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Biotin accounts for less than half of all biotin and biotin metabolites in the cerebrospinal fluid of children^1,2,3

¹ From the Departments of Biochemistry and Molecular Biology (AB, SLS, and DMM), Pathology (DAE), and Pediatrics (DAE and DMM), University of Arkansas for Medical Sciences, Little Rock, AR

² Supported by grant no. R37 DK 36823 from the National Institutes of Health.

³ Reprints not available. Address correspondence to DM Mock, Department of Biochemistry and Molecular Biology #516, University of Arkansas for Medical Sciences, Little Rock, AR 72205. E-mail: mockdonaldm{at}uams.edu.

	ABSTRACT

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Background: Biotin is likely transported into cerebral spinal fluid (CSF) via one or more specific transporters. Concentrations of biotin in CSF measured by using modern analytic techniques that are specific for biotin and biotin metabolites have not previously been reported.

Objectives: We aimed to accurately measure the concentration of biotin and major biotin metabolites, biotin sulfoxide (BSO) and bisnorbiotin (BNB), in the CSF of children.

Design: Concentrations of biotin were determined initially as total avidin-binding substances (TABS) in CSF obtained by lumbar puncture from 55 children. Biotin, BSO, and BNB were quantitated by HPLC and an avidin-binding assay in CSF samples from a subset of 11 children.

Results: Concentrations of TABS in CSF averaged 1.6 nmol/L with substantial variability (SD = 1.3 nmol/L). CSF concentrations of biotin and biotin analogs varied widely, but substantial amounts of BSO were detected in every sample. Biotin accounted for 42 ± 16%, BSO for 41 ± 12%, and BNB for 8 ± 14% of the total. It was surprising that the molar sum of biotin, BSO, and BNB on average was >200-fold the TABS concentrations from the same CSF sample. Using several analytic approaches, we found no masking of detection, nor did we find degradation of biotin or BSO. Gel electrophoresis and streptavidin Western blot detected several biotinylated proteins in CSF.

Conclusions: Biotin appears to be bound to protein covalently, reversibly, or both, and this binding likely accounts for the increase in detectable biotin after HPLC. Protein-bound biotin may play an important role in biotin nutriture of the brain.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Biotin is a water-soluble vitamin that plays an essential biochemical role in all mammals. In humans, biotin acts as a coenzyme for 5 carboxylases: propionyl-coenzyme A (CoA) carboxylase, 3-methyl-crotonyl carboxylase, pyruvate carboxylase, and 2 isoenzymes of acetyl-CoA carboxylase. These biotin-dependent carboxylases catalyze vital reactions in important metabolic pathways involving gluconeogenesis, fatty acid synthesis, catabolism of branched-chain amino acids, and metabolism of some neurotransmitters (1, 2). Mammals fill their biotin requirement through diet and perhaps through absorption of the biotin produced by the normal microflora of the intestine (3, 4).

As do all other mammalian cells, neurons require biotin for normal function. In humans, biotin deficiency causes neurologic symptoms including seizures, hypotonia, developmental delay, ataxia, visual problems (eg, optic atrophy), and sensorineural hearing loss.

Children with inborn errors of biotin metabolism, such as holocarboxylase synthetase deficiency (infantile multiple carboxylase deficiency) and individual carboxylase deficiencies (eg, propionyl-CoA carboxylase deficiency), are treated with high doses of biotin for months or years. Some respond well to this therapy; others do not. For example, in the case of holocarboxylase synthetase deficiency, defects in binding affinity (Michaelis constant, K_m) respond well to biotin therapy, whereas defects in maximum velocity (V_max) have a more limited response. To optimize therapy, biotin must reach the central nervous system, which could be indirectly assessed by measuring the biotin concentration in the cerebrospinal fluid (CSF).

For proper interpretation, a normal range of biotin content in CSF should be established. In previous studies of the concentration of biotin in CSF, biotin was analyzed by using an avidin-binding assay without metabolite separation (5). Investigations in our group's laboratory have provided evidence that the presence of avidin-binding substances in addition to biotin can confound the ability of avidin-binding assays to quantitate biotin in serum, plasma, and urine (6, 7).

