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Biomedical subjects

D M Mock

Publications and source records attributed to D M Mock.

At least 19 recordsLinked to original sources
PubMed1992

Rayon balls and disposable-diaper material selectively adsorb creatinine.

One method for the collection of urine samples from infants involves absorption of the urine on cotton balls placed in the diaper. Such samples are not timed and excretions are often expressed per mg of urinary creatinine. An assumption in this method is that the creatinine concentration is not changed by the absorption process. The concentration of creatinine in urine samples was measured before and after absorption of the urine by cotton balls, rayon balls, or diaper material over a range of wetness. For urine from both adults and infants, absorption on rayon balls and diaper material caused an important artifactual decrease in the concentration of creatinine. The effect was particularly striking in lightly wetted samples; the mean percent decrease was only 3 +/- 2% for cotton but was 9 +/- 5% for rayon (n = 10) and 13 +/- 4% for diaper material (n = 7). These data provide evidence that rayon balls and diaper material (and to a lesser extent cotton balls) selectively adsorb creatinine from human urine.

Adsorption↗
PubMed1992

Distribution of biotin in human plasma: most of the biotin is not bound to protein.

Estimates of the plasma concentration of biotin differ considerably. Variation in detectability of biotin bound covalently to protein is one potential source of disagreement. In this study we determined the amount of biotin covalently bound to plasma protein. First, greater than 99% of free and reversibly bound biotin was removed by dialysis; then greater than 90% of covalently bound biotin was released by acid hydrolysis. For plasma samples from 11 normal adults, the ratio of covalently bound biotin to free biotin was 0.15 +/- 0.09 (mean +/- SD). Taking into account the additional biotin that is reversibly bound to protein, this study provides evidence that approximately 12% of total biotin in plasma is covalently bound, 7% is reversibly bound, and 81% is free. We conclude that covalently bound biotin cannot account for the reported sixfold increase in biotin detected after acid hydrolysis. We speculate that the reported increase was an artifact caused by substances produced during acid hydrolysis of plasma.

Adult↗
PubMed1992

Biotin in human milk: methods, location, and chemical form.

For infants, no Recommended Dietary Allowance for biotin has been published; the estimated safe and adequate intake seems to be based on measurements of human milk. However, published estimates of the biotin content disagree substantially. We sought to address several of the potential sources of disagreement by defining the conditions for the collection, storage, and subcompartment distribution of biotin in human milk using the [125I]avidin assay. Composition of the collection vessel (glass vs. common plastics) had no effect on biotin content of human milk. The biotin content of milk did not change during storage at room temperature for at least 1 wk, at 5 degrees C for at least 1 mo, or at -20 degrees C or -70 degrees C for at least 1.5 y. Biotin in the cell pellet and fat fraction accounted for less than 5% of that in the skim fraction. Of the biotin in the skim fraction, none (less than 3%) was reversibly bound to macromolecules, and less than 5% was covalently bound to macromolecules. We conclude that assay of free biotin will detect almost all of the biotin present in a sample of mature human milk.

Adult↗
PubMed1992

Secretory patterns of biotin in human milk.

The current recommendation for safe and adequate daily dietary intake of biotin for infants is based on measurements of biotin concentration in human milk and calculations of biotin intake that tacitly assume that biotin content of human milk is reasonably uniform for a given subject. This assumption of uniformity was tested by examining the effects of several factors on the biotin concentration. The degree of breast emptying had little effect on biotin concentration. However, in three of five individuals studied, the biotin concentration varied significantly over 24 h. In two of five subjects, there was a consistent difference between breasts of approximately 16%. In the first 18 d postpartum, the milk concentration of biotin increased steadily in four of the eight individuals studied, remained low in two and increased erratically in two. Rather than reaching a stable plateau in mature milk, biotin concentration varied substantially in most of the subjects. These observations provide evidence that an adequate scheme for estimating total biotin intake of the breastfed infant will require sampling from both breasts frequently over the 24-h cycle and frequently as a function of time postpartum.

Analysis of Variance↗
PubMed1992

Biotin deficiency in rats: disturbances of leucine metabolism are detectable early.

