Biotin as a masking agent in chorionic gonadotropin assays utilizing biotinylated antibodies.

Biotin interference in streptavidin/biotin-based immunoassays has been recently recognized as a confounding factor in clinical settings. Depending on the nature of the assay, the presence of excess biotin in patient samples can cause falsely high or low results. One of the platforms known to be affected, Roche Cobas, is widely used in anti-doping laboratories to test for intact chorionic gonadotropin (hCG) in urine. While biotin levels in blood have been well studied, less is known about urinary biotin due to its limited clinical significance. Having analyzed over 4,000 urine samples, we have established a reference range for urinary biotin with a median concentration of approximately 12 ng/ml. However, a significant number of samples contain much higher amounts, with a maximum approaching 10 µg/ml, suggesting biotin supplementation. Consequently, the tolerance of hCG STAT assay towards biotin was investigated over a wide concentration range. The apparent hCG concentration was found to decrease almost linearly as biotin increased from 100 to 1,000 ng/ml, with only 10% of the expected value reported by the assay as biotin reached 1,000 ng/ml. Further increase of biotin resulted in a progressive, albeit more moderate, decline in measured hCG concentration. To avoid a false negative result in the context of anti-doping analysis, it is highly recommended to monitor biotin in urine and perform diafiltration before hCG measurement in samples with elevated biotin to remove the interference.

Thiamine phosphokinase deficiency and mutation in TPK1 presenting as biotin responsive basal ganglia disease.

The product of thiamine phosphokinase is the cofactor for many enzymes, including the dehydrogenases of pyruvate, 2-ketoglutarate and branched chain ketoacids. Its deficiency has recently been described in a small number of patients, some of whom had a Leigh syndrome phenotype. The patient who also had a Leigh phenotype was initially found to have a low concentration of biotin in plasma and massive urinary excretion of biotin. Despite treatment with biotin and thiamine, her disease was progressive. Mutations c.311delG and c.426G > C were found in the TPK1 gene.

Assessment of biotin interference with qualitative point-of-care hCG test devices.

Biotin is commonly used as a dietary supplement for the claimed benefits of promoting healthy hair and nail growth and is available without a prescription at doses up to 10mg/capsule. Biotin-mediated interference in immunoassay testing is an emerging issue for clinical laboratories and previous studies have indicated that biotin at regularly encountered doses may interfere with these assays. In this study, we evaluated the effect of supplemental biotin on seven POC urine hCG test devices using purified biotin and urine collected from four volunteers consuming 10mg biotin/day. Six of the seven devices showed no evidence of biotin interference as each device's control line remained clearly detectable at all biotin concentrations tested. However, the QuickVue device control line demonstrated a marked decrease in intensity when used to test solutions containing >5µg/mL biotin. The absence of a control line during patient testing has the potential to delay care due to the generation of an invalid test result and lead to additional unnecessary testing. It is not realistic to measure urinary biotin concentrations in every patient undergoing qualitative urine hCG testing but biotin supplementation should be considered if repeat testing on a patient sample generates an invalid test result.

Effects of Fatty Liver Induced by Excess Orotic Acid on B-Group Vitamin Concentrations of Liver, Blood, and Urine in Rats.

Fatty liver is caused when rats are given orotic acid of the pyrimidine base in large quantities. The lack of B-group vitamins suppresses the biosynthesis of fatty acids. We investigated how orotic acid-induced fatty liver affects the concentrations of liver, blood, and urine B-group vitamins in rats. The vitamin B6 and B12 concentrations of liver, blood, and urine were not affected by orotic acid-induced fatty liver. Vitamin B2 was measured only in the urine, but was unchanged. The liver, blood, and urine concentrations of niacin and its metabolites fell dramatically. Niacin and its metabolites in the liver, blood, and urine were affected as expected. Although the concentrations of vitamin B1, pantothenic acid, folate, and biotin in liver and blood were decreased by orotic acid-induced fatty liver, these urinary excretion amounts showed a specific pattern toward increase. Generally, as for the typical urinary excretion of B-group vitamins, these are excreted when the body is saturated. However, the ability to sustain vitamin B1, pantothenic acid, folate, and biotin decreased in fatty liver, which is hypothesized as a specific phenomenon. This metabolic response might occur to prevent an abnormally increased biosynthesis of fatty acids by orotic acid.

Biotin-deficient diet induces chromosome misalignment and spindle defects in mouse oocytes.

