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Understanding of biotin pharmacokinetics and regulation of metabolism is essential for the determination of the biotin requirement for humans. Using Landrace-Cambrough pigs as a model, we initially demonstrated that biotin binding to protein accounts for only a small percentage of the total biotin in plasma. A physiologic amount of [14C]biotin was administered intravenously to three pigs; nine blood samples were collected over 48 h. Plasma concentrations of 14C-labeled metabolites were negligible for the first 2 h after biotin infusion. Disappearance curves of total 14C and of [14C]biotin were similar; both fit a triexponential function consistent with a three-compartment, open model. To characterize the rapid early phase of disappearance more precisely, a physiologic amount of [14C]biotin was administered intravenously to five pigs; eight blood samples were collected over the first hour and 16 total samples over 48 h. Again a triexponential function provided an excellent fit. The mean half-life values (+/- 1 SD) for the three phases were 0.11 +/- 0.07, 1.43 +/- 0.42 and 22 +/- 4 h. The [14C]biotin accumulated primarily in the liver, kidney and muscle. When administered intravenously at tracer doses to three pigs, [3H]biotin exhibited similar early pharmacokinetics; however, substantial quantities of a 3H-labeled metabolite appeared after 1 h. These studies provide evidence that egress of biotin from plasma is more rapid than previously appreciated. The slower second and third phases may represent transport into the cytosol, biotransformation into intermediates and covalent binding to intracellular proteins. Similar pharmacokinetics are likely to be seen in humans.

This study determined the effects of supplemental dietary biotin (0, 10, or 20 mg/d) on performance of Holstein cows (n = 45; 18 primiparous and 27 multiparous). Treatments started at 14 d prepartum and continued until 100 d in milk (DIM). Blood samples were taken at 14 d prepartum, and blood and milk samples were taken at calving, and 30, 60, and 100 DIM. Dry matter intake during lactation was not different across treatments (19.7 kg/d). Milk production linearly increased with biotin supplementation (36.9, 37.8, and 39.7 kg/d for 0, 10, and 20 mg/d of supplemental biotin, respectively). Biotin supplementation did not affect milk fat and true protein percentages or fat yield but linearly increased true protein yield. Supplemental biotin increased concentrations of biotin in plasma and milk at all time points. Concentrations of biotin in plasma and milk (colostrum) at calving were higher than at other time points for cows fed supplemental biotin. In an ancillary experiment, plasma biotin concentrations were not as high when cows were fed 20 mg/d of supplemental biotin for 14 d during the middle of their dry period as when cows were fed 20 mg/d of biotin for the last 14 d of gestation. This suggests that events associated with parturition altered plasma biotin concentrations. Plasma concentrations of glucose, insulin, nonesterified fatty acids, and molar proportions of ruminal volatile fatty acids were not affected by biotin supplementation. Biotin supplementation had no effect on change in body weight or condition score. Supplemental biotin linearly increased milk and protein yields, however, the mode of action that caused these increases was not determined.

High performance liquid chromatography followed by post-column reaction detection in the far-red spectral region provides added sensitivity and selectivity. A homogeneous fluorescence energy transfer assay in the competitive mode based on the binding of biotin and streptavidin was developed as an on-line post-column reaction detection system. The labels used for energy transfer were R-Phycoerythrin conjugated to biotin and Cyanine 5 labeled with streptavidin. The energy transfer peak was measured at 670 nm and excitation was achieved using the 488 nm line of an argon ion laser. The biotin concentration in plasma ultrafiltrate ranged from 0.024 to 6.12 ng/mL (n = 6). The precision of the two controls, 0.24 and 2. 44 ng/mL, was found to be 18.70% and 9.92% relative standard deviation respectively. Accuracy was 10.47% and 1.95% difference from spiked, respectively (n = 6). The limit of detection was 21.70 pg/mL (8.90 x 10(-11)M) calculated based on a factor of 2x the standard deviation of the blank (n = 6). The correlation coefficient for the calibration curve was found to be 0.9995. Recovery from plasma ultrafiltrate at 2.44 ng/mL was 103.40% (n = 6). Detection selectivity was indicated by the absence of background fluorescence in six different plasma samples collected from six individual donors. Endogenous levels were detected in two of the six pools of plasma ultrafiltrates., (Copyright 2000 John Wiley & Sons, Ltd.)

A beta1-40 peptide radiopharmaceuticals could be used to image A beta brain amyloid in transgenic mouse models of Alzheimer's disease should the A beta peptide radiopharmaceutical be made transportable through the blood-brain barrier (BBB) in vivo. The present studies used the RI7-217 rat monoclonal antibody to the mouse transferrin receptor as a BBB drug targeting vector for the delivery to brain of A beta1-40 radiolabeled with either 125-Iodine or 111-Indium. The A beta peptide radiopharmaceutical is conjugated to the RI7 MAb using avidin biotin technology, wherein the A beta1-40 peptide radiopharmaceutical is monobiotinylated (bio) and bound to a conjugate of the RI7 MAb and streptavidin (SA). The [125 I]-bio-A beta1-40 or the [111 In]-bio-A beta1-40 either free or bound to the RI7/SA conjugate was injected intravenously into anesthetized adult mice and plasma pharmacokinetics and organ uptake were measured over the next 60 minutes. The A beta1-40 peptide radiopharmaceutical radiolabeled with 111-Indium was the preferred formulation, compared to peptide labeled with 125-Iodine, because there was a greater metabolic stability and reduced artifactual organ uptake of metabolites associated with the use of the 111-Indium nuclide. However, biotinylated A beta1-40 peptide radiopharmaceuticals conjugated to the RI7/SA brain drug targeting system were metabolically unstable in mice in vivo owing to active biotinidase activity. Future work involving brain drug targeting in mice that utilizes avidin biotin technology will need to incorporate biotin analogues that are resistant to biotinidase.

