Biotin in urine

Biotin is widely distributed in many foods. It is synthesized by intestinal flora, and in balance studies the total output of biotin in urine plus faeces is three- to sixfold greater than the intake, reflecting bacterial synthesis. It is not known to what extent this is available to the host (see also Problem 10.4). 

A variety of biotin-requiring microorganisms have been used to assay biotin Saccharomyces cerevisiae (H6), Lactobacillus casei (S2), Lactobacillus arabinosus (now L. plantarum ATCC No. 8014) (W14), Micrococcus sodonensis (Al), Neurospora crassa (H10), and Rhizobium tri-folii (W7). None have been applied successfully for assaying biotin in biologic fluids. Because the flagellate Ochromoms danica had a specific and sensitive biotin requirement (A2), it was utilized as a reagent for biotin in blood, serum, urine, brain, and liver tissue (B3b). 

Urine was diluted 1 40 with buffer without enzyme. Biotin is free in urine, making enzymatic hydrolysis unnecessary. The solution was... 

Figure 12-2. Metabolic pathways involving the four biotin-dependent carboxylases. The solid rectangular blocks indicate the locations of the enzymes ACC, acetyl-CoA carboxylase PMCC, P-methylcrotonyl-CoA carboxylase PC, pyruvate carboxylase PCC, propionyl-CoA carboxylase. Isolated deficiencies of the first three carboxylases (mitochondrial) have been established (isolated ACC deficiency has not been confirmed). At least the activities of the three mitochondrial carboxylases can be secondarily deficient in the untreated multiple carboxylase deficiencies, biotin holocarboxylase synthetase deficiency and biotinidase deficiency. Lowercase characters indicate metabolites that are frequently found at elevated concentrations in urine of children with both multiple carboxylase deficiencies. The isolated deficiencies have elevations of those metabolites directly related to their respective enzyme deficiency.

The affected infants have a normal plasma concentration of biotin and excrete normal amounts of biotin in the urine. Skin fibroblasts have extremely low activities of aU four biotin-dependent carboxylases when they are cultured in media containing approximately physiological concentrations of biotin. But, culture with considerably higher concentrations of biotin results in normal activity of aU four carboxylases. The defect is in the affinity of holocarboxylase synthetase for biotin (its is 20- to 70-fold higher than normal). 

Biotin forms part of several enzyme systems and is necessary for normal growth and body function. Biotin functions as a cofactor for enzymes involved in carbon dioxide fixation and transfer. These reactions are important in the metaboHsm of carbohydrates, fats, and proteins, as well as promotion of the synthesis and formation of nicotinic acid, fatty acids, glycogen, and amino acids (5—7). Biotin is absorbed unchanged in the upper part of the small intestine and distributed to all tissues. Highest concentrations are found in the Hver and kidneys. Little information is available on the transport and storage of biotin in humans or animals. A biotin level in urine of approximately 160 nmol/24 h or 70 nmol/L, and a circulating level in blood, plasma, or serum of approximately 1500 pmol/L seems to indicate an adequate supply of biotin for humans. However, reported levels for biotin in the blood and urine vary widely and are not a reHable indicator of nutritional status. 

Unidentified biotin metabolites were analyzed and identified in urine from healthy adults by TLC. Three unknown biotin metabolites were identified as... 

Schrijver J, van Breederode N, van ben Berg H, Bitsch R. Biotin in whole blood by microbiological assay biotin in plasma or urine by RIA. In Fidanza F, ed. Nutritional status assessment A manual for population studies. London Chapman 8c Hall, 1991 296-303. 

Unidentified biotin metabolites were analyzed and identified in urine from healthy adults by TLC. " Three unknown biotin metabolites were identified as biotin sul-fone (Rf values 0.49 and 0.17), bisnorbiotin methyl ketone (Rf values 0.78 and 0.29), and tetranorbiotin-/-sulfoxide (/ f values 0.22 and 0.01) by derivatization with -demethylaminocinnamaldehyde after TLC on microcellulose with 1-butanol-acetic acid-water (4 1 1) and 1-butanol as solvents, respectively." ... 

Reduced activities of carboxylase enzymes can cause a metabolic block of certain substrates and a use of alternative pathways for catabolism. Therefore, 3-hydroxyisovaleric acid and 3-methylcrotonyl glycine are formed consequently to a shunt of 3-methylcrotonyl carboxylase counterbalancing its activity decrease. Marginal biotin deficiency experimentally induced by 20 days of free biotin diets in human increased 3-hydroxyisovaleric acid excretion in urine above the upper limit of normal. The normal urinary excretion of 3-hydroxyisovaleric acid in healthy adults is 112 38 pmol per 24 hours (Mock et al. 1997). This suggests that 3-hydroxyisovaleric acid urinary excretion is a good indicator of marginal biotin deficiency. 

Biotin deficiency causes disturbances in a variety of carboxylase-mediated metabolic reactions. As a result, such a deficiency may induce ketolactic acidosis and organic aciduria (Zempleni et al. 2008). Organic acids such as 3-methylcrotonylglycine, 3-hydroxyvaleric acid or methylcitric acid are excreted in urine in case of biotin deficiency (Figure 43.2). 

The percent of the represented by bisnorbiotin increased relative to biotin as the total radioactivity in urine decreased exponentially with time. Aeration of homogenates expectedly increased the amount of sulfoxides. Also, trace amounts of C02 and [ C]urea were de-... 

Bioassays are generally sensitive enough to measure biotin in blood and urine. However, the bacterial bioassays (and perhaps the eukaryotic bioassays as well) suffer interference from unrelated substances and variable growth response to biotin analogues. Bioassays give conflicting results if biotin is bound to protein. 

Biocytin is e-N-biotinyl-L-lysine, a derivative of D-biotin containing a lysine group coupled at its e-amino side chain to the valeric acid carboxylate. It is a naturally occurring complex of biotin that is typically found in serum and urine, and probably represents breakdown products of recycling biotinylated proteins. The enzyme biotinidase specifically cleaves the lysine residue and releases the biotin component from biocytin (Ebrahim and Dakshinamurti, 1986, 1987). 

Vitamins are chemically unrelated organic compounds that cannot be synthesized by humans and, therefore, must must be supplied by the diet. Nine vitamins (folic acid, cobalamin, ascorbic acid, pyridoxine, thiamine, niacin, riboflavin, biotin, and pantothenic acid) are classified as water-soluble, whereas four vitamins (vitamins A, D, K, and E) are termed fat-soluble (Figure 28.1). Vitamins are required to perform specific cellular functions, for example, many of the water-soluble vitamins are precursors of coenzymes for the enzymes of intermediary metabolism. In contrast to the water-soluble vitamins, only one fat soluble vitamin (vitamin K) has a coenzyme function. These vitamins are released, absorbed, and transported with the fat of the diet. They are not readily excreted in the urine, and significant quantities are stored in Die liver and adipose tissue. In fact, consumption of vitamins A and D in exoess of the recommended dietary allowances can lead to accumulation of toxic quantities of these compounds. 

The organic acid analysis in the urine of this child was consistent with biotin deficiency or multiple carboxylase deficiency. Biotin deficiency usually can be excluded unless there is a history of dietary indiscretion, such as consuming a diet containing raw eggs or few biotin-containing foods, or there is a history of prolonged parenteral hyperalimentation without biotin supplementation. Low serum biotin concentrations can be useful in differentiating... 

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