Increased abnormal oocytes due to meiotic chromosome misalignment and spindle defects lead to elevated rates of infertility, miscarriage, and trisomic conceptions. Here, we investigated the effect of biotin deficiency on oocyte quality. Three-week-old female ICR mice were fed a biotin-deficient or control diet (0, 0.004 g biotin/kg diet) for 21 days. On day 22, these mouse oocytes were analyzed by immunofluorescence. Due to biotin, undernutrition increased the frequency of abnormal oocytes (the biotin deficient vs. control: 40 vs. 16%). Next, the remaining mice in the biotin-deficient group were fed a control or biotin-deficient diet from day 22 to 42. Although biotin nutritional status in the recovery group was restored, the frequency of abnormal oocytes in the recovery group was still higher than that in the control group (48 vs. 18%). Our results indicate that steady, sufficient biotin intake is required for the production of high-quality oocytes in mice.

Pregnancy and lactation alter biomarkers of biotin metabolism in women consuming a controlled diet.

BACKGROUND: Biotin functions as a cofactor for several carboxylase enzymes with key roles in metabolism. At present, the dietary requirement for biotin is unknown and intake recommendations are provided as Adequate Intakes (AIs). The biotin AI for adults and pregnant women is 30 µg/d, whereas 35 µg/d is recommended for lactating women. However, pregnant and lactating women may require more biotin to meet the demands of these reproductive states. OBJECTIVE: The current study sought to quantify the impact of reproductive state on biotin status response to a known dietary intake of biotin. METHODS: To achieve this aim, we measured a panel of biotin biomarkers among pregnant (gestational week 27 at study entry; n = 26), lactating (postnatal week 5 at study entry; n = 28), and control (n = 21) women who participated in a 10- to 12-wk feeding study providing 57 µg of dietary biotin/d as part of a mixed diet. RESULTS: Over the course of the study, pregnant women excreted 69% more (vs. control; P < 0.001) 3-hydroxyisovaleric acid (3-HIA), a metabolite that accumulates during the catabolism of leucine when the activity of biotin-dependent methylcrotonyl-coenzyme A carboxylase is impaired. Interestingly, urinary excretion of 3-hydroxyisovaleryl-carnitine (3-HIA-carnitine), a downstream metabolite of 3-HIA, was 27% lower (P = 0.05) among pregnant (vs. control) women, a finding that may arise from carnitine inadequacy during gestation. No differences (P > 0.05) were detected in plasma biotin, urinary biotin, or urinary bisnorbiotin between pregnant and control women. Lactating women excreted 76% more (vs. control; P = 0.001) of the biotin catabolite bisnorbiotin, indicating that lactation accelerates biotin turnover and loss. Notably, with respect to control women, lactating women excreted 23% less (P = 0.04) urinary 3-HIA and 26% less (P = 0.05) urinary 3-HIA-carnitine, suggesting that lactation reduces leucine catabolism and that these metabolites may not be useful indicators of biotin status during lactation. CONCLUSIONS: Overall, these data demonstrate significant alterations in markers of biotin metabolism during pregnancy and lactation and suggest that biotin intakes exceeding current recommendations are needed to meet the demands of these reproductive states. This trial was registered at clinicaltrials.gov as NCT01127022.

Metabolomic analysis reveals metabolic changes caused by bisphenol A in rats.

Bisphenol A (BPA) is a widely used material known to cause adverse effects in humans and other mammals. To date, little is known about the global metabolomic alterations caused by BPA using urinalysis. Sprague-Dawley rats were orally administrated BPA at the levels of 0, 0.5 µg/kg/day and 50 mg/kg/day covering a low dose and a reference dose for 8 weeks. We conducted a capillary electrophoresis in tandem with electrospray ionization time-of-flight mass spectrometry based nontargeted metabolomic analysis using rat urine. To verify the metabolic alteration at both low and high doses, reverse transcription-polymerase chain reaction (RT-PCR) and western blotting were further conducted to analyze hepatic expression of methionine adenosyltransferase Iα (Mat1a) and methionine adenosyltransferase IIα (Mat2a). Hepatic S-adenosylmethionine (SAMe) was also analyzed. A total of 199 metabolites were profiled. Statistical analysis and pathway mapping indicated that the most significant metabolic perturbations induced by BPA were the increased biotin and riboflavin excretion, increased synthesis of methylated products, elevated purine nucleotide catabolism, and increased flux through the choline metabolism pathway. We found significantly higher mRNA and protein levels of Mat1a and Mat2a, and significantly higher SAMe levels in rat liver at both low and high doses. These two genes encode critical isoenzymes that catalyze the formation of SAMe, the principal biological methyl donor involved in the choline metabolism. In conclusion, an elevated choline metabolism is underlying the mechanism of highly methylated environment and related metabolic alterations caused by BPA. The data of BPA-elevated accepted biomarkers of injury indicate that BPA induces DNA methylation damage and broad protein degradation, and the increased deleterious metabolites in choline pathway may also be involved in the toxicity of BPA.