In the present study a novel Ab-avidin fusion protein has been constructed to deliver biotinylated compounds across the blood brain barrier. This fusion molecule consists of an Ab specific for the transferrin receptor genetically fused to avidin. The Ab-avidin fusion protein (anti-TfR IgG3-CH3-Av) expressed in murine myeloma cells was correctly assembled and secreted and showed both Ab- and avidin-related activities. In animal models, it showed much longer serum half-life than the chemical conjugate between OX-26 and avidin. Most importantly, this fusion protein demonstrated superior [3H]biotin uptake into brain parenchyma in comparison with the chemical conjugate. We also delivered a biotinylated 18-mer antisense peptide-nucleic acid specific for the rev gene of HIV-1 to the brain. Brain uptake of the HIV antisense drug was increased at least 15-fold when it was bound to the anti-TfR IgG3-CH3-Av, suggesting its potential use in neurologic AIDS. This novel Ab fusion protein should have general utility as a universal vehicle to effectively deliver biotinylated compounds across the blood-brain barrier for diagnosis and/or therapy of a broad range of CNS disorders such as infectious diseases, brain tumors as well as Parkinson's and Huntington's diseases.

The complex(153)Sm(III)DTPA-bis-biotin was prepared with a 99% radiochemical purity and a specific activity of 370 MBq/mg employing a molar ratio of DTPA-bis-biotin/Sm from 2 to 4 at pH 8.0. In vitro studies demonstrated that the complex is stable after dilution in saline and in human serum. Avidity of labeled biotin for avidin was not affected by the labeling procedure. Pharmacokinetic data of (153)Sm(III)DTPA-bis-biotin in normal mice showed that blood clearance is biexponential during the time interval from 0 to 24 h and that 3 h postinjection 92 +/- 4.32% of the dose is eliminated in the urine. To have further evidence which could sustain that (153)Sm(III)DTPA-bis-biotin is stable in solution as a real coordination complex, (152)Sm(III)DTPA-bis-biotin was obtained in macroscopic quantities and its characterization was done by IR, TGA, and conductivity measurements. The results indicated that the complex was chemically pure, where the Sm(3+) ion is neutralized by three carboxylate groups of the DTPA-bis-biotin ligand and coordinated to it. Using the Force Field method followed by ab initio calculations, the DTPA-bis-biotin and the Sm(III)DTPA-bis-biotin molecules were done. Accordingly, the coordination sphere of Sm(III) was totally satisfied with nitrogen and oxygen donors; the best coordination number was 9. The conformation geometry of both compounds is presented.

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.

The study was designed to test the ability of sequential applications of biotin-containing ointment to increase serum biotin levels. Twenty atopic dermatitis patients (mean age, 20.5 yr) and 11 healthy subjects (mean age, 25.5 yr) volunteered to participate in this study. The diagnosis of atopic dermatitis was established dermatologically. Seven grams per day of ointment containing 0.3% biotin and 1-4 g per day of steroid ointment were both applied sequentially. The healthy subjects applied only biotin ointment. The biotin concentration was determined microbiologically. Before biotin treatment, the average serum biotin level was significantly lower in atopic dermatitis patients than in healthy subjects. The percutaneous application of biotin-containing ointment caused a significant increase in the serum biotin concentration in both healthy subjects (from 41.5 +/- 10.0 to 50.2 +/- 9.2 nmol/L) and in atopic dermatitis patients (from 27.9 +/- 17.4 to 50.7 +/- 21.6 nmol/L), especially in patients whose initial level was low, and also could be effective in regulating the atopic allergic response involving eosinophils. In conclusion, biotin appears to be readily absorbed through both normal and dermatitis-affected human skin.

Although estimated average requirements for biotin have been proposed, the human requirements for biotin in specific populations and at various ages remain uncertain, in part because indicators of biotin status have not been validated. With the use of improved methods for measuring biotin and metabolites, a recent study indicated that decreased urinary excretion of biotin and bisnorbiotin is an early and sensitive indicator of biotin deficiency, but decreased serum concentration of biotin is not. Increased urinary excretion of 3-hydroxyisovaleric acid (3-HIA), a leucine metabolite that is excreted in increased quantities with deficiency of the biotin-dependent enzyme beta-methylcrotonyl-CoA carboxylase, is also an early and sensitive indicator of biotin deficiency. When these indicators were assessed longitudinally in 13 pregnant women, biotin excretion was not significantly decreased early in pregnancy but did decrease significantly from early to late pregnancy. Excretion of 3-HIA was abnormally increased in about three-fourths of the women studied in both early and late pregnancy. Thus, each indicator detected biotin deficiency late in pregnancy, but assessment of biotin status for the two indicators conflicted early in pregnancy. Preliminary results from a trial assessing response of 3-HIA excretion to biotin treatment indicate that biotin status is indeed impaired both early and late in pregnancy.

One hypothesis to explain the age-dependent clearance of red blood cells (RBCs) from circulation proposes that denatured/oxidized hemoglobin (hemichromes) arising late during an RBC's life span induces clustering of the integral membrane protein, band 3. In turn, band 3 clustering generates an epitope on the senescent cell surface leading to autologous IgG binding and consequent phagocytosis. Because dog RBCs have survival characteristics that closely resemble those of human RBCs (ie, low random RBC loss, approximately 115-day life span), we decided to test several aspects of the above hypothesis in the canine model, where in vivo aged cells of defined age could be evaluated for biochemical changes. For this purpose, dog RBCs were biotinylated in vivo and retrieved for biochemical analysis at various later dates using avidin-coated magnetic beads. Consistent with the above hypothesis, senescent dog RBCs were found to contain measurably elevated membrane-bound (denatured) globin and a sevenfold enhancement of surface-associated autologous IgG. Interestingly, dog RBCs that were allowed to senesce for 115 days in vivo also suffered from compromised intracellular reducing power, containing only 30% of the reduced glutathione found in unfractionated cells. Although the small quantity of cells of age >/=110 days did not allow direct quantitation of band 3 clustering, it was nevertheless possible to exploit single-cell microdeformation methods to evaluate the fraction of band 3 molecules that had lost their normal skeletal linkages and were free to cluster in response to hemichrome binding. Importantly, band 3 in RBCs >/=112 days old was found to be 25% less restrained by skeletal interactions than band 3 in control cells, indicating that the normal linkages between band 3 and the membrane skeleton had been substantially disrupted. Interestingly, the protein 4.1a/protein 4.1b ratio, commonly assumed to reflect RBC age, was found to be maximal in RBCs isolated only 58 days after labeling, implying that while this marker is useful for identifying very young populations of RBCs, it is not a very sensitive marker for canine senescent RBCs. Taken together, these data argue that several of the readily testable elements of the above hypothesis implicating band 3 in human RBC senescence can be validated in an appropriate canine model.