The present study sought to accurately measure the concentration of biotin and 2 other major biotin metabolites, bisnorbiotin (BNB) and biotin sulfoxide (BSO), in the CSF of children by using HPLC and an avidin-binding assay (HPLC/avidin-binding assay). The present study also investigated the potential mechanism of an observed discrepancy between total avidin-binding substances (TABS) measured by using a direct avidin-binding assay and the molar sum of biotin and the 2 metabolites measured by HPLC avidin-binding assay.

	SUBJECTS AND METHODS

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Study population characteristics
CSF was obtained from children undergoing lumbar puncture for routine diagnostic purposes and was cultured for routine microbiological diagnosis and subjected to cell counting to assess inflammation and neoplasia. CSF remaining after these clinically indicated procedures was provided in a de-identified form for the present research project. Inclusion criteria included sterility on bacterial culture and no evidence of inflammation or neoplasia based on cell count. Samples from children with nutritional disorders and diseases with predictable malnutrition (eg, inflammatory bowel disease) were excluded. The subjects' ages ranged from 9 mo to 18 y. Samples were stored at –70 °C until they were assayed.

The study was approved by the Institutional Review Board of the University of Arkansas for Medical Sciences. The Institutional Review Board approved the study as exempt; the samples used were pre-existing and stripped of all identifiers except diagnosis, and therefore, the Board did not require informed consent.

Quantitation of biotin and biotin metabolites in cerebrospinal fluid
TABS were quantitated in the CSF of 55 children by using a previously described sequential solid-phase avidin-binding assay (8). In CSF from a subset of 11 children, biotin and biotin metabolites were separated by HPLC using a C18 reversed-phase column as previously described (9). Briefly, 1-mL aliquots of CSF were fractionated by using HPLC. The HPLC fractions collected at specific retention times were determined to contain specific metabolites by comparison to retention times of authentic radiolabeled biotin metabolites synthesized as previously described (9). Our group (6, 9, 10) has reported that, for both unlabeled biotin and [³H]-biotin, recovery by HPLC is >95%. Biotin, BNB, and BSO were the only significant avidin-binding substances detected in CSF (see Results); they were quantitated against authentic standards with the use of an avidin-binding assay as previously described (8). BSO exists as 2 stereoisomers referred to as the d-isomers and l-isomers. Our current HPLC method does not reliably separate these isomers. Thus, BSO in this report refers to the sum of the 2 isomers.

Methods for investigation of the underestimation of biotin in cerebrospinal fluid
Evaluation of a masking effect
To evaluate the possibility that the detection of free biotin was masked by an unknown CSF component that blocks binding to avidin, [³H]-biotin was added to CSF and allowed to incubate for 1 h at room temperature. An additional aliquot of the same CSF sample without the addition of any [³H]-biotin served as a control. TABS were quantitated by using the avidin-binding assay, and differences between samples were compared with the amount of biotin added, which was independently quantitated by scintillation counting of [³H].

Evaluation of conversion effects or microbial uptake or both
To evaluate the possibility that the detection of biotin metabolites in CSF resulted from in vitro degradation of biotin or from the uptake of biotin by any potential microbial infectious organisms during storage in the clinical laboratory, [³H]-biotin was added to an aliquot of CSF and incubated for 72 h at 4 °C (the typical maximum storage period for samples in the hospital laboratory before acquisition for this study). The CSF containing [³H]-biotin was further characterized with respect to its chemical nature by subjecting the sample to HPLC as described above. Fractions eluting from the HPLC column were collected as 1-mL aliquots and quantitated by scintillation counting. The identity of radioactive substances in HPLC fractions was inferred from a comparison of their retention times with those of radiolabeled biotin and biotin metabolites synthesized and characterized as described previously (8).

Confirmation of identification of biotin metabolites
To confirm that an early-eluting avidin-binding substance at the BSO fraction was indeed BSO—rather than biotin bound either covalently or reversibly to another molecule, eg, a protein or an oligopeptide, which could result in possible co-elution with the BSO fraction—we re-chromatographed the avidin-binding substance from the BSO fraction. The HPLC fractions were collected and assayed for avidin-binding substances. The identities of the detected avidin-binding substances were inferred from a comparison of their retention times with those established for radiolabeled biotin and biotin metabolites (9).