Measurement of acylcarnitine substrate to product ratios specific to biotin-dependent carboxylases offers a combination of indicators of biotin status in humans.

This work describes a novel liquid chromatography tandem MS (LC-MS/MS) method for the determination of ratios of acylcarnitines arising from acyl-CoA substrates and products that reflect metabolic disturbances caused by marginal biotin deficiency. The urinary ratios reflecting reduced activities of biotin-dependent enzymes include the following: 1) the ratio of 3-hydroxyisovalerylcarnitine : 3-methylglutarylcarnitine (3HIAc : MGc) for methylcrotonyl-CoA carboxylase; 2) the ratio of propionylcarnitine:methylmalonylcarnitine (Pc : MMc) for propionyl-CoA carboxylase (PCC); and 3) the ratio of acetylcarnitine : malonylcarnitine (Ac : Mc) for acetyl-CoA carboxylase. To demonstrate the suitability of the LC-MS/MS method for biomonitoring, we measured the 3 ratios for 7 healthy adults at various time points (d 0, 14, and 28) during the induction of marginal biotin through the consumption of egg white. The mean change in the Pc : MMc ratio relative to d 0 was 5.3-fold by d 14 (P = 0.0049) and 8.5-fold by d 28 (P = 0.0042). The mean change in the 3HIAc : MGc ratio was 2.8-fold by d 14 (P = 0.0022) and 3.8-fold by d 28 (P = 0.0001). The mean change in the Ac : Mc ratio was 2.9-fold by d 14 (P = 0.03) and 4.7-fold by d 28 (P = 0.02). The results suggest that simultaneous assessment of ratios of multiple biotin-dependent pathways offers insight into the complex metabolic disturbances caused by marginal biotin deficiency. We hypothesize that one or a combination of the ratios might be more sensitive or robust with respect to other nutrient deficiencies or confounding metabolic processes.

Measurement of 3-hydroxyisovaleric acid in urine from marginally biotin-deficient humans by UPLC-MS/MS.

Measurement of 3-hydroxyisovaleric acid (3HIA) in human urine has been shown to be a useful indicator of biotin status for a variety of clinical situations, including pregnancy. The work described herein presents a novel UPLC-MS/MS method for accurate and precise quantitation of urinary 3HIA. This method utilizes sample preparation prior to quantitation that has been simplified compared to the previous GC-MS method. To demonstrate the suitability of the UPLC-MS/MS method for human bio-monitoring, this method was used to measure 3-HIA in 64 human urine samples from eight healthy adults in whom marginal biotin deficiency had been induced experimentally by egg white feeding. 3HIA was detected in all specimens; the mean concentration [±standard deviation (SD)] was 80.6 ± 51 µM prior to inducing biotin deficiency. Mean excretion rate for 3HIA (expressed per mol urinary creatinine) before beginning the biotin-deficient diet was 8.5 ± 3.2 mmol 3HIA per mol creatinine and the mean increased threefold with deficiency. These specimens had been previously analyzed by GC-MS; the two data sets showed strong linear relationship with a correlation coefficient of 0.97. These results provide evidence that this method is suitable for bio-monitoring of biotin status in larger populations.

Development and characterization of a mouse with profound biotinidase deficiency: a biotin-responsive neurocutaneous disorder.

Biotinidase deficiency is the primary enzymatic defect in biotin-responsive, late-onset multiple carboxylase deficiency. Untreated children with profound biotinidase deficiency usually exhibit neurological symptoms including lethargy, hypotonia, seizures, developmental delay, sensorineural hearing loss and optic atrophy; and cutaneous symptoms including skin rash, conjunctivitis and alopecia. Although the clinical features of the disorder markedly improve or are prevented with biotin supplementation, some symptoms, once they occur, such as developmental delay, hearing loss and optic atrophy, are usually irreversible. To prevent development of symptoms, the disorder is screened for in the newborn period in essentially all states and in many countries. In order to better understand many aspects of the pathophysiology of the disorder, we have developed a transgenic biotinidase-deficient mouse. The mouse has a null mutation that results in no detectable serum biotinidase activity or cross-reacting material to antibody prepared against biotinidase. When fed a biotin-deficient diet these mice develop neurological and cutaneous symptoms, carboxylase deficiency, mild hyperammonemia, and exhibit increased urinary excretion of 3-hydroxyisovaleric acid and biotin and biotin metabolites. The clinical features are reversed with biotin supplementation. This biotinidase-deficient animal can be used to study systematically many aspects of the disorder and the role of biotinidase, biotin and biocytin in normal and in enzyme-deficient states.