In 23 patients with chronic renal failure (CRF), 23 patients on chronic intermittent hemodialysis treatment (DP), 22 patients after renal transplantation (RT) and 40 normal persons (NP), Biotin plasma levels and the urinary excretion were analysed and compared to the dietary Biotin intake. Unsupplemented DP had lower intake of Biotin than the CRF, RT, NP and DP with supplementation. DP excreted only 1.6-6.3% of the daily intake as compared to 39.7% in NP, 27.6% in CRF and 24.3% in RT. In unsupplemented DP patients, Biotin plasma levels were elevated by 4 times and in supplemented patients by 6 times compared to NP. During hemodialysis treatment, the Biotin plasma level dropped by about 30% in DP with and by 33% in DP without vitamine supplementation. However, after 44 hours, the initial concentration was reached again in those receiving vitamine supplementation (99% of basal level) and in DP without substitution (97% of basal level). Only in male DP significantly higher Biotin plasma levels before HD were detected irrespective of the supplementation dose as compared to female patients (30 micrograms and 300 micrograms Biotin after each dialysis session). Biotin plasma concentration did not vary with respect to the underlying renal disease, the serum creatinine concentration and the length and frequency of dialysis treatment, including the type of dialyzer (low- vs high flux) used and the blood flow rate (QB 180-260 vs 270-280 vs 300 ml/min). There were no major effects of the age of the patients (< 60 years vs > 60 years), the BMI, nicotine abuse, or alcohol intake on Biotin blood concentration. Our results showed normal Biotin plasma levels which reflect a normal functional status and exclude a functional deficit, therefore there is no reason for a regular Biotin supplementation in patients with chronic renal failure.

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.

We studied the uptake of biotin into human peripheral blood mononuclear cells (PBMC) using [3H]biotin and studied the catabolism of biotin in PBMC using [14C]biotin. Over 30 min, [3H]biotin uptake was greater at 37 degrees C than at 25 degrees C (KT = 2.6 +/- 0.4 nM, maximal velocity = 2.9 +/- 0.2 fmol . 10(6) cells-1 . 30 min-1). Ouabain reduced [3H]biotin uptake to 65% of control values, suggesting that biotin uptake is Na-K-ATPase dependent. Unlabeled biotin and biotin analogs reduced the uptake of [3H]biotin to 22-70% of control values, suggesting the presence of a competition for a structurally specific biotin transporter. When endocytosis by PBMC was stimulated by various acyl glycerols, [3H]biotin uptake was 40-73% of control values; these data are consistent with the hypothesis that stimulated endocytosis reduces biotin transporter density on the cell surface. During a 168-h incubation, PBMC did not catabolize [14C]biotin.

Biotin deficiency is known to affect immune function in both humans and experimental animals. In this study, we determined the effect of biotin deficiency on 4-wk-old Balb/cAnN mice during 20 wk of experimentation. The growth rate of mice slowed significantly during the first 6 wk of consumption of a diet designed to induce biotin deficiency; thereafter, from weeks 7 to 20 there was progressive weight loss in the mice receiving the biotin-deficient diet. In the livers of biotin-deficient mice, the specific activities of two biotin-dependent enzymes--pyruvate carboxylase and propionyl-CoA carboxylase--decreased by as much as 75% and 80%, respectively, and in spleen lymphocytes the specific activities of these two enzymes decreased by 63% and 75%, respectively. With respect to the effects of biotin deficiency on the immune system, we observed statistically significant changes in both the absolute number of spleen cells and in the proportions of spleen cells carrying different phenotypic markers: after 16 wk the percentage of cells expressing surface immunoglobulin (sIg) decreased from 47% (control and supplemented) to 27% (deficient) and CD3+ cells increased from 42% (control and supplemented) to 54% (deficient). The mitogen-induced proliferation of spleen cells from deficient mice was lower than that of spleen cells from the control mice. These findings suggest that biotin could have an important role in lymphocyte maturation and responsiveness to stimulation, and consequently in the capacity of the immune system to respond to an antigenic challenge.

Objective: Biotin plays important roles in carbohydrate and lipid metabolism as well as in the decarboxylation of amino acids. In this study, to determine the biotin status in elderly people, we determined the levels of biotin and other biochemical variables in their serum., Methods: Blood was collected from 685 elderly people aged 65 years and over (284 men and 401 women) and from 2,004 reference people. Biotin levels in the serum were microbiologically quantified by the agar plate method and other biochemical variables were recorded using the autoanalyzer., Results: The serum biotin level in elderly people was 10.2 +/- 7.20 pmol/ml (2.5 +/- 1.76 ng/ml), the distribution of which was skewed to the right compared to the reference group (9.4 +/- 1.43 pmol/ml) (2.3 +/- 0.35 ng/ml). However, serum biotin levels did not change with age in the elderly people and no sex-related differences were detected. On the basis of the correlation coefficients among the biochemical variables in the serum, biotin levels were correlated positively with the total cholesterol level. A negative correlation was found between the biotin level and the serum albumin, triiodothyronine, phosphate, and calcium levels. On the other hand, 5.8% of the elderly people had biotinyl IgG in their serum, which did not differ from the percentage in the reference group (4.1%). However, in the elderly people the level of biotinyl IgG positivity was significantly lower in women than in men (4.1% vs. 8.1%). In the biotinyl IgG positive elderly, the levels of parathyroid hormone and total cholesterol were lower, whereas the uric acid level was higher compared to the biotinyl IgG negative elderly., Conclusions: Although serum biotin levels are not affected by aging, the serum biotin levels of elderly people vary greatly.