3-Methylcrotonyl-CoA originates from catabolism of leucine and is normally metabolized to acetyl-CoA. However, in biotin deficiency, reduced hepatic activity of the biotin-dependent enzyme methylcrotonyl-CoA carboxylase causes the enzyme's substrate 3-methylcrotonyl-CoA to be shunted via an alternate pathway to 3-hydroxyisovaleric acid (3-HIA), which is excreted at increased rates in the urine. In a previous study, unequivocal separation in 3-HIA excretion rates between biotin-deficient and control animals was not apparent until d 35 of feeding a diet that induced biotin deficiency. The present study tested the hypothesis that abnormal 3-HIA excretion could be detected earlier in the course of biotin deficiency if 3-HIA were more accurately measured using a method that incorporated an improved extraction regimen, deuterated 3-HIA as internal standard, and unlabeled 3-HIA as external standard. Biotin deficiency was induced in rats by feeding a diet containing avidin; control rats received the same diet and biotin injections. With the more accurate method, unequivocal detection of deficiency was possible in all deficient rats by d 16. This study provides evidence that, in rats, reduction of analytical error allows earlier detection of biotin deficiency and that disturbances of leucine metabolism occur earlier than previously appreciated.

Animals↗
PubMed1991

Skin manifestations of biotin deficiency.

This article reviews current knowledge concerning the dermatologic manifestations of biotin deficiency. Biotin is a water-soluble vitamin that acts as an essential cofactor for four carboxylases, each of which catalyzes an essential step in intermediary metabolism. For example, acetyl-CoA carboxylase catalyzes the rate-limiting step in fatty acid elongation. In infants, children, and adults, deficiency of biotin causes alopecia and a characteristic scaly, erythematous dermatitis distributed around body orifices. The rash closely resembles that of zinc deficiency. Candida albicans often can be cultured from the skin lesions. Biotinidase deficiency, an inborn error, causes biotin deficiency, probably as a consequence of unpaired intestinal absorption, cellular salvage, and renal reclamation of biotin; biotinidase deficiency causes dermatologic manifestations similar to biotin deficiency. There is evidence that impaired fatty acid metabolism secondary to reduced activities of the biotin-dependent carboxylases (especially acetyl-CoA carboxylase) plays an etiologic role in the dermatologic manifestations of biotin deficiency. Candida infections secondary to impaired immune function might also contribute to the dermatitis of biotin deficiency.

Adult↗
PubMed1990

Effect of aging on intestinal biotin transport in the rat.

The influence of aging of biotin intestinal transport was examined in Fisher 344 rats (3- 12- and 24-month old) using a brush border membrane vesicle (BBMV) technique. In all age groups examined, transport of biotin as a function of concentration was saturable in the presence of a Na+ gradient (out greater than in), but was lower and linear in the presence of a choline gradient (out greater than in.). The Vmax of the biotin transport process was found to be significantly (p less than 0.01) higher in 24-month-old rats compared to 3- and 12-month old rats. On the other hand, the apparent Km of the biotin transport system was similar in all age groups. We also measured plasma levels of biotin in the different age groups. The mean plasma levels of biotin in 24-month-old rats was found to be significantly higher than that of 3-month (p less than 0.005) and 12-month (p less than 0.025) old rats. These results demonstrate that aging is associated with an increase in biotin intestinal transport. This increase appears to be due to changes in the activity (and/or number) but not the affinity of the biotin transport system. Furthermore, an increase in plasma biotin levels was observed with aging, which might be a consequence to the increase in the vitamin's intestinal transport.

Aging↗
PubMed1990

Studies of the reversible binding of biotin to human plasma.

In this study we sought to determine the extent of the reversible binding of [3H]biotin to macromolecules (presumably proteins) of human plasma under physiologic conditions during physiologic time intervals ranging from minutes to days. Fresh, heparinized human plasma and [3H]biotin were incubated in 5% CO2, 95% N2 at 37 degrees C. After reaching equilibrium, free biotin was separated from bound biotin by centrifugal ultrafiltration (1500 x g for 60 min) using membranes with a molecular weight cutoff of 30,000. In a sample from a single individual, we detected a mean retentate:ultrafiltrate ratio of 1.20 +/- 0.03 (mean +/- SD, n = 14 determinations). In a group of 10 healthy adults, the mean retentate:ultrafiltrate ratio was 1.17 +/- 0.02; thus, approximately 8% of the biotin in the combined pool of free and reversibly bound biotin is bound. No consistent change in retentate:ultrafiltrate ratio was detected for total biotin concentrations ranging between 500 fmol/mL and 2 nmol/mL, but the ratio decreased to 1.1 at 400 nmol of biotin per mL. Thus, the biotin-binding system of human plasma appears to be a low affinity, high capacity system. A similar biotin-binding system was detected in experiments with purified human serum albumin. These results provide evidence that the majority of added [3H]biotin does not bind to plasma protein under physiologic conditions. To the extent that added [3H]biotin can be assumed to reflect reversible binding of endogenous unlabeled biotin, we infer that endogenous, reversibly bound biotin can account for no more than one-tenth of the increase in biotin detected after acid hydrolysis of plasma.

