Avidin-binding Substances Research Articles

Biotin Accounts for Only Half of the Total Avidin-Binding Substances in Human Serum

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 avidinbinding substances detected. To provide a range of values for serum concentrations of biotin, bisnorbiotin, and biotin sulfoxide, HPLC was used to separate avidinbinding 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 avidinbinding 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.

Case Study: Concentrations of biotin in blood of Holstein bull calves supplemented with biotin and pantothenic acid

<h2>ABSTRACT</h2> Pantothenic acid interferes with biotin absorption in nonruminants. The objective of this study was to determine whether pantothenic acid affects the concentration of biotin in the blood of young calves. Weaned bull calves (n = 16) were fed ad libitum a pelleted starter including no supplemental B-vitamins and hay for 2 wk before the beginning and until the end of the experiment. Water was available ad libitum at all times. Two wk after weaning, calves were blocked by age and randomly assigned to 4 treatments according to a randomized complete block design. Treatments consisted of administering, for 14 d, a daily gelatin capsule containing no B-vitamins, 10 mg of biotin, 240 mg of pantothenic acid, and 10 mg of biotin + 240 mg of pantothenic acid. Expeller soybean meal was used as a carrier of the vitamins in the capsules. Blood samples were collected by venipuncture of the jugular vein at d 0 and 14. Concentrations of avidin-binding substances (ABS) in plasma were determined by a single-step competitive enzyme-binding assay. The concentrations of ABS in plasma were similar for all treatments (<i>P</i> > 0.24) and did not change after dosing vitamins over time (3.47 and 3.07 ng/mL for d 0 and 14, respectively; SEM=1.5 ng/mL; <i>P</i> > 0.63). Plasma concentrations of ABS were substantially greater than those previously reported for lactating dairy cows (0.7–2.1 ng/mL). Based on these data, we cannot conclude whether or not an interaction exists between biotin and pantothenic acid in regards to biotin absorption.

Plasma Levels of Biotin Metabolites Are Elevated in Hemodialysis Patients with Cramps.

Patients with renal failure undergoing hemodialysis (HD) are susceptible to muscle cramps during and after HD. Muscle cramps are defined as the sudden onset of a prolonged involuntary muscle contraction accompanied by severe pain. Through HD, water-soluble vitamins are drawn out with water. Since biotin, a water-soluble vitamin, plays an essential role as one of the coenzymes in producing energy, we have hypothesized that deficiency of biotin may be responsible for HD-associated cramps. We previously reported that biotin administration ameliorated the muscle cramps, despite the elevated plasma biotin levels before HD and biotin administration, as judged by an enzyme-linked immunosorbent assay (ELISA). However, the ELISA measures not only biotin but also total avidin-binding substances (TABS) including biotin metabolites. In the present study, we determined biotin in HD patients as well as healthy controls, using a newly developed method with ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). The plasma samples were collected from 28 HD patients (16 patients with cramps and 12 patients without cramps) before HD and biotin administration and from 11 controls. The results showed that the accumulation of biotin and TABS in plasma of HD patients compared to controls. Importantly, the levels of biotin metabolites, i.e. TABS subtracted by biotin, increased significantly in patients with cramps over those without cramps. Moreover, the levels of biotin metabolites were significantly higher in patients with a poor response to administered biotin, compared to those with a good response. We propose that accumulated biotin metabolites impair biotin's functions as a coenzyme.

Effect of biotin and pantothenic acid on performance and concentrations of avidin-binding substances in blood and milk of lactating dairy cows