Biotin is an important vitamin for cellular function and growth and, therefore, essential for fetal development. The fetus is exclusively dependent on maternal biotin supply. Since biotin is not produced within the body, maternal biotin levels depend on dietary intake. In order to investigate the biotin status of the human fetus, we measured the plasma biotin levels in 15 pregnant women and their fetuses who underwent amniocentesis and fetal blood sampling at 18-24 weeks of gestation for prenatal diagnosis of thalassemia. Maternal biotin was found to be 131 +/- (SD) 102 ng/l and fetal biotin 784 +/- 327 ng/l (p < 0.0001). Our findings are indicative of an active transport mechanism of biotin through the placenta in favor of the fetus.

Clear immunoscintigraphy with radiolabeled monoclonal antibodies (MAbs) requires a high tumor tissue/blood ratio of radioactivity. In this study, we attempted to obtain a high tumor tissue/blood ratio by the active removal of radiolabeled MAb from the circulation, using the avidin-biotin system. Biotinylated 125I-labeled MAb A7 was injected intravenously into nude mice bearing a human colon cancer (WiDr) xenograft. Avidin was injected 24 h later. The tumor tissue/blood ratio of radioactivity was almost four times that of controls. These results suggest that biotinylated 125I-labeled MAb A7 and avidin are potentially useful for the rapid immunodetection of human colon cancer.

Biotinidase deficiency is an autosomal-recessive disorder of biotin recycling. Children with profound biotinidase deficiency usually have neurological and cutaneous symptoms in early childhood, but they may not develop symptoms until adolescence. We now report on a man and a woman with profound biotinidase deficiency who are asymptomatic and who were diagnosed only because their biotinidase-deficient children were identified by newborn screening. These adults have never exhibited symptoms of the disorder and are homozygous for two different mutations resulting in different aberrant enzymes. There is no evidence of an increased dietary intake of biotin to explain why they have remained asymptomatic. Although these adults may still be at risk for developing symptoms, they could represent a small group of individuals with profound biotinidase deficiency who will never develop clinical problems. Their lack of symptoms suggests that there are probably epigenetic factors that protect some enzyme-deficient individuals from developing symptoms. These individuals broaden the spectrum of expression of biotinidase deficiency.

Using serum biotin concentration as the indicator, a previous study reported biotin deficiency resulting from long-term anticonvulsant therapy. However, serum biotin may not be a good indicator of tissue biotin status. Using better indicators of biotin status in anticonvulsant-treated subjects, we found increased urinary excretion of biotin catabolites and 3-hydroxyisovaleric acid, an organic acid produced in greater quantities secondary to reduced activity of a biotin-dependent carboxylase. We conclude that anticonvulsant treatment led to increased biotin catabolism and probably to reduced biotin status.

We sought to determine whether the biliary excretion of biotin contributes substantially to the overall excretion of the vitamin in mammals, and hence, whether metabolism by gut microorganisms could account for some metabolism of biotin administered parenterally. [carbonyl-14C]Biotin was injected intravenously into six rats; bile and urine were collected for 24 h after injection. In a study of five pigs, serum and bile were analyzed for endogenous biotin and metabolites. In rat bile and urine, biotin, bisnorbiotin, biotin-d,l-sulfoxide, bisnorbiotin methyl ketone and two unidentified compounds were quantitated. In bile, these six compounds accounted for only 1.9 +/- 0.2% of the administered 14C, but in urine they accounted for 60.6 +/- 4.1%. The metabolite and time profiles in bile were also strikingly different from those in urine. Only biotin, bisnorbiotin and biotin-d,l-sulfoxide were quantitated in pig bile and serum. The concentrations of biotin, bisnorbiotin and biotin-d,l-sulfoxide in bile were 6.9-14.7 times the concentrations in serum. However, the bile to serum ratios of biotin and metabolites were >99% less than those of bilirubin, which is actively excreted. These data provide evidence that the biliary excretion of biotin and metabolites is quantitatively negligible.

The effect of biotin administration on the glucose tolerance of streptozotocin (STZ)-induced diabetic Wistar rats was investigated. STZ-induced diabetes was induced by intraperitoneal injection of streptozotocin (45 mg/kg body weight as a single dose). The impaired glucose tolerance in response to an oral glucose load (1.8g per kg body weight) in STZ-induced diabetic rats (STZ-rat) was partially improved by intraperitoneal administration of biotin for 15 days (100 micrograms/rat/day). However, a recovery in the STZ-rat's insulin secretion was not found after biotin administration. To help clarify the mechanism underlying the improvement in glucose tolerance seen with biotin treatment, glucokinase and hexokinase activities were determined in the liver and pancreas. In STZ-rats that had received biotin (STZ-biotin rats), glucokinase activity was higher by 3.4-fold in liver and by 2.4-fold in pancreas than in the STZ-rats. The biotin level of STZ-rats was significantly lower in the liver and pancreas than that of the control rats (no STZ administration); but in STZ-biotin rats, the level in these organs recovered to the control level. These results demonstrate that injected biotin can improve glucose handling without increasing insulin secretion in STZ-rats.