Adult↗
PubMed1990

Evidence for a pathogenic role of omega 6 polyunsaturated fatty acid in the cutaneous manifestations of biotin deficiency.

Abnormalities in fatty acid composition have been detected in biotin deficiency in humans and in the rat. However, the pathogenetic role of these abnormalities, if any, in the cutaneous manifestations of biotin deficiency is not clear. In a nutrient interaction experiment, we tested the hypothesis that an abnormality of omega 6 polyunsaturated fatty acid (PUFA) metabolism plays a pathogenetic role in the cutaneous manifestations. Five rats were fed an egg white diet that induces biotin deficiency; these rats developed the characteristic cutaneous abnormalities of biotin deficiency. Five additional rats were pair-fed the egg white diet and received Liposyn [77% linoleic acid (18:2 omega 6)]; these rats did not develop the cutaneous manifestations of biotin deficiency. The two groups had similar decreases in the rates of urinary excretion of biotin, the serum concentrations of biotin, the amounts of biotin in liver, and the hepatic activities of two biotin-dependent carboxylases; the two groups had similar increases in the rates of urinary excretion of 3-hydroxyisovaleric acid. These observations provide evidence that (a) some abnormality in omega 6 PUFA metabolism has a pathogenetic role in the cutaneous manifestations of biotin deficiency and (b) this pathogenetic role can not be explained as a difference in biotin nutritional status due to a biotin-sparing effect of Liposyn.

Animals↗
PubMed1989

Quantification of urinary 3-hydroxyisovaleric acid using deuterated 3-hydroxyisovaleric acid as internal standard.

Deficiency of biotin at the tissue level can be assessed indirectly by measuring the urinary excretion of 3-hydroxyisovaleric acid. This paper describes the application of an improved method of quantifying urinary 3-hydroxyisovaleric acid using unlabeled and uniformly deuterated 3-hydroxyisovaleric acid. These compounds were synthesized by a modification of the lithioacetic acid method for generation of beta-hydroxy acids. Elemental analysis, nuclear magnetic resonance, gas chromatographic and gas chromatographic/mass spectrometric data demonstrated that the compounds are greater than 95% pure. Mass spectrometry confirmed the identity of the unlabeled compound, demonstrated that the deuterated compound is uniformly labeled, and offered insight into the pattern of mass fragmentation. The method for determination of the concentration of 3-hydroxyisovaleric acid in rat urine uses gas chromatographic/mass spectrometric quantification of the di-trimethylsilyl derivative with the deuterated compound as the internal standard. Results provide evidence that this method is more accurate than a previously published method that did not utilize the unlabeled and deuterated standards.

Animals↗
PubMed1989

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.

Adult↗
PubMed1989

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.

Acids↗
PubMed1989

Regulation of intestinal biotin transport in the rat: effect of biotin deficiency and supplementation.

The effect of biotin deficiency and supplementation at pharmacological doses on the intestinal transport of the vitamin was examined in the rat using a brush-border membrane vesicle (BBMV) technique. Transport of biotin in both jejunal and ileal BBMV was significantly (P less than 0.05-0.01) higher in biotin-deficient rats compared with control (pair-fed) rats. This increase in biotin transport appeared to be specific because transport of D-glucose was similar in the two rat groups. The increase in biotin transport in the deficient rats appeared to be mediated through a marked increase (146-230%) in the Vmax of the biotin transport process (with minimal change in the apparent Km), suggesting an increase in the number of the transport carriers. In contrast, supplementation at pharmacological doses of biotin caused significant (P less than 0.05-0.01) and specific decrease (suppression) in biotin transport compared with (unsupplemented) controls. The suppression of biotin transport in the supplemented rats appeared to be mediated through a marked decrease (58%) in the Vmax of the biotin transport process (with minimal change in the apparent Km), suggesting a decrease in the number of the transport carriers. These results provide evidence that biotin transport in the intestine is regulated by the level of the vitamin in the diet (and/or body stores). Furthermore, the results demonstrate the ability of the small intestine to adapt to the challenge of deficiency of an essential nutrient, a capability that may be crucial for the survival of the animal.