Evaluation of the possibility that biotin is bound reversibly or covalently to macromolecules
To assess whether biotin is bound to macromoloecules, ultrafiltration of CSF pooled from 10 children was performed by centrifugation for 1 h at 5000 x g in 10-kDa devices (Centricon; Millipore, Milford, MA). Biotin was quantitated in the ultrafiltrate and retentate by using an avidin-binding assay as described previously (8). Total biotin covalently bound to macromolecules was measured by releasing biotin with the use of acid hydrolysis. Released biotin was quantitated by using an avidin-binding assay as described previously (8, 11).

Analysis of cerebrospinal fluid proteins
CSF proteins were resolved electrophoretically by using an adaptation of the method of Lewis et al (12) and were stained with colloidal Coomassie blue stain (Invitrogen, Carlsbad, CA) according to the manufacturer's recommendations.

Electrophoretically resolved proteins were electroblotted onto a polyvinyldifluoride membrane for 1 h at 30 V. The membrane was blocked, washed, and incubated with streptavidin-peroxidase conjugate (Roche Diagnostics, Indianapolis, IN) as previously described (13). The membrane was developed by using an immunopure metal-enhanced diaminobenzidine substrate kit (Thermo Scientific, Waltham, MA) according to the manufacturer's recommendations.

Statistical analysis
Central tendency is expressed as the mean. Variability is expressed as the SD with the use of STATVIEW software (version 5.0.1; SAS Inc, Cary, NC).

	RESULTS

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Results for CSF samples from 55 children along with diagnostic classification are shown in Table 1. The mean (±SD) concentration of TABS was 1.6 ± 1.3 nmol/L. TABS values ranged from undetectable (0.022 nmol/L) to 5.9 nmol/L.

Table 2

By adding biotin independently identified and quantified as [³H]-biotin to CSF before the avidin-binding assay, we further evaluated whether an underestimation of biotin in CSF as measured by direct avidin-binding assay could be the result of a potential masking effect. The measured increase in biotin by the avidin-binding assay accounted for 100% of the added biotin, which provides evidence against masking.

We also evaluated the possibility that factors or microbes present in CSF may have degraded or taken up biotin during storage in the clinical laboratory, thus artifactually decreasing measured biotin concentration and producing the observed biotin metabolites. When [³H]-biotin was incubated in CSF and then subjected to HPLC, the recovery of total radioactivity was 97%, which provides evidence against biotin loss. No conversion of the radiolabeled biotin to biotin metabolites was observed by HPLC, which provides evidence against artifactual degradation.

Re-chromatography of the BSO fraction resulted in the recovery of 99% of the avidin-binding substance. The substance continued to elute at the retention time for BSO. This observation provides evidence that the avidin-binding substance is indeed BSO, rather than biotin bound to a larger molecule.

We also tested the possibility that significant amounts of biotin in CSF are bound to macromolecules. By centrifugal ultrafiltration, we separated free biotin from any biotin that may be bound to macromolecules with molecular weight of >10 kDa. Concentrations of biotin in the ultrafiltrate and retentate were quantified with the use of an avidin-binding assay. The ratio of retentate value to ultrafiltrate value was 1.2 ± 0.05. These data suggest that only 20% of biotin could be bound to macromolecules. However, the approach does not rule out the possibility that biotin may be bound to macromolecules in a way that masks detectability by an avidin-binding assay (eg, bound inside the protein).

We further evaluated the possibility that biotin is bound to macromolecules and released during acidic HPLC. One CSF sample underwent acid hydrolysis, and the total avidin-binding substances increased 30-fold. This increase accounted for most, but not all, of the 90-fold increase observed for this particular sample after HPLC. This finding suggested that acid hydrolysis and acidic HPLC release biotin from macromolecules (possibly CSF proteins) to which biotin was bound covalently or reversibly in a location in the macromolecule that was not accessible to avidin.

We visualized proteins present in CSF and further assessed whether any of these proteins were biotinylated. As expected, there were many proteins, and they appeared in large amounts in CSF (Figure 1). Several biotinylated proteins bands were detected, the most prominent of which were at 12–15, 55, 60–65, 70–75, and >100 kDa (Figure 2). It is interesting that the location of the protein band with the highest concentration in CSF coincided with a band of biotinylated proteins.