This study assessed biotin nutritional status longitudinally during pregnancy as judged by urinary excretion of biotin and biotin metabolites and by serum concentration of biotin. 3-Hydroxyisovaleric acid excretion was also assessed because increased excretion of that acid reflects decreased tissue activity of the biotin-dependent enzyme, methylcrotonyl-CoA carboxylase. Thirteen women provided untimed urine samples during both early and late pregnancy. Twelve nonpregnant women served as controls. Biotin and metabolites were determined by a combined HPLC/avidin-binding assay. 3-Hydroxyisovaleric acid was determined by gas chromatography/mass spectrophotometry. Significance of changes from early to late pregnancy was tested by paired t test; to compare nonpregnant controls with early and late pregnancy, ANOVA was used. During early pregnancy, biotin excretion was not significantly different than controls; however, 3-hydroxyisovaleric acid excretion was significantly increased relative to controls (P < 0.0001) and was greater than the upper limit of normal in 9 of 13 women. From early to late pregnancy, biotin excretion decreased in 10 of 13 women (P < 0.01); by late pregnancy, biotin excretion was less than normal in six women. During late pregnancy, 3-hydroxyisovaleric acid remained significantly increased relative to controls (P < 0.0001). Serum concentrations of biotin were significantly greater than those of controls during early pregnancy (P < 0.0001) and decreased in each woman from early to late pregnancy (P < 0.0001). These data provide evidence that biotin status decreases during pregnancy.

The use of streptavidin-conjugated antibody to pretarget tumors in animals and patients, prior to administration of radiolabeled biotin, has provided encouraging results, in part because of the high affinity of biotin for streptavidin and the rapid whole-body clearance of biotin. However, binding of endogenous biotin to streptavidin may interfere with the clinical potential of this approach. This report evaluates the effect of endogenous biotin on an antibody-streptavidin conjugate in a mouse tumor model. Tumored nude mice were depleted of endogenous biotin by sequential intraperitoneal injections of streptavidin. The assay of serum biotin levels indicated less than 0.5 ng of biotin per mL of serum in treated mice versus 4 ng per mL in untreated animals. Flow cytometric analysis was used on single-cell suspensions of tumor from animals receiving streptavidin-conjugated IgG to detect the presence of the antibody on the cell membrane (with fluoroisothiocyanate-conjugated goat anti-mouse antibody), and to detect biotin binding sites on streptavidin (with biotin-phycoerythrin). Both treated and untreated mice demonstrated the presence of antibody on tumor cells through 48 h postadministration, but only in treated animals were biotin binding sites observed. These results in the mouse model suggest that the small concentration of streptavidin delivered to a tumor via a specific antibody may be saturated with endogenous biotin and therefore not able to be targeted subsequently with radiolabeled biotin.

To assess the utility of various indicators of biotin status, marginal biotin deficiency was induced experimentally in normal adults. Ten subjects consumed a diet that contained enough avidin to bind seven times more biotin than that in the diet. Blood and 24-h urine samples were collected before the diet began and twice weekly thereafter for 20 d. The urinary excretion and serum concentration of biotin and its two principal inactive metabolites bisnorbiotin and biotin sulfoxide were determined after HPLC separation with an avidin-binding assay. The urinary concentration of 3-hydroxyisovaleric acid, an indicator of reduced activity of a biotin-dependent enzyme, was quantitated by gas chromatography-mass spectrometry. The urinary excretion of 3-hydroxyisovaleric acid increased significantly (P < 0.0001). For all subjects, the urinary excretion of both biotin and bisnorbiotin decreased significantly (P < 0.0001 for each). In contrast, the mean serum concentration of biotin did not decrease significantly (P = 0.06). These data provide evidence that the urinary excretion of 3-hydroxyisovaleric acid and the urinary excretion of biotin are early and sensitive indicators of biotin deficiency and that the serum concentration of biotin is not.

In addition to the pharmacokinetic interest, serum concentrations of biotin and biotin metabolites are important because biotin in serum might interfere with assays that use avidin-biotin detection systems. With acute and chronic oral administration of biotin the serum concentration of biotin increases. Because of limited specificity of bioassays or avidin-binding assays used in previous studies, the proportion of the increase attributable to biotin metabolites (if any) remains unknown. To address these questions 15 adults consumed 1,200 microg biotin daily for 14 days. Blood samples were obtained before biotin ingestion and at 3 hours after biotin ingestion on the first day ("acute supplementation") and the fourteenth day ("chronic supplementation"). Biotin, bisnorbiotin, and biotin sulfoxide were measured with a chemically specific high-pressure liquid chromatography/avidin-binding assay. Serum concentrations of biotin, bisnorbiotin, and biotin sulfoxide increased approximately fiftyfold with acute supplementation of biotin; each increased further with chronic supplementation. With acute supplementation the proportion of the total attributable to metabolites did not decrease significantly, suggesting that pathways for biotin catabolism are not easily saturated. With chronic supplementation the proportion of the total attributable to metabolites did not increase significantly, suggesting that biotin catabolism was not substantially induced. We conclude that on a mole basis the contribution of biotin metabolites is important, and we provide an estimate of the biotin and biotin metabolite concentration that might be encountered in individuals who self-select large biotin supplements.

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.

Biotinyl-3-[211 At]astatoanilide ([211 At]AtBA) was prepared in more than 80% yield by destannylation. In vitro, [211 At]AtBA exhibited a high affinity for streptavidin, and was stable after incubation in human serum, cerebrospinal fluid and distilled water, whereas it was rapidly degraded in mouse serum. HPLC analysis showed that the main degradation pathway in mouse serum was the cleavage of [211 At]astatoaniline. In mice, [211 At]AtBA and its 125I-labeled analogue cleared rapidly from most tissues; however, there was some evidence for dehalogenation of both tracers.