Animals↗
PubMed1988

Isolation and characterization of highly purified streptavidin obtained in a two-step purification procedure from Streptomyces avidinii grown in a synthetic medium.

A method is described for isolation of streptavidin from cultures of Streptomyces avidinii grown in a synthetic culture medium for 6-10 days. Streptavidin is precipitated directly from culture supernatant fluid using 80% ammonium sulfate, and the precipitate is dialyzed against water and centrifuged at 40,000 X g for 60 min. The absorbency coefficient at 280 nm of purified streptavidin was estimated to be 31.7142 +/- 0.1806 for a 1% solution. The protein appeared to be greater than 90% homogeneous by gel permeation chromatography and polyacrylamide gel electrophoresis. No biotin-binding molecules less than 70 kDa in size were detected at any step during the purification of streptavidin. Streptavidin was able to maintain a stable crosslink between two biotinylated molecules in a solid-phase assay. Streptavidin purified by this method was stable in 50% glycerol/water at -20 degrees C for more than 1 year. Lyophilization or iodination did not produce apparent damage to the protein.

Bacterial Proteins↗
PubMed1988

A study of the interaction of avidin with 2-anilinonaphthalene-6-sulfonic acid as a probe of the biotin binding site.

The environment of the biotin binding site on avidin was investigated by determining the fluorescence enhancement of a series of fluorescent probes that are anilinonaphthalene sulfonic acid derivatives. Of the compounds tested, 2-anilinonaphthalene-6-sulfonic acid (2,6-ANS) exhibited the greatest enhancement under the conditions used (which would reflect both molar fluorescence enhancement and binding affinity) and exhibited more than 95% reversal upon addition of biotin. Thus, 2,6-ANS was chosen for more detailed characterization of the interaction with avidin. Only a single class of binding sites for 2,6-ANS was identified; the mean value for the Kd was 203 +/- 16 microM (X +/- 1 S.D.), and the molar ratio of 2,6-ANS binding sites to biotin binding sites was approx. 1. These results provide evidence that the biotin binding site and the 2,6-ANS binding site are at least partially overlapping, but the possibility that the probe binding site is altered by a conformational change induced by biotin binding cannot be excluded. At excitation = 328 nm and emission = 408 nm, the molar fluorescence of the bound probe was 6.8 +/- 1.0 microM-1 and that of the free probe was 0.061 +/- 0.008 microM-1 giving an enhancement ratio (molar fluorescence of bound probe/molar fluorescence of free probe) of 111 +/- 22. Upon binding, the wavelength of maximum fluorescence decreases. These findings also provide evidence that the fluorescence enhancement associated with the interaction of 2,6-ANS and avidin reflects the environment of the biotin binding site. The Kosower's Z factor, an empirical index of apolarity, was 82.1 for the 2,6-ANS binding site on avidin. This value reflects a degree of apolarity that is similar to apolar environments observed for substrate binding sites on several enzymes; although not the dominant factor, this environment may contribute to the strong binding of biotin.

Anilino Naphthalenesulfonates↗
PubMed1988

Biotin transport and metabolism in the central nervous system.

The mechanisms by which biotin enters and leaves brain, choroid plexus and cerebrospinal fluid (CSF) were investigated by injecting [3H]biotin either intravenously or intraventricularly into adult rabbits. [3H] biotin, either alone or together with unlabeled biotin was infused at a constant rate into conscious rabbits. At 180 minutes, [3H]biotin had entered CSF, choroid plexus, and brain. In brain, CSF, and plasma, greater than 90% of the nonvolatile 3H was associated with [3H]biotin. The addition of 400 mumol/kg unlabeled biotin to the infusion syringe decreased the penetration of [3H]biotin into brain and CSF by approximately 70 percent. Two hours after an intraventricular injection, [3H]biotin was cleared from the CSF more rapidly than mannitol and minimal metabolism of the [3H]biotin had occurred in brain. However, 18 hours after an intraventricular injection, approximately 35% of the [3H]biotin remaining in brain had been covalently incorporated into proteins, presumably into carboxylase apoenzymes. These results show that biotin enters CSF and brain by saturable transport systems that do not depend on metabolism of the biotin. However, [3H]biotin is very slowly incorporated covalently into proteins in brain in vivo.

Animals↗