Effects of biotin deficiency on embryonic development in mice.

OBJECTIVE: The purpose of this investigation was to determine the effects of biotin deficiency on maternal metabolism and embryonic development in pregnant mouse dams. METHODS: The pregnant mice were randomly assigned to one of three dietary groups and given a biotin-deficient diet, biotin-supplemented diet, or biotin-control diet during gestation. On days of gestation (dgs) 0, 4, 8, 12, and 16, organic acids including 3-hydroxyisovaleric acid in urine were discovered by high-performance liquid chromatography, and the biotin level in the serum and urine was determined by a bioassay. On dg 18, fetuses were examined for morphologic development. RESULTS: In the biotin-deficient group, biotin excretion in urine decreased on dg 4 and was subsequently below the lower limit, whereas the urinary concentration of 3-hydroxyisovaleric acid increased after dg 12. In contrast, the biotin concentration in urine significantly increased on dgs 4, 8 and 12 in the biotin-supplemented group, but decreased on dg 16 in the biotin-supplemented and biotin-control groups. The urinary excretion of pyruvic acid in the biotin-deficient group was significantly higher than that in the biotin-supplemented group throughout the entire gestation. These concentrations in urine significantly increased on dg 16 compared with dg 0. The inhibition of embryonic development and external malformations such as cleft palate (100%), micrognathia (100%), and micromelia (91.4%) were also detected in biotin-deficient fetuses. CONCLUSION: These findings indicated that, as the requirement of biotin increases during gestation and/or embryonic development, a large amount of biotin is necessary for maintaining normal reproductive performance during the late stage of gestation.

Changes in vitamin status of Indian Antarctic expeditioners during a one-month stay in Austral Summer.

OBJECTIVE: Antarctic expeditioners face extremes of environmental conditions along with isolation which affect normal human activity at a polar station. Diets of polar expeditioners consist of products that have been kept in storage for more than a year. Processing and preservation adversely affect the nutritive value of the food products, especially water-soluble vitamins. This study was conducted to determine water-soluble vitamin status of Antarctic expeditioners consuming processed canned food. METHODS: Twenty-two healthy male volunteers age 26 to 56 years (39.5+/-8.5, mean+/-SD) participated in the study. The study was conducted in 3 phases: at Goa, India (phase I), 48 hours after arriving in Antarctica (phase II) and after 1 month in Antarctica (phase III). Water-soluble vitamin status in erythrocytes was assessed at each phase with evaluation of riboflavin, thiamine, and pyridoxine status. Urinary N-methyl nicotinamide and methylmalonic acid (MMA) levels were measured to assess niacin and vitamin B(12) status. Blood plasma assays were used to assess ascorbic acid status. RESULTS: No significant changes in riboflavin, thiamine, and pyridoxine status in erythrocytes and urinary excretion levels were observed after 1 month in Antarctica. Vitamin C levels decreased significantly (P<.001) after 1 month in Antarctica compared with basal values (1.31+/-0.076 mg/dL during phase I to 0.81+/-0.063 mg/dL during phase III). However, these levels were still within the normal reference range. CONCLUSION: This study found no water-soluble vitamin deficiencies in participants consuming processed and canned food after 1 month in Antarctica.

Development of an egg-white bioassay for monitoring biotin levels in urine and serum.

This article reports on the development of a simple and cost-effective bioassay for the detection of biotin in urine and serum, based on the very selective binding of avidin and biotin. Avidin was allowed to react without isolating it from egg white. Egg white was treated with the dye HABA, which binds to avidin. Upon subsequent treatment with biotin, HABA is released due to the high affinity of biotin to avidin. The amount of HABA released is proportional to the amount of biotin used.

Lymphocyte propionyl-CoA carboxylase and its activation by biotin are sensitive indicators of marginal biotin deficiency in humans.