Using biochemical indices thiamin, riboflavin, pyridoxine, ascorbic acid, retinol, alpha-tocopherol, folic acid, cyanocobalanin, carotene and biotin status were assessed in a sample of 243 young British adults. Using conventional definitions the number of individuals whose vitamin status can be described as adequate, marginal or deficient was determined. Vitamin status was related to alcohol intake and smoking. The status of ascorbic acid, cyanocobalamin, alpha-tocopherol, folic acid and in males retinol, was adequate in the majority of cases. In the cases of riboflavin and pyridoxine the status of a substantial minority was either borderline or deficient. The thiamin and biotin status of a minority of both sexes, and retinol in the females, was marginal. In males alcohol was associated with better retinol, ascorbic acid, alpha-tocopherol and pyridoxine status and lower levels of biotin. Lower carotene values were associated with drinking alcohol. In both sexes those who smoked had lower ascorbic acid, folic acid and carotene values. In males smoking was additionally associated with lower riboflavin and biotin; in females cyanocobalamin and alpha-tocopherol were lower. As all the subjects were students volunteers, care should be taken in generalizing the data to other groups.

The Otsuka Long-Evans Tokushima Fatty (OLETF) rat, serving as a spontaneously diabetic model with non-insulin-dependent diabetes mellitus (NIDDM), exhibits impaired glucose tolerance (IGT) at about 16 weeks of age. In this study, we investigated whether or not biotin, a water-soluble vitamin, improved the IGT of OLETF rats. To this end, we administered diets containing one of three levels of biotin, a high-biotin diet (BH), a normal-biotin diet (BN) and a basal-biotin diet (BB), to OLETF rats up to 24 weeks of age. An oral glucose tolerance test (OGTT) was performed four times between 13 and 22 weeks of age. The administration of a BH corrected the IGT of OLETF rats. Upon further investigation, we found that insulin secretion in the OLETF-BH rats was decreased to a significant extent, signaling that the hyperinsulinemia typical to the OLETF-BH rats had clearly improved. Body weights were significantly lower in the OLETF-BH group than in the other OLETF groups, even though the OLETF-BH rats showed a significantly higher average daily food intake. The body weight gain of the OLETF-BH rats followed the same tendency as the control-LETO (Long Evans Tokushima Otsuka) rats (LETO-BB and LETO-BN). These results demonstrate that a high-level biotin diet can improve the glucose handicap in NIDDM rats.

We modified Bayer's method of micro-plate assay for quantitation of biotin concentration. Biotin concentration in the solution and serum which cannot be quantitated directly by a microorganism assay (bio-assay), was easily determined by this method, which showed a high affinity of streptavidin for biotin this method and had a wide measurement range (0.9-60,000 pg/ml). We measured the concentration of biotin in 150 sera from 44 patients (21 males and 23 females) with active hepatitis (high level of both GOT and GPT, over 100 IU/l), 15 patients (7 males and 8 females) with inactive hepatitis (positive HCV-Ab but within normal limits of both GOT and GPT level), 17 patients (8 males and 9 females) with hepatoma and liver cirrhosis and 71 healthy persons (34 males and 37 females). The biotin concentration of sera in the healthy persons was 243.5 +/- 184.6 pg/ml, there being no sex difference. The biotin concentration in sera was higher in the patients than in healthy persons. It was high in the hepatoma and cirrhosis group (4,394.0 +/- 6,176.3), the active hepatitis group (2,397.4 +/- 2,785.5), and the inactive hepatitis group (1,873.2 +/- 1,523.7). These findings suggest that the biotin concentration is not significantly correlated with an escape enzyme such as GOT and GPT. These findings suggest that a high biotin concentration reflects other mechanisms such as escape from damaged liver cells.

This study of 36 rats with rat colon adenocarcinoma transplants was carried out to investigate the efficacy of a new method of whole blood immunoadsorption (WBIA) in removing biotinylated monoclonal antibodies (MAbs) directly from unseparated blood, in order to increase 'the tumor/normal-tissue uptake ratio', as compared with extracorporeal immunoadsorption (ECIA) of antibodies from plasma. Compared with the ECIA system, the overall volume of the WBIA system (comprising only a pump, an adsorption column, a drop-chamber and tubings) was less (3.6 vs. 6.2 ml), and procedure duration 2 h less. The 17 rats undergoing the WBIA procedure, started 12 h after i.v. injection of 4.0-4.5 MBq 125I-BR96-biotin, manifested neither hemolysis nor any other complication; no signs of organ edema were found at dissection; whole body and blood radioactivity values were reduced by 51% and 89.5%, respectively. The WBIA method was as effective as ECIA, but technically simpler, safer and more reliable.

Biotin deficiency is well known as a cause of hyperammonemia, but there has been no report on the effect of biotin deficiency on hepatic ureagenesis. In this study, we examined the changes in the activities and gene expression of urea cycle enzymes using rats fed raw egg white as a model of biotin deficiency. All rats were made biotin-deficient by feeding them an avidin-containing diet for 6 wk. The rats were divided into two groups at the beginning of this experiment: biotin-deficient rats (BD rats) and biotin-supplemented rats (BS rats) which were treated with biotin once a day at a dose of 1 mg per rat intraperitoneally. The plasma ammonia concentration of the BD rats (92.8 +/- 12 mumol/L) was significantly higher than that of BS rats (63.9 +/- 16 mumol/L, P < 0.05). The activities of ornithine transcarbamylase (OTC) was significantly lower in the liver of the BD (110.2 +/- 5.5) rats than in the BS rats (154 +/- 3.8 U/mg protein, P < 0.01). Activities of the other urea cycle enzymes were not significantly different in the two groups. OTC gene expression in the liver of BD rats was 40% lower than in BS rats (P < 0.05). These data suggest that biotin deficiency decreases OTC activity and the amount of OTC mRNA.