View larger version (77K): [in this window] [in a new window]   FIGURE 1.. In-gel colloidal Coomassie protein stain showed a large number of proteins at various molecular weights (MW). Lane 1, MS marker; Lane 2, 10 µg cerebrospinal fluid from one subject; Lane 3, 10 µg CSF from a second subject; Lane 4, 20 ng biotinylated bovine serum albumin (BSA) serving as control; Lane 5, 40 ng BSA serving as control.

View larger version (27K): [in this window] [in a new window]   FIGURE 2.. Western blot probed by streptavidin and metal-enhanced diaminobenzidine substrate provided evidence that biotin in cerebrospinal fluid (CSF)is bound to proteins. Biotinylated proteins () can be seen at several particular molecular weights (MW). Lane 1, biotinylated MW marker; Lane 2, biotinylated bovine serum albumin serving as control; Lane 3, bovine serum albumin serving as control; Lane 4, 25 µg CSF from subject in lane 2 of Figure 1; Lane 5, 25 µg CSF from subject in lane 3 of Figure 1.

	DISCUSSION

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

In CSF from 55 children, we found that the average TABS concentration was 1.6 nmol/L (range: 0.02–5.9 nmol/L). The concentrations of TABS in CSF reported in the present study are approximately twice those reported by Lombard and Mock (14) for fasting concentrations of TABS in plasma from children. As reported by those authors using the same avidin-binding assay as used in the present study, the average TABS concentration detected was 0.83 nmol/L (range: 0.56–1.08 nmol/L). Anagnostouli et al (5) reported biotin concentrations in CSF from adults using a similar avidin-binding assay. Those investigators found mean concentration of biotin in CSF to be 0.56 ± 0.31 nmol/L. Our findings with respect to the concentrations of biotin in the CSF of adults are similar to but moderately greater than those reported by Anagnostouli et al. However, they found that the concentrations of biotin in adult CSF are only one-third of those reported for fasting concentrations of biotin in serum from adults. Such a difference may be related to differences in subjects (eg, timing of samples or biotin intake) or assay differences.

Our finding that the concentrations of TABS in CSF are approximatelytwice those reported for plasma is similar to the finding of Lo et al (15) in rats, obtained by using an avidin-binding assay. Those investigators reported that biotin concentrations in rat CSF are 2.5 times those in serum concentrations.

We found substantially different results when CSF was subjected to HPLC under acidic conditions and then assayed. The total of biotin plus the 2 major metabolites, BNB and BSO, was an average of 240 times the concentrations of TABS. For an avidin-binding assay to measure biotin specifically, biotin must be the only avidin-binding metabolite in the CSF. The present studies provide evidence that it is not. The equilibrium-binding constant for avidin varies among the biotin metabolites (16). BSO and BNB have binding affinities to avidin that are less than the affinity of biotin and hence that are likely to be less efficiently detected. As a consequence, these metabolites will be underestimated when measured in a mixture with biotin and quantitated against a biotin standard curve (7). However, on the basis of our group's previous reports (6, 9, 17), the true concentrations of BSO and BNB were only 3-fold the amounts found in analyses against a biotin standard curve. This 3-fold increase cannot explain the 240-fold concentration observed in the current study.

Approximately half of the TABS found is attributable to biotin metabolites rather than to biotin per se. In contrast to the relative concentrations of metabolites in the plasma and urine of adults (9, 18), BSO (rather than BNB) is the major biotin metabolite in the CSF of children, which suggests that sulfur oxidation (rather than β-oxidation of the valeric acid side chain) is the primary metabolic pathway for biotin in the central nervous system. The specific metabolic pathways leading to BSO and BNB found in human CSF have not been elucidated. The results from a previous study (19) provided evidence that biotin enters CSF and brain by saturable transport systems that do not depend on metabolism of biotin. According to a study by Melville (20), it seems likely that biotin metabolites BNB and BSO in CSF originate from the catabolism of biotin in brain tissue and that this catabolism occurs in both children and adults.

The large range in the concentrations of biotin, BNB, and BSO in the 11 persons reported in this study is far greater than the analytic imprecision of the method. We used diagnosis to create subgroups of these 11 subjects, 6 of whom had acute lymphoblastic leukemia, and, even in this diagnostic subgroup, we observed large variability. These subgroup data suggest that the variation likely reflects either true biological variability among persons or heterogeneity of subjects (eg, drug therapies) or both. Our group (21, 22) has shown in both rats and human subjects that medications potentially used in these children can accelerate biotin metabolism. We speculate that higher concentrations of biotin metabolites could be the result of the induction of accelerated biotin biotransformation. However, the pattern observed in urinary metabolites did not confirm this possibility; drug induction generally favored an increase in BNB over one in BSO (21).