We hypothesized that pantothenic acid reduces the absorption of biotin in lactating dairy cows. Therefore, the objective of this study was to evaluate the plausible interaction between biotin and pantothenic acid on production performance and concentration of avidin-binding substances (ABS), an indicator of biotin concentration, in blood and milk of lactating dairy cows. Eight primiparous and 16 multiparous Holstein cows were assigned to 1 of 4 diet sequences in a replicated 4×4 Latin square design with 18-d periods. Cows were housed in a freestall barn and fed once daily (0730 h) by means of a Calan gate system (American Calan Inc., Northwood, NH). Treatments consisted of a control diet that contained no B-vitamins, a biotin diet that contained 0.87 mg of biotin per kilogram of dry matter (DM), a pantothenic acid diet that contained 21 mg of pantothenic acid per kilogram of DM, and a biotin plus pantothenic acid diet that contained 0.87 mg of biotin and 21 mg of calcium pantothenic acid per kilogram of DM. Four different concentrates were prepared in a commercial feed mill. These concentrates were mixed with corn silage and grass hay and delivered ad libitum as a total mixed ration. Biotin supplementation did not affect DM intake, milk yield, or milk fat, protein, lactose, and milk-urea-nitrogen concentrations. Fat, protein, and lactose yields were not affected by treatments. The fat-to-protein ratio was <1 and similar among all treatments. Biotin supplementation did not increase the concentration of ABS in plasma. The supplementation of pantothenic acid did not affect the concentration of ABS in plasma when either supplemented alone or in combination with biotin. Biotin supplementation increased the concentration of ABS in milk relative to control. Contrary to our hypothesis, the supplementation of pantothenic acid did not decrease the concentration of ABS in milk relative to the control. When cows were supplemented with both biotin and pantothenic acid, the concentration of ABS in milk was similar to that of cows supplemented with biotin alone. In conclusion, pantothenic acid did not affect the concentrations of ABS in plasma and milk, suggesting that increasing dietary supply of pantothenic acid did not inhibit biotin absorption.

Biotin accounts for less than half of all biotin and biotin metabolites in the cerebrospinal fluid of children

Background:Biotin is likely transported into cerebral spinal fluid (CSF) via one or more specific transporters. Concentrations of biotin in CSF measured by using modern analytic techniques that are specific for biotin and biotin metabolites have not previously been reported. Objectives:We aimed to accurately measure the concentration of biotin and major biotin metabolites, biotin sulfoxide (BSO) and bisnorbiotin (BNB), in the CSF of children. Design:Concentrations of biotin were determined initially as total avidin-binding substances (TABS) in CSF obtained by lumbar puncture from 55 children. Biotin, BSO, and BNB were quantitated by HPLC and an avidin-binding assay in CSF samples from a subset of 11 children. Results:Concentrations of TABS in CSF averaged 1.6 nmol/L with substantial variability (SD = 1.3 nmol/L). CSF concentrations of biotin and biotin analogs varied widely, but substantial amounts of BSO were detected in every sample. Biotin accounted for 42 ± 16%, BSO for 41 ± 12%, and BNB for 8 ± 14% of the total. It was surprising that the molar sum of biotin, BSO, and BNB on average was >200-fold the TABS concentrations from the same CSF sample. Using several analytic approaches, we found no masking of detection, nor did we find degradation of biotin or BSO. Gel electrophoresis and streptavidin Western blot detected several biotinylated proteins in CSF. Conclusions:Biotin appears to be bound to protein covalently, reversibly, or both, and this binding likely accounts for the increase in detectable biotin after HPLC. Protein-bound biotin may play an important role in biotin nutriture of the brain.

Distribution of Biotin in Cerebrospinal Fluid of Children: Most of the Biotin is Bound to Protein

Direct measurement of total avidin binding substances (TABS) in CSF underestimates the amount of true biotin as measured by HPLC/avidin‐binding assays. We investigated the mechanism of the underestimation as follows: 1) To evaluate whether detection of free biotin was masked by an unknown CSF component that blocks binding to avidin, biotin (independently quantified as 3H‐biotin) was added to CSF. TABS assay detected 100% of the added biotin, providing evidence against masking. 2) To determine if in vitro conversion of biotin to metabolites confounded detection, 3H‐biotin was incubated for 72 h in CSF at 4°C. No conversion of 3H‐biotin to biotin metabolites was observed by HPLC, providing evidence against conversion. 3) To evaluate the possibility that biotin is bound to a macromolecule and released during HPLC, CSF was acid hydrolyzed. Detectable biotin increased approximately 30 fold, providing evidence in favor of this mechanism. The 30 fold increase accounts for most, but not all, of the 90 fold increase observed for this sample after HPLC. Biotin‐binding proteins in CSF were further characterized by SDS‐PAGE, western blot, and streptavidin detection. Three bands were detected. These studies provide evidence that reversible and/or covalent binding to proteins causes substantial underestimation of CSF biotin. We speculate that this phenomenon may have confounded previous studies. NIH DK 36823

