A direct streptavidin-binding assay does not accurately quantitate biotin in human urine.

In human urine, the biotin concentration assayed directly using an avidin-binding assay (ABA) apparently overestimates "true" biotin concentration as measured by HPLC separation of biotin from biotin metabolites followed by ABA. Because biotin metabolites account for about half of biotin plus biotin metabolites in human urine, we speculate that the error might arise from biotin metabolites. We sought to test the following hypothesis: biotin measured by direct ABA routinely exceeds true biotin in urine due to biotin metabolites; however, if urinary biotin is quantitated using a streptavidin-binding assay (SABA) that does not detect biotin metabolites, results will agree with true biotin. An assay for biotin that uses europium coupled to streptavidin and time-resolved fluorescence was developed and validated. Urine samples were obtained from biotin-deficient, normal and biotin-supplemented adults. In 133 urine samples from 26 subjects, biotin by direct ABA correlated positively and significantly with biotin measured after HPLC separation (P < 0.001; r = 0.78). However, biotin by direct ABA routinely exceeded true biotin. The magnitude of the overestimate correlated strongly with biotin metabolites; r = 0.80 and P < 0.0001. In 92 samples from nine subjects, biotin by direct SABA correlated positively and significantly with true biotin (P = 0.001; r = 0.73) but exceeded true biotin by more than analytical error in 62 of the 92 samples. The error did not correlate significantly with total biotin metabolites. In 62 samples analyzed by both assays, biotin by direct SABA correlated weakly (r = 0.69) but significantly (P < 0.0001) with biotin by direct ABA. These studies provide evidence that direct SABA does not accurately quantitate biotin. Although the errors from direct ABA arise primarily from metabolites, the errors from direct SABA cannot be attributed primarily to biotin metabolites. Whether these interfering substances are biotin metabolites or other unknown substances, the substances are likely separated from the biotin fraction by HPLC.

Analytical techniques for determining biotin.

Biotin is a vitamin of the B-complex, which plays an important biochemical role in every living cell. In the recent years, the interest in this vitamin has been rekindled, mainly due to its association with serious human disorders, such as the inherited syndrome multiple carboxylase deficiency, which can be successfully treated with biotin administration. Diagnosis of biotin deficiency as well as monitoring of biotin levels in biological fluids of patients receiving biotin treatment is crucial. Equally important is the determination of biotin levels in pharmaceutical preparations as well as in food and food supplement products, which constitute the main source of biotin in humans. Several analytical methods for measuring biotin in various samples, e.g. human fluids, pharmaceutical formulations, food material etc., have been reported in the literature. In this review, the most representative of these methods are presented, and their characteristics are evaluated.

Cerebrospinal fluid levels of biotin in various neurological disorders.

OBJECTIVES: To analyse biotin concentrations in human cerebrospinal fluid (CSF) and serum from controls without evidence of nutritional or neurological disorders and patients with common neurological disorders. PATIENTS AND METHODS: Cerebrospinal fluid was obtained from patients by lumbar puncture, serum was prepared from freshly drawn whole blood and biotinidase in samples was inhibited before being analysed for biotin by radioligand assay. RESULTS: Assay characteristics were within an acceptable range (intra-and interassay coefficient of variations were 8.8 and 12.0 respectively, recovery: 91-114% and sensitive, lowest standard concentration 15 ng/l). Significantly lower values for biotin were found in patients with multiple sclerosis (both CSF and serum) in comparison to the controls. Significantly reduced values for cerebrospinal fluid biotin were found in epileptics compared to controls, whereas, in serum the difference was approaching significance. No significant differences were observed in other groups of patients. CONCLUSION: There is a significant reduction in cerebrospinal fluid biotin in epileptics and patients with multiple sclerosis compared to controls. In epileptics this may be related to competition between biotin and anticonvulsants bearing carbamide ring for absorption. Reduction of biotin levels in patients with multiple sclerosis could be attributed to intestinal malabsorption caused by the underlying disease or a biotin-binding immunoglobulin which may be involved in multiple sclerosis pathogenesis.

Biotin recycling impairment in phenylketonuric children with seborrheic dermatitis.

OBJECTIVE: To investigate the effect of a therapeutic diet on serum biotin levels and to explain the seborrheic dermatitis in phenylketonuric (PKU) patients on a "loose" diet. DESIGN: Forty-seven patients were divided into two groups: group A (n=21) demonstrated good compliance to a special diet and group B (n=26) were on a "loose" diet. Most of the patients in group B (20/26), who suffered from mild seborrheic dermatitis, were requested to return to phenylalanine (Phe)-restricted diet for at least 15 days. Seventy-nine healthy children of comparable age were used as controls. Biotin serum levels and plasma biotinidase activity were measured in patients as well as controls. In addition, biotinidase activity was evaluated in vitro after incubation with various concentrations of Phe. RESULTS: Biotin levels in group A patients (636+/-118 ng/L) were statistically significantly elevated (P < 0.01) compared with those of group B patients before (412+/-184 ng/L) and after (501+/-160 ng/L) 15 days on a Phe-restricted diet, as well as with those of controls (337+/-290 ng/L). Furthermore, biotinidase activities were decreased in group B patients (4.2+/-1.68 nmol/min/L) compared with those of group A patients (6.4+/-0.7 nmol/min/L) and controls (6.10+/-0.8 nmol/min/L). Additionally, biotinidase activities in the patients of group B were restored to normal (5.78+/-0.81 nmol/min/L), with a simultaneous remission of their skin lesions, after 15 days on a Phe-restricted diet. Moreover, the in vitro findings showed a 51% inhibition of biotinidase activity when incubated with Phe (20 mg/dL). CONCLUSIONS: It is suggested that the high biotin levels in group A patients reflect the intake of water-soluble biotin of vegetable origin. In contrast, the low biotinidase activity in group B patients may be attributed to their high Phe plasma levels, which acts as an enzyme inhibitor, as shown by the in vivo and in vitro results. Consequently, the observed seborrheic dermatitis in PKU children (group B) is associated with an impairment of biotin recycling.

Indirect enzyme-linked method for determining biotin in human serum.

An indirect enzyme-linked assay was developed for quantifying biotin concentrations in human sera. Biotin standard solutions or unknown samples are preincubated with streptavidin-conjugated horseradish peroxidase (streptavidin-HRP) and added to plates coated with biotinylated bovine IgG (B-IgGb). The concentration of the streptavidin-HRP is such that the streptavidin binding sites are sufficient to bind apparently all the biotin present in samples, whereas, the remaining sites are inversely proportional to the amount of biotin in analysed sample. These sites could subsequently interact with the immobilized B-IgGb providing signal. The assay demonstrated dynamic range 5 to 640 ng/L, detection limit 2 ng/L, intra- and interassay C.V., 1.6-3.9% and 3.7-7.2% respectively, recovery 100-114% and linear recovery 90-117%. Serum biotin determined: healthy individuals 66 to 600 ng/L, pregnant women (> or = 36 weeks) 60 to 360 ng/L, and patients under chronic haemodialysis 0.56 to 1.62 micrograms/L. The method described is among those few which have been experimentally evaluated for their capabilitity of assessing biotin in human sera.