BACKGROUND: Marginal biotin deficiency may be a human teratogen. A biotin status indicator that is not dependent on renal function may be useful in studies of biotin status during pregnancy. A previous study of experimental biotin deficiency suggested that propionyl-coenzyme A carboxylase (PCC) activity in peripheral blood lymphocytes (PBLs) is a sensitive indicator of biotin status. OBJECTIVE: We examined the utility of measuring PCC activity and the activation of PCC by biotin in detecting marginal biotin deficiency. DESIGN: Marginal biotin deficiency was induced in 7 adults (3 women) by egg-white feeding for 28 d. Blood and urine were obtained on days 0, 14, and 28 (depletion phase) and 44 and 65 (repletion phase). PBLs were incubated with (activated) or without (control) biotin before PCC assay. The activation coefficient of PCC is the ratio of PCC activity in activated PBLs to that in control PBLs. The significance of differences for all measurements was tested by repeated-measures analysis of variance with Fisher's post hoc test and Bonferroni correction. RESULTS: Changes in the urinary excretion of biotin and of 3-hydroxyisovaleric acid confirmed that marginal biotin deficiency was successfully induced. By day 14, PCC activity had decreased (P < 0.0001) to below the lower limit of normal in all subjects. By day 28, the activation coefficient of PCC had increased significantly (P = 0.003) and was above the upper limit of normal in 6 of 7 subjects. CONCLUSION: PCC activity is the most sensitive indicator of biotin status tested to date. In future pregnancy studies, the use of lymphocyte PCC activity data should prove valuable in the assessment of biotin status.

A simple binding assay for the direct determination of biotin in urine.

BACKGROUND: We have devised a simple assay to detect adequate biotin intake, which uses an alternative configuration from most existing assays. METHODS: The assay depends on the competition of streptavidin peroxidase for immobilized biotin or soluble biotin in standards or samples. Immobilized streptavidin peroxidase is detected using tetramethylbenzidine, and the plates are read at 450 nm. The assay was normalised by determining the biotin/creatinine ratio in the urine of healthy adults. Urinary biotin excretion was measured in unsupplemented pregnant women. The half-life of biotin excretion after a single oral supplement was determined for healthy volunteers. RESULTS: Urinary biotin excretion in unsupplemented pregnant women was 2.9+/-1.9 micromol/mol creatinine (mean+/-S.D.) and was significantly lower (p<0.001) than those of healthy males and females, which were 9.0+/-5.4 and 7.0+/-2.1 micromol/mol creatinine (mean+/-S.D.), respectively. The half-life of a single oral biotin supplement was 30-40 h, with excretion returning to basal levels at 70 h. CONCLUSION: We have devised a novel binding assay for the direct determination of total biotin excretion in urine, which is suitable for routine clinical laboratory. The assay is inexpensive, simple, rapid, and could be fully automated.

Biotin deficiency in an infant fed with amino acid formula.

Biotin deficiency is rarely encountered in an infant on weaning from breast and formula feeding. It is characterized by alopecia and scaly, erythematous dermatitis distributed around the body orifices. We report a 5-month-old Japanese infant with typical skin lesions who had been diagnosed as a neonate with dyspepsia and fed only an amino acid formula. Serum and urine levels of biotin were below the normal range, but zinc and biotinidase were within normal range. Urinary excretion of 3-methylcrotonylglycine, 3-hydroxyisovaleric acid, and methylcitric acid was significantly elevated. Daily oral supplementation with 1 mg of biotin resulted in dramatic improvement of the periorificial dermatitis and hair growth together with a complete disappearance of the organic aciduria. Our case shows that the characteristic skin manifestations are the most important clue to the diagnosis of biotin deficiency and demonstrated that urinary excretion of biotin and organic aciduria, rather than the serum concentration of biotin, are the sensitive indicators for evaluating the patient's status of biotin deficiency.

Measurement of 3-hydroxyisovaleric acid in urine of biotin-deficient infants and mice by HPLC.

We developed an assay for measuring urinary 3-hydroxyisovaleric acid (3-HIA) using HPLC after derivatization with 2-nitrophenylhydrazine hydrochloride (2-NPH . HCl). The derivatized 3-HIA was extracted into n-hexane and separated isocratically on a C8 reversed-phase column for fatty acids (YMC-Pack FA). We used this method to measure 3-HIA in urine extracts from mice fed a biotin-deficient diet for >4 wk and in an infant who was fed a special Japanese formula and was suspected of being biotin deficient. Urinary 3-HIA could be assayed within the range of 0.42-8.5 mmol/L with high accuracy by this method, as an indicator of biotin deficiency. Therefore, the HPLC method for 3-HIA described here may be a useful tool clinically as well as in the research laboratory.