Changes in Measures of Biotin Status Do Not Reflect Milk Yield Responses When Dairy Cows Are Fed Supplemental Biotin

A sensitive indicator of biotin status for lactating dairy cows is necessary to understand factors that affect milk yield responses to biotin supplementation. 3-Hydroxyisovaleric acid (3HIA) is an alternative metabolite in the pathway of Leu catabolism when the biotin-dependent enzyme methylcrotonyl-coenzyme A carboxylase is limiting. We evaluated urinary excretion of 3HIA as a determinant of biotin status in lactating dairy cows. We hypothesized that high-producing cows would have a greater biotin requirement and excrete more 3HIA than low-producing cows and that biotin supplementation would decrease 3HIA excretion. Twenty high-producing and 20 low-producing Holstein cows (43±5 and 23±4 kg/d of milk, respectively) were fed diets that contained either 0 or 0.96 mg/kg of supplemental biotin. On d 16 cows were given an intraruminal infusion of 1.4mol of isovaleric acid and urine was sampled. Biotin supplementation did not affect basal urinary excretion of 3HIA. The infusion of isovaleric acid increased urinary excretion of 3HIA (maximum at 8h after infusion), but biotin supplementation did not attenuate this increase. The increase in urinary 3HIA excretion was less for low-producing cows than for high-producing cows. Biotin increased yields of milk and milk components in high-producing cows but had no effect in low-producing cows. However, potential measures of biotin status (concentrations of avidin-binding substances in the plasma, milk, and urine, and urinary 3HIA excretion) responded similarly to biotin supplementation for both high- and low-producing cows. A sensitive indicator of biotin status for lactating dairy cows is still needed.

Balance and serum concentration of biotin in sheep fed alfalfa meal-based diets with increasing level of concentrate.

The objective of this study was to test the hypothesis that serum biotin concentration and biotin balance (consumed - [urinary output + fecal output]) measured as total avidin-binding substances (biotin + biotin metabolites) are responsive to changes in the proportions of dietary alfalfa meal and concentrate fed to sheep. Eight sheep (initial BW = 40 kg) consumed a pelleted alfalfa meal-based diet that had 95:5, 48:52, 23:77, or 9:91% alfalfa meal:concentrate ratios (DM basis) in a replicated 4 x 4 Latin square design with 20-d periods (10 d of acclimation, 7 d of adaptation, and a 3-d collection period with jugular blood drawn on the last day). Replacing alfalfa meal with concentrate in the pelleted diets decreased dietary concentrations of biotin proportionally. As the percentage of alfalfa meal in the diet decreased, there was a linear decrease in daily DM intake (1,128 to 901 g of DMI/d; P < 0.01), with a linear (P < 0.01) and quadratic (P < 0.01) increase in the apparent total-tract DM digestibility of diets (51.0 to 80.0%). The biotin consumed decreased with alfalfa meal proportion in the diet (linear, P < 0.01). Both fecal biotin concentration (linear, P < 0.01) and fecal biotin output (quadratic, P < 0.05) increased, reaching peaks at 23% alfalfa meal. Fecal biotin output was not correlated with biotin intake, DMI, or intake of digestible DM. Mean urinary output, urinary biotin concentration, urinary biotin output, and serum biotin concentration were not affected by treatments. Means of biotin balance were negative and revealed the same trends among treatments as did fecal output. Biotin balance was a quadratic (P < 0.05) function of decreasing alfalfa meal in the diet, with more negative values at the alfalfa meal:concentrate ratio of 23:77. Results suggest that the greatest synthesis of biotin in the total digestive tract occurs with diets of either 52 or 77% concentrate for sheep; however, research addressing the significance of biotin metabolites on biotin balance and plasma biotin pool is needed.