Biotinylation of erythrocytes has been developed in rabbits as a tool to retrieve labeled cells following various periods in circulation. This retrieval capability allows biochemical studies to be conducted on red blood cells (RBC) that have aged for desired times in vivo. However, because erythrocyte life span is much shorter in rabbits than in humans, and because cell removal is measurably age-independent in rabbits, we have sought to validate the same protocol in dogs, whose cell life span and age-dependent removal characteristics are similar to humans'. Canine RBC were biotinylated in vivo by infusion of N-hydroxysuccinimidyl biotin dissolved in dimethylacetamide or dimethylsulfoxide. Cell life spans were evaluated using 14C-cyanate labeling followed by scintillation counting or avidin-FITC labeling followed by flow cytometry. Both methods gave identical results. The life span of the biotin-conjugated cells was found to be normal (approximately 110 days), and the stability of the biotin ligand was adequate for efficient retrieval of cells using avidin-coated magnetic beads (magnetic cell sorting [MACS]). From each isolation, approximately 20 microL of packed biotinylated cells of approximately 90% purity (i.e., 10% contamination by unlabeled cells) could be harvested. On average, approximately 60% of the biotinylated cells in any sample could be retrieved. Either multiple isolations or use of larger collection columns will facilitate collection of cell numbers sufficient for biochemical tests. After incorporating several modifications in the previous biotinylation protocol that were required for adaptation to the dog, the methodology can be used to study red cell senescence in an animal that has several pertinent similarities to humans.

An enzyme-linked assay was developed to quantify serum biotin concentrations in experimental animals and humans. With this assay the effect on serum biotin concentration after intravenous injection of streptavidin or the addition of avidin to food was studied in rabbits and dogs. Intravenous injection of streptavidin reduced serum biotin values quickly but temporarily, in a dose-dependent manner in both species. Addition of avidin to the diet lowered biotin values approximately four- to fivefold in the rabbits, with resultant biotin concentrations similar to those of humans.

Proof of principle has been shown for a rapid, quantitative, homogeneous immunoassay capable of analyzing whole-blood samples. The assay was performed with test cards and a small instrument designed for use at the point of care. The immunoassay has an immunological "front end" combined with a coagulation cascade chemistry "back end" and is made possible by combining two patented technologies: (a) a serine protease inhibitor [Porter and Bruhnke, Photochem and Photobiol 1990; 51(1):37] and (b) paramagnetic iron oxide particles (PIOP) in a mixture of buffers and coagulation assay components supplied as a dry film in a test-card reaction chamber [Oberhardt et al., Clin Chem 1991;37:520]. A model steric-hindrance immunoassay based on these technologies was established for the measurement of biotin. The calibration curve was developed by measuring plasma samples supplemented with biotin. The reagents were inhibited biotinylated thrombin, anti-biotin monoclonal antibody, and PIOP.

This study involved a macroscopic evaluation of hoof quality in 152 Lipizzaner horses (130 from Austria and 22 from other countries) and a controlled double blind trial of the effects of biotin on hoof horn growth and quality over 19 months in 42 stallions from the Spanish Riding School (SRS) in Vienna. Using a grading system that incorporated evaluation of horn wall, white line, sole and frog, the macroscopic study revealed the following: 90% of the Austrian Lipizzaners had soft white lines and crumbling, fissured horn at the bearing border of the walls; 39% of the stallions of the SRS, > 4-years-old, had medium to severe hoof horn changes. Daily administration of 20 mg biotin to a test group of horses (n = 26) and a placebo to a control group (n = 16) showed that after 9 months the test group had significantly improved compared to the beginning of the trial and the placebo group (P < 0.01). In the test group, further improvement was observed during the following 5 months and, subsequently, the same good level of hoof condition was maintained over 3 further years of observation. Growth rate of the horn wall was equal in the biotin and placebo group, being 7 mm/28 days, giving a wall renewal period of 11 months. Mean plasma biotin level of untreated horses was 350 ng/l; plasma levels of biotin supplemented horses were > 1000 ng/l. It was concluded that continuous dietary supplementation with biotin at a daily dose of 20 mg is indicated to improve and maintain hoof horn quality in horses with less than optimum quality hoof.

In studies using an avidin-binding assay to measure the serum or plasma concentration of biotin, biotin is sometimes assumed to be equal to the avidin-binding substances detected. To provide a range of values for serum concentrations of biotin, bisnorbiotin, and biotin sulfoxide, HPLC was used to separate avidin-binding substances in human serum, and the chromatographic fractions were assayed for avidin-binding substances (biotin and biotin metabolites). In sera from 15 normal fasting adults, substantial concentrations of avidin-binding substances other than biotin were detected. Two of the principal substances were identified as bisnorbiotin and biotin sulfoxide based on their chromatographic properties. The serum concentrations of bisnorbiotin and biotin sulfoxide varied widely among the individuals. In three subjects, the concentration of bisnorbiotin exceeded that of biotin. The presence of avidin-binding substances in addition to biotin may have confounded previous measurements of the concentration of biotin in serum, plasma, and blood when avidin-binding assays were used. Because bioassay methods for biotin sometimes use organisms for which one or more of these biotin metabolites are growth factors, measurements of biotin in blood using some bioassays are likely to overestimate the concentrations of biotin.

We studied the effect of a supplement of biotin (10 mg/d) or a placebo under double-blind conditions on plasma biotin concentrations and lymphocyte propionyl CoA carboxylase (PCC) and pyruvate carboxylase (PC) in 22 children with severe protein-energy malnutrition (PEM) (5 with kwashiorkor, 10 with marasmus, and 7 "sugar babies"). There were significant differences between the malnourished and control subjects only for PCC, although not among the three PEM types. Six of the patients had both PC and PCC activities below the lowest of the normal control subjects; there was no correlation between biotin concentrations and carboxylase activities in individual patients. In response to biotin supplementation, the greatest change in lymphocyte carboxylase activities was detected in patients who had abnormally decreased initial carboxylase activities, but the response was not related to initial plasma biotin concentration. These results indicate that these enzyme deficiencies are the result of a nutritionally determined biotin deficiency, that carboxylases and especially PCC are better indicators of the biotin status in individual patients than is the plasma biotin concentration, and that in some malnourished patients biotin deficiency may be rate-limiting in their nutritional homeostasis.

