotin
metabolites rather than interfering substances (Fig. 1)
.
Biocytin (biotinyl-
-lysine), bisnorbiotin,
biotin-d,l-sulfoxide and various synthetic biotin
derivatives bind less tightly than biotin to avidin (Green 1975
, Mock et al. 1993
, Wright et al. 1950
). Figure 2
shows calibration curves of biotin, bisnorbiotin and
biotin-d,l-sulfoxide in the avidin-binding assay. We
measured the avidin binding of 15 naturally occurring biotin
metabolites. The avidin binding of biotin metabolites was compared with
biotin and expressed as a binding ratio (binding ratio = avidin
binding of biotin metabolite/avidin binding of equimolar biotin).
Table 1
shows the binding ratios of some of the biotin metabolites; biotin
metabolites had smaller avidin affinities than biotin. Some biotin
metabolites may not be detectable at physiologic concentrations because
of their small avidin affinity. We concluded the following:
1) biotin compounds in biological samples should be
separated chromatographically before avidin-binding assays are used;
2) avidin-binding assays should be calibrated using
authentic standards of biotin metabolites rather than biotin alone; and
3) avidin-binding assays will detect most but not
necessarily all biotin metabolites.
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); 2) binding to avidin
(Mock 1997
); 3) Rf values on TLC in
two different solvent systems (Zempleni et al. 1997b
); and
4) derivatization with
p-dimethylaminocinnamaldehyde, a compound that reacts with
N1 of the biotin molecule to produce an intense color with
an absorption maximum at 533 nm (McCormick and Roth 1970
). Using these
techniques, we identified biotin sulfone, bisnorbiotin methyl ketone (a
side product of the ß-oxidation of biotin) and
tetranorbiotin-l-sulfoxide in human urine, as well as
biotin, bisnorbiotin and biotin-d,l-sulfoxide (Zempleni et al. 1997b
). The peak previously denoted as unknown #1 (Fig. 1)
is
biotin sulfone. Bisnorbiotin methyl ketone is peak #3. In urine from
six normal adults, biotin and bisnorbiotin were the two major biotin
compounds; they accounted for a combined 84% of the molar total
(Table 2
).The newly identified biotin sulfone and bisnorbiotin methyl ketone
accounted for a combined 12%; biotin-d,l-sulfoxide
accounted for 4%; tetranorbiotin-l-sulfoxide was not
quantitated because it did not bind to avidin sufficiently. Given the
small subject number, it is not surprising that there are some small
differences in metabolite profile from our previously published values
for 10 normal subjects (Mock et al. 1993
).
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ORIGIN OF BIOTIN METABOLITES IN HUMAN URINE |
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TOPABSTRACTINTRODUCTIONPATHWAYS OF BIOTIN CATABOLISMANALYSIS OF BIOTIN AND...ORIGIN OF BIOTIN METABOLITES...BIOAVAILABILITY OF BIOTIN IN...REFERENCES |
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Hence, initially we sought to determine whether the biliary excretion
of biotin is quantitatively important. [14C]Biotin was
administered intravenously to rats, and bile and urine were collected
in timed intervals over 24 h (Zempleni et al. 1997a
). In urine, we
recovered 60.6 ± 4.1% of the dose; in bile, we recovered only
1.9 ± 0.2%. We concluded that the biliary excretion of biotin is
quantitatively minor and that exposure of intravenously administered
biotin to intestinal microorganisms is likely to be small.
We hypothesized that if the concentration of biotin metabolites in
human urine increases after intravenous administration of biotin, the
metabolites must originate from catabolism in human tissues.
Alternatively, if the metabolites originate from microbial catabolism
of dietary biotin, their urinary concentration would remain unchanged
after intravenous biotin administration. Biotin (18.5 µmol,
equivalent to 15–45 times the recommended dietary intake) was
administered intravenously to six adults; untimed single void urines
were collected before and after biotin administration (Zempleni et al. 1997b
). The urinary excretion was expressed in units of nmol/h.
Excretion of biotin sulfone, bisnorbiotin,
biotin-d,l-sulfoxide and bisnorbiotin methyl ketone
increased 21–130 times after biotin injection. Thus, it seems likely
that biotin metabolites in human urine originate from biotin catabolism
in human tissues.
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BIOAVAILABILITY OF BIOTIN IN HUMANS |
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TOPABSTRACTINTRODUCTIONPATHWAYS OF BIOTIN CATABOLISMANALYSIS OF BIOTIN AND...ORIGIN OF BIOTIN METABOLITES...BIOAVAILABILITY OF BIOTIN IN...REFERENCES |
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).
Amounts that were ~50% of the intravenous dose and each of the two
larger oral doses of biotin were recovered in urine as biotin plus
biotin metabolites; ~100% of the smallest oral dose was recovered in
urine (Fig. 3
, upper
panel).Because the recovery of two large oral doses is about the same as the
recovery of the intravenous dose, it seems likely that these largest
oral doses were absorbed completely. Of course, the 100% recovery of
the smallest oral dose does not imply 200% bioavailability. Rather,
dietary biotin in the self-chosen diet probably accounts for the
increased urinary biotin and metabolite excretion following the
smallest test dose.
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). In the postdose urines, biotin per se accounted for
>50% of the avidin-binding substances (Fig. 3
, lower
panel). The percentage of biotin was significantly greater after
the intravenous dose compared with the three oral doses. In addition,
the percentage of biotin was significantly greater after the largest
oral dose compared with the 8.2-µmol dose. Bisnorbiotin accounted for
13–23% of total biotin, biotin-d,l-sulfoxide for 5–13%,
bisnorbiotin methyl ketone for 3–9% and biotin sulfone for 1–3%.