Identification of biotin sulfone, bisnorbiotin methyl ketone, and tetranorbiotin-l-sulfoxide in human urine

In previous studies using the HPLC and avidin-binding assay, five unidentified avidin-binding substances were observed in human urine. The present study investigated the identity of these substances. Urine was collected before and after intravenous administration of 18.5 mumol biotin to healthy adults. Unknown substances 1 and 3 were initially identified as biotin sulfone and bisnorbiotin methyl ketone, respectively, by coelution with authentic standards on HPLC. Identities were confirmed by thin-layer chromatography and by derivatization with p-dimethyl-aminocinnamaldehyde. As expected for biotin metabolites, the urinary excretion of biotin sulfone and bisnorbiotin methyl ketone increased with biotin administration. The urinary excretion of biotin sulfone increased 21-fold from 0.2 nmol/h before to 4.2 nmol/h after administration; the excretion of bisnorbiotin methyl ketone increased 130-fold from 0.4 to 51.8 nmol/h. At presumed steady state in free-living subjects (n = 6), biotin sulfone and bisnorbiotin methyl ketone accounted for 3.6% and 7.9% of total biotin excretion, respectively. Traces of tetranorbiotin-l-sulfoxide were also identified by using thin-layer chromatography and derivatization with p-dimethylaminocinnamaldehyde. However, tetranorbiotin-l-sulfoxide was not detectable in urine by the HPLC and avidin-binding assay because this metabolite has weak avidin-binding affinity. We conclude that biotin sulfone and bisnorbiotin methyl ketone are present in measurable quantities in human urine; their quantitation should allow more accurate studies on human biotin metabolism and turnover.

Biotin and Biotin Analogs in Human Urine: Biotin Accounts for only Half of the Total

In studies using avidin-binding assays to measure the urinary excretion of biotin, biotin is sometimes assumed to be equal to the detectable avidin-binding substances present. High performance liquid chromatography was used to separate avidin-binding substances in human urine, and the chromatographic fractions were assayed for avidin-binding substances (biotin and biotin analogs) by a sensitive, specific assay based on binding of biotin to [125I]avidin. In a study of ten normal adults, substantial amounts of avidin-binding substances other than biotin were detected, two of which were bisnorbiotin and biotin sulfoxide. These biotin analogs were initially identified by their chromatographic properties, and identities were confirmed by chemical conversion. The presence of avidin-binding substances in addition to biotin may have confounded previous measurements of the urinary excretion of biotin using avidin-binding assays. Because bioassay methods for biotin often use organisms for which one or more of these biotin analogs are growth factors, measurements of biotin in urine using some bioassay methods are likely to overestimate the concentrations of biotin.

Biotin Analysis of Commercial Vitamin and Other Nutritional Supplements

Because previous observations suggest that biotin may be present in vitamin preparations not labeled as containing biotin, we determined the biotin content of several over-the-counter vitamin and nutritional supplements said to contain biotin (group 1) and several in which biotin content was not specified (group 2). Biotin concentration was measured using the 125I-avidin assay which detects total avidin-binding substances. Water extracts were assayed for free biotin and acid hydrolysates were assayed for total biotin. The results of the 125I-avidin assay agreed with the stated biotin content for most vitamin and nutritional supplements in group 1. Biotin was the only avidin-binding substance in the preparations from group 1, based on reversed-phase chromatography. However, some vitamin and nutritional supplements in group 2 contained nutritionally significant amounts of biotin, particularly if the supplement contained liver or yeast extract. Total biotin was significantly higher than free biotin in one supplement; the difference was attributable to release of biotin rather than a biotin analog. We conclude that biotin may be present in some vitamin and nutritional supplements not labeled as containing biotin; biotin intake might be under-estimated if the subject is receiving a nutritional supplement containing extracts of liver or yeast.

Biotin nutritional status of vegans, lactoovovegetarians, and nonvegetarians

Urinary excretion of biotin (total avidin-binding substances) was measured in adults and children who were adhering to one of the following self-selected diets: strict vegetarian (vegan), lactoovovegetarian, or mixed (containing meat and dairy products as well as plant-derived foods). In a subset of subjects, plasma biotin concentrations were also measured. In adults the biotin excretion rate was significantly greater in the vegan group than in either the lactoovovegetarian or the mixed-diet groups; the latter were not significantly different from one another. In children the biotin excretion rates in both the vegan group and the lactoovovegetarin group were significantly greater than in the mixed-diet group. A similar trend (vegan greater than lactoovovegetarian greater than mixed) was detected in the plasma concentrations of biotin of adults and children but differences were not generally statistically significant. These observations provide evidence that the biotin nutritional status of vegans is not impaired.