Values of water-soluble vitamins in blood and urine of Japanese young men and women consuming a semi-purified diet based on the Japanese Dietary Reference Intakes.

We investigated the levels of water-soluble vitamins except for vitamin B6 in the blood and urine of Japanese college male (n = 10) and female (n = 10) students. They consumed for 7 d a semi-purified diet based on Japanese Dietary Reference Intakes to assess the Recommended Dietary Allowances (RDA) for water-soluble vitamins and to present some new normal values for blood and urine levels of water-soluble vitamins in Japanese. The blood and the 24-h urine samples were collected on the last day of the experiment and measured. The values of total vitamin B1 in whole blood, total vitamin B2 in whole blood, total cyanocobalamin in serum, total nicotinamide in whole blood, total pantothenic acid in whole blood, total folates in serum, total biotin in serum, and ascorbic acid in plasma were 104+/-17 pmol/mL (mean+/-SD), 216+/-25 pmol/mL, 0.34+/-0.05 pmol/mL, 59.1+/- 5.0 nmol/ mL, 2.45+/-0.37 nmol/mL, 15.6+/-4.6 pmol/mL, 8.3+/-0.5 pmol/mL, and 62+/-10 nmol/mL, respectively, in males, and 90+/-23, 234+/-18, 0.67+/-0.20, 61.9+/-6.0, 2.48+/-0.30, 30.2+/-8.6, 8.4+/-0.3, and 67+/-14, respectively, in females. There was a significant difference in the values of cyanocobalamin and total folates between men and women. The urinary excretion of vitamin B1, vitamin B2, cyanocobalamin, sum of the catabolic metabolites of nicotinamide, pantothenic acid, folates, biotin, and ascorbic acid were 665+/-114 nmol/d, 562+/-325 nmol/d, 93+/-31 pmol/d, 84+/-26 micromol/d, 9.3+/-2.3 micromol/d, 19.4+/-2.8 nmol/d, 83+/-18 pmol/d, and 148+/-51 micromol/d, respectively, in males, and 495+/-212, 580+/-146, 145+/-49, 83+/-19, 16.9+/-1.3, 22.7+/-2.7, 83+/-23, and 140+/-51, respectively, in females. There was a significant difference in the urinary excretion of cyanocobalamin, pantothenic acid and total folates between men and women. These values will be useful for the nutritional assessment of water-soluble vitamins for Japanese, although the examination period was short. In future, an experiment with various age groups and re-evaluation by repeated experiments will provide more accurate values.

Smoking accelerates biotin catabolism in women.

BACKGROUND: Smoking accelerates the degradation of many nutrients, including lipids, antioxidants, and certain B vitamins. Accelerated biotin catabolism is of concern in women because marginal biotin deficiency is teratogenic in mammals. OBJECTIVE: The objective was to assess the effect of smoking on the biotin status of women. DESIGN: A preliminary study of 7 women and 3 men examined the urinary concentrations of biotin and its metabolites biotin sulfoxide and bisnorbiotin in smokers. The interpretation of the results of this study was limited by the lack of a contemporaneous control group; consequently, we conducted a cohort-controlled study. Smoking women (n = 8) and nonsmoking control subjects (n = 15) provided 24-h urine samples; excretion rates of biotin, the biotin metabolites, and 3-hydroxyisovaleric acid were determined. Increased urinary excretion of 3-hydroxyisovaleric acid, which reflects a reduced activity of the biotin-dependent enzyme 3-methylcrotonyl-Co A carboxylase, is a sensitive indicator of biotin depletion at the tissue level. RESULTS: Compared with control subjects from previous studies, the smoking women in the preliminary study excreted significantly less urinary biotin (P = 0.02). Moreover, the ratio of urinary biotin sulfoxide to biotin increased (P = 0.04) in these women. In the cohort-controlled study, the urinary excretion of biotin decreased by 30% (P = 0.04), and the ratios of urinary bisnorbiotin and biotin sulfoxide to biotin increased significantly, which indicated accelerated catabolism in smokers. Moreover, the urinary excretion of 3-hydroxyisovaleric acid was greater in the smokers than in the control subjects (P = 0.04), which indicated biotin depletion in the smokers at the tissue level. CONCLUSION: These data provide evidence of accelerated biotin metabolism in smoking women, which results in marginal biotin deficiency.