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Distribution of Biotin in Cerebrospinal Fluid of Children: Most of the Biotin is Bound to Protein
Author(s) -
Bogusiewicz Anna,
Stratton Shawna L,
Ellison Dale A,
Mock Donald M
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.1104.4
Subject(s) - biotin , avidin , biotin deficiency , streptavidin , chemistry , biochemistry , high performance liquid chromatography , biotinylation , chromatography
Direct measurement of total avidin binding substances (TABS) in CSF underestimates the amount of true biotin as measured by HPLC/avidin‐binding assays. We investigated the mechanism of the underestimation as follows: 1) To evaluate whether detection of free biotin was masked by an unknown CSF component that blocks binding to avidin, biotin (independently quantified as 3 H‐biotin) was added to CSF. TABS assay detected 100% of the added biotin, providing evidence against masking. 2) To determine if in vitro conversion of biotin to metabolites confounded detection, 3 H‐biotin was incubated for 72 h in CSF at 4°C. No conversion of 3 H‐biotin to biotin metabolites was observed by HPLC, providing evidence against conversion. 3) To evaluate the possibility that biotin is bound to a macromolecule and released during HPLC, CSF was acid hydrolyzed. Detectable biotin increased approximately 30 fold, providing evidence in favor of this mechanism. The 30 fold increase accounts for most, but not all, of the 90 fold increase observed for this sample after HPLC. Biotin‐binding proteins in CSF were further characterized by SDS‐PAGE, western blot, and streptavidin detection. Three bands were detected. These studies provide evidence that reversible and/or covalent binding to proteins causes substantial underestimation of CSF biotin. We speculate that this phenomenon may have confounded previous studies. NIH DK 36823