Further assessment of the cause of the large observed variability is limited by study design. Other unknown factors may have affected subject heterogeneity. For example, subjects may have received biotin supplementation, but this information was not available because of the use of de-identified samples. If subjects were supplemented, variability in biotin and metabolites concentrations in CSF may be the consequence of greater biotin intake (23, 24).

Our investigation of the mechanism leading to underestimation of the amount of true biotin in CSF by direct avidin-binding assay yielded evidence to support the following 4 conclusions. First, masking effects did not occur. Second, artifactual conversion of the radiolabeled biotin to biotin metabolites (either by degradation or uptake by microbes) did not occur. Third, incorrect identification of biotin metabolites did not occur. Fourth, artifactual conversion of biotin sulfoxide to a more-oxidized metabolite such as biotin sulfone did not occur.

We hypothesize that the increase in measurable biotin and biotin metabolites arises, in large part, from the release of biotin from biotin-binding macromolecules such as proteins. Ultrafiltration experiments suggested that most of the biotin is bound in a way that is not accessible to avidin (eg, in the interior of the macromolecules) and, as such, that it is "hidden" from the assay. Acid hydrolysis experiments produced a 30-fold increase of the biotin concentration in CSF, which is consistent with a mechanism in which most of the biotin in CSF is bound to the interior of macromolecules.

In blotting experiments, we detected several biotinylated proteins, which provides support for the hypothesis that biotin in CSF may be bound to proteins. The identities of these proteins are under investigation. Previous studies suggested that substantial amounts of biotin are bound to biotinidase (25, 26). Studies by Frank et al (27) have indicated that human albumin and β-globulin can bind biotin. A biotin-binding glycoprotein in human serum has also been reported (28; D Gehrig, F Leuthardt, unpublished observations, 1976), and Nagamine et al (29) identified biotin-binding immunoglobulin in human serum.

In summary, these data provide evidence that CSF contains amounts of biotin, BSO, and BNB that substantially exceed the amount detected by a direct avidin-binding assay. The present studies provide evidence that the release of biotin that was reversibly or covalently bound to protein explains most of the increases in detectable biotin after HPLC separation of biotin and biotin analogs. We conclude that most of the biotin in CSF is bound to protein.

	ACKNOWLEDGMENTS

 
The authors' responsibilities were as follows—DAE (Study Coordinator): collected all clinical samples;AB, SLS, and DMM: designed the study; AB and SLS: performed all laboratory measurements and techniques; AB: drafted the manuscript and prepared the tables: SLS: prepared the figures; all authors: edited the manuscript; and DMM (Principal Investigator): was responsible for the final version of the manuscript. None of the authors had a personal or financial conflict of interest

	REFERENCES

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

 