Generally, the percentage excretions of biotin metabolites were smaller
than baseline percentages and smaller for intravenous administration
compared with oral. Also, there was a tendency for the percentage
excretion of biotin metabolites to be smaller after the 81.9-µmol
dose compared with the two smaller oral doses.
The smaller percentage excretion of biotin metabolites after
intravenous biotin administration and after the largest oral dose
compared with the two smaller oral doses is probably due to the rapid
urinary excretion of biotin when large serum biotin concentrations are
achieved. Saturation of ß-oxidation or sulfur oxidation is unlikely
to occur because of the enzymatic capacities of these pathways (Lee et al. 1972
).
We concluded that biotin is absorbed nearly completely even at pharmacologic doses. The percentage excretion of biotin metabolites tends to increase when smaller doses of biotin are administered and when they are given orally.
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FOOTNOTES |
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1 Presented at the symposium "Nutrition,
Biochemistry and Molecular Biology of Biotin" as part of Experimental
Biology 98, April 18–22, 1998, San Francisco, CA. The symposium was
sponsored by the American Society for Nutritional Sciences and was
supported in part by an educational grant from Roche Vitamins and Fine
Chemicals. Published as a supplement to The Journal of
Nutrition. Guest editor for the symposium publication was Donald
Mock, University of Arkansas for Medical Sciences, Arkansas Children's
Hospital, Little Rock, AR.
2 Supported by National Institutes of Health grant
DK 36823 (to D.M.M.).
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REFERENCES |
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TOPABSTRACTINTRODUCTIONPATHWAYS OF BIOTIN CATABOLISMANALYSIS OF BIOTIN AND...ORIGIN OF BIOTIN METABOLITES...BIOAVAILABILITY OF BIOTIN IN...REFERENCES |
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1. Bitsch R., Salz I., Hötzel D.. Studies on bioavailability of oral biotin doses for humans. Int. J. Vitam. Nutr. Res. 1989;59:65-71.[Medline]
2. Clevidence B., Marshall M., Canary J. J.. Biotin levels in plasma and urine of healthy adults consuming physiological levels of biotin. Nutr. Res. 1988;8:1109-1118.
3. Green N. M.. Avidin. Adv. Protein Chem. 1975;29:85-133.[Medline]
4. Lee H. M., Wright L. D., McCormick D. B.. Metabolism of carbonyl-labeled 14C-biotin in the rat. J. Nutr. 1972;102:1453-1464.
5.
Lee Y. C., Joiner-Hayes M. G., McCormick D. B.. Microsomal oxidatin of -thiocarboxylic acids to sulfoxides. Biochem. Pharmacol. 1970;19:2825-2832.[Medline]
6. McCormick D. B., Roth J. A.. Specificity, stereochemistry, and mechanism of the color reaction between p-dimethylaminocinnamaldehyde and biotin analogs. Anal. Biochem. 1970;:226-236.
7. McCormick D. B., Wright L. D.. The metabolism of biotin and analogues. Florkin M. Stotz E. H. eds. Metabolism of Vitamins and Trace Elements 1971:81-110 Elsevier Publishing Company Amsterdam, The Netherlands.. .
8. Mock D. M.. Determinations of biotin in biological fluids. McCormick D. B. Suttie J. W. Wagner C. eds. Vitamins and Coenzymes, Part 1 1997:265-275 Academic Press San Diego, CA.. .
9. Mock D. M., Lankford G. L., J. Cazin J.. Biotin and biotin analogs in human urinebiotin accounts for only half of the total. J. Nutr. 1993;123:1844-1851.
10. Mock D. M., Lankford G. L., Mock N. I.. Biotin accounts for only half of the total avidin-binding substances in human serum. J. Nutr. 1995;125:941-946.
11.
Mock D. M., Wang K.-S., Kearns G. L.. The pig is an appropriate model for human biotin catabolism as judged by the urinary metabolite profile of radioisotope-labeled biotin. J. Nutr. 1997;127:365-369.
12.
Wang K.-S., Mock N. I., Mock D. M.. Biotin biotransformation to bisnorbiotin is accelerated by several peroxisome proliferators and steroid hormones in rats. J. Nutr. 1997;127:2212-2216.
13. Wang K.-S., Patel A., Mock D. M.. The metabolite profile of radioisotope-labeled biotin in rats indicates that biotin metabolism is similar to that in humans. J. Nutr. 1996;126:1852-1857.
14. Wright L. D., Valentik K. A., Nepple H. M., Cresson E. L., Skeggs H. R.. Affinity of avidin for biocytin. Proc. Soc. Exp. Biol. Med. 1950;74:273-274.
15.
Zempleni J., Green G. M., Spannagel A. U., Mock D. M.. Biliary excretion of biotin and biotin metabolites quantitatively minor in rats and pigs. J. Nutr. 1997;127:1496-1500.
16.
Zempleni J., McCormick D. B., Mock D. M.. Identification of biotin sulfone, bisnorbiotin methyl ketone, and tetranorbiotin-l-sulfoxide in human urine. Am. J. Clin. Nutr. 1997;65:508-511.
17. Zempleni J., McCormick D. B., Stratton S. L., Mock D. M.. Lipoic acid (thioctic acid) analogs, tryptophan analogs, and urea do not interfere with the assay of biotin and biotin metabolites by high-performance liquid chromatography/avidin-binding assay. J. Nutr. Biochem. 1996;7:518-523.
18. Zempleni J., Mock D. M.. Biotin. Song W. O. Beecher G. R. eds. Modern Analytical Methodologies on Fat and Water-Soluble Vitamins 1999 John Wiley & Sons New York, NY (in press).. .
19. Zempleni J., Mock D. M.. Bioavailability of biotin given orally to humans in pharmacologic doses. Am. J. Clin. Nutr. (in press). 1999;.
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