An improved agar plate method of biotin bioassay using Lactobacillus plantarum ATCC 8014 and bromocresol purple was established to determine biotin levels in human serum and urine. Samples were treated with 4.5 N H2SO4 to liberate free biotin, autoclaved for 1 h and neutralized by 4.5 N NaOH, then 10 microliters was added to wells in each plate. The biotin levels were measured in 190 serum and 59 urine samples, and the means were 2.7 +/- 0.53 ng/ml and 12.4 +/- 5.56 ng/mg of creatinine, respectively. The intra-assay coefficient varience (CV) were 3.2 (n = 20) and 1.3% (n = 23), respectively. The recovery of biotin added (10 ng/ml) to serum was 110.7%, and to urine was 99.6%. These findings suggest that this assay is sufficiently accurate and reproducible for routine use in the clinical laboratory. The excretion of orally administered biotin was also demonstrated by the method.

Background: The objectives of this research were 1) to determine whether two populations of platelets may be labeled with different levels of biotin and followed concurrently in vivo by flow cytometry and 2) to determine whether the level of biotinylation affects the in vivo platelet recovery and survival., Study Design and Methods: Two platelet aliquots were biotinylated under conditions that resulted in either a lower or a higher number of biotin molecules per platelet. After transfusion, the two populations were distinguished and quantitated by flow cytometry., Results: In five animals, recoveries were 69.8 +/- 27.0 percent for low-biotin platelets and 72.6 +/- 26.7 percent for high-biotin platelets. For each animal, the recoveries agreed closely. Life span, determined by the multiple-hit method, was 2.68 +/- 0.63 days for low-biotin platelets and 2.58 +/- 0.69 days for high-biotin platelets. These values for recovery and life span are consistent with those measured in rabbits by using radioisotope labels., Conclusion: Platelet biotinylation offers a nonisotopic method for direct comparison of alternative harvest and storage conditions. It also offers the potential for simultaneous evaluation of the in vivo characteristics of platelets from at least two donors.

Objective: The objective of the present study was to establish whether copper (Cu)-deficient rats fed a diet containing fructose as their sole carbohydrate source require more biotin than the recommended 2 mg/kg diet when egg-white serves as the dietary protein., Methods: Eighty weanling male Sprague-Dawley rats were randomly divided into 8 groups according to type of dietary carbohydrate (starch or fructose), level of Cu (0.6 micrograms Cu/g diet or 6.0 micrograms Cu/g diet) and level of biotin (2 mg/kg diet or 10 mg/kg diet)., Results: Regardless of the level of dietary biotin, Cu-deficient rats fed a fructose-containing diet exhibited growth retardation, anemia, atrophied pancreata, enlarged hearts and similar death rates. The remaining Cu-deficient rats fed fructose were emaciated and sick regardless of dietary biotin levels. The concentration of biotin in serum and biotin content of liver of rats fed fructose were higher than corresponding values from rats fed starch., Conclusion: Cu-deficient rats fed fructose are not deficient in biotin compared to published normal values. Supplementation of 10 mg/biotin/kg diet did not improve morbidity or mortality and therefore was not beneficial.

Biotin-binding immunoglobulin (BBI) was recently identified in human serum and has been suggested to have a significant association with allergic and autoimmune disorders. Attempts were made to evaluate the clinical significance of BBI in autoimmune thyroid disorders. Prevalence of BBI was significantly higher in Graves' disease (47%) than in Hashimoto's disease (8%) and healthy controls (10%). The BBI consisted of heterogeneous subtypes with respect to binding of several immunoglobulin classes. Sera in Graves' disease showed predominantly IgG-binding BBI, whereas healthy subjects had IgM-binding BBI. Thyroid stimulating hormone receptor antibody (TRAb) level was significantly higher in the BBI non-detected group than in the detected group. There was no significant relationship between BBI prevalence and thyroid hormone concentrations, anti-thyroglobulin antibody (TGAb) or anti-thyroid microsomal antibody (McAb) titers. In addition, biotin levels in peripheral blood and red blood cells and biotinidase activity did not differ in the BBI detected and non-detected groups. The present results suggest that BBI is associated with autoimmune dysfunction in Graves' disease.

Antibodies against biotinylated proteins have been identified in 10% of individuals tested (6/60). These antibodies bind readily to biotinylated proteins (50% inhibitory concentration = 0.59 mumol/L) but only modestly to free biotin or biocytin. It is unlikely that any clinical consequences occur as a result of these antibodies, because the affinity for free biotin is too low (50% inhibitory concentration = 0.51 mmol/L) to affect the normal level of free plasma biotin, 0.5 nmol/L. The pathogenesis of this antibiotin immune response is unclear. Repeat testing of several individuals 5 months apart indicated that the antibiotin response was stable. In addition, 51 of the individuals tested for antibiotin antibodies were also examined for antiavidin antibodies. Whereas five were positive for antiavidin, only one individual was positive for both antibiotin and antiavidin antibodies. The presence of an antibiotin antibody is unlikely to affect the in vivo use of biotinylated proteins or cells in human subjects because its affinity for biotinylated proteins is modest and the level of biotinylation for in vivo studies is intentionally low. However, these antibodies may affect clinical or laboratory assays based on the biotin-avidin system where an antibiotin antibody may either positively or negatively affect the specific assay.

Single oral (p.o.) or intravenous (i.v.) doses of biotin were given to four cattle (400-450 kg body weight) in two consecutive tests two weeks apart. Dosages were p.o. 20, 40, 80 or 160 and i.v. 5, 10, 20, 40 mg biotin per 300 kg body weight. A three-compartment model was used to describe the course of serum concentrations with time. After i.v. administration, terminal half-lives of about 8 h were found. Areas under the curves were linearly related to both the p.o. and the i.v. doses. The systemically available fraction of the p.o. dose was 50 to 60%. On the basis of kinetic parameters, the biotin uptake via the feed was estimated to be 2.5 mg/day, which was about half of that estimated to be in the hay consumed. The data suggest that there was no relevant ruminal synthesis of biotin.