1. Perry TL, Haworth JC, Robinson BH. Brain amino acid abnormalities in pyruvate carboxylase deficiency. J Inherit Metab Dis 1985;8:63–6.[Medline]
2. Wallace JC, Jitrapakdee S, Chapman-Smith A. Pyruvate carboxylase. Int J Biochem Cell Biol 1998;30:1–5.[Medline]
3. Said HM. Cellular uptake of biotin: mechanisms and regulation. J Nutr 1999;129(suppl):490S–3S.[Medline]
4. Wolf B. Disorders of biotin metabolism. In: Scriver CR, Beaudet AL, Sly WS, Valle D, eds. The metabolic and molecular basis of inherited disease. 8th ed. New York, NY: McGraw-Hill, Inc, 2001;3151–77.
5. Anagnostouli M, Livaniou E, Nyalala JO, et al. Cerebrospinal fluid levels of biotin in various neurological disorders. Acta Neurol Scand 1999;99:387–92.[Medline]
6. Mock DM, Lankford GL, Mock NI. Biotin accounts for only half of the total avidin-binding substances in human serum. J Nutr 1995;125:941–6.[Abstract/Free Full Text]
7. Zempleni J, Mock DM. Advanced analysis of biotin metabolites in body fluids allows a more accurate measurement of biotin bioavailability and metabolism in humans. J Nutr 1999;129(suppl):494S–7S.[Medline]
8. Mock DM. Determinations of biotin in biological fluids. In: McCormick DB, Suttie JW, Wagner C, eds. Methods in enzymology. New York, NY: Academic Press, 1997;265–75.
9. Mock DM, Lankford GL, Cazin J Jr. Biotin and biotin analogs in human urine: Biotin accounts for only half of the total. J Nutr 1993;123:1844–51.[Abstract/Free Full Text]
10. Mock DM, Nyalala JO, Raguseo RM. A direct streptavidin-binding assay does not accurately quantitate biotin in human urine. J Nutr 2001;131:2208–14.[Abstract/Free Full Text]
11. Mock DM, Malik MI. Distribution of biotin in human plasma: most of the biotin is not bound to protein. Am J Clin Nutr 1992;56:427–32.[Abstract/Free Full Text]
12. Lewis B, Rathman S, McMahon RJ. Detection and quantification of biotinylated proteins using the STORM 840 optical scanner. J Nutr Biochem 2003;14:196–202.[Medline]
13. Apoga D, Ek B, Tunlid A. Analysis of proteins in the extracellular matrix of the plant pathogenic fungus Bipolaris sorokiniana using 2-D gel electrophoresis and MS/MS. FEMS Microbiol Lett 2001;197:145–50.[Medline]
14. Lombard KA, Mock DM. Biotin nutritional status of vegans, lacto-vegetarians and non-vegetarians. Am J Clin Nutr 1989;50:486–90.[Abstract/Free Full Text]
15. Lo W, Kadlecek T, Packman S. Biotin transport in the rat central nervous system. J Nutr Sci Vitaminol (Tokyo) 1991;37:567–72.[Medline]
16. Green NM. Avidin. Adv Protein Chem 1975;29:85–133.[Medline]
17. Zempleni J, Mock DM. Biotin. In: Song WO, Beecher GR, eds. Modern analytical methodologies in fat and water-soluble vitamins. Baltimore, MD: Wiley, 2001;459–66.
18. Mock DM. Biotin analogs account for half of the total avidin-binding substances in human plasma. Clin Res 1989;37:955A (abstr).
19. Spector R, Mock DM. Biotin transport and metabolism in the central nervous system. Neurochem Res 1988;13:213–9.[Medline]
20. Melville DB. Biotin sulfoxide. J Biol Chem 1954;208:495–501.[Free Full Text]
21. Wang K-S, Mock NI, Mock DM. Biotin biotransformation to bisnorbiotin is accelerated by several peroxisome proliferators and steroid hormones in rats. J Nutr 1997;127:2212–6.[Abstract/Free Full Text]
22. Mock DM, Dyken ME. Biotin catabolism is accelerated in adults receiving long-term therapy with anticonvulsants. Neurology 1997;49:1444–7.[Abstract/Free Full Text]
23. Mock DM, Mock NI. Serum concentrations of bisnorbiotin and biotin sulfoxide increase during both acute and chronic biotin supplementation. J Lab Clin Med 1997;129:384–8.[Medline]
24. Zempleni J, Mock DM. Bioavailability of biotin given orally to humans in pharmacologic doses. Am J Clin Nutr 1999;69:504–8.[Abstract/Free Full Text]
25. Wolf B. Biotinidase: its role in biotinidase deficiency and biotin metabolism. J Nutr Biochem 2005;16:441–5.[Medline]
26. Chauhan J, Dakshinamurti K. Role of human serum biotinidase as biotin-binding protein. Biochem J 1988;256:265–70.[Medline]
27. Frank O, Luisada-Opper AV, Feingold S, Baker H. Vitamin binding by human and some animal plasma proteins. Nutr Rep Int 1970;1:161–8.
28. Vallotton M, Hess-Sander U, Leuthardt F. Fixation spontanee de la biotine a une proteine dans le serum humain. (Spontaneous fixation of biotin to a protein in human serum.) Hel Chim Acta 1965;48:126–33 (in French).
29. Nagamine T, Takehara K, Fukui T, Mori M. Clinical evaluation of biotin-binding immunoglobulin in patients with Graves' disease. Clin Chim Acta 1994;226:47–54.[Medline]

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