Biotin determination

The literature contains very little on methods for biotin determination in foods. Microbiological assays of biotin predominate in food work (19,180). Table 23 summarizes a recent HPLC method for the analysis of biotin in infant formula. 

Methylcobalamin, 516 5-Melhyl-cytosine, deaminatitm, 894 Methylglyoxal, 836 Methylmalonic acid (MMA), 434, 522 MethylmalcHiy. CoA, 434, 517, 518 Mevalonic acid, 327, 328 Mg-ATP complex, 795-796 Micelles, 25,27-29 MLcroaulophagy, 444 Microbiological assays, 508 biotin determination, 541 folate status by, 509 thiamin status, 607 Microcytic anemia, 5H Microsomal ethanol-oxidizing system, 247 Microvilli, 58 Milk... 

Ion chromatography (IC) using Dowex 1X2 has played an important role in biotin determinations of the past, being an essential component of biotin discovery and purification. Nowadays, high-performance IC platforms with preparative columns have replaced earlier techniques and remain an important... 

The usual methods for biotin determination are based on the biochemical properties of the vitamin and do not involve chromatographic separation. Depending on whether avidin is used, they can be classified into two categories. 

This technique has been developed by Aizawa and coworkers. The goal is to build a convenient and specific detector using an enzymatic activity as signal. In the case of biotin determination, avidin coupled with catalase is bound to a membrane bearing covalently linked HABA residues. Addition of biotin destroys quantitatively the HABA-avidin-catalase complex. Washing the membrane and measurement of the remaining catalase activity afforded a sensitive (0.5 ng) and convenient titration of biotin (95). 

Table 2 Comparison of the Detection Limits of the Main Biotin Determination Methods... 

T Pukuii, K linuma, J Oizumi, Y Izumi. Agar plate method using Lactobacillus plantamm for biotin determination in serum and urine. J Nutr Sci Vitaminol 40 491-498, 1994. 

These methods were the first developed for biotin analysis and are still accepted as official methods because of their high sensitivity (nanogram level). The species most commonly employed for biotin determination are some Lactobacilli such as L. arabinosus (ATCC 2112), L. plantarum (ATCC 8014), and L casei (ATCC 7469) Ochromonas danica, Neurospora crassa, and Saccharomyces cerevisae (ATCC 4228) are also employed. Kloeckera brevis (ATCC 9774) is useful for a radiometric-microbiological determination based on the measurement of " C02 formed by the test microorganism from " C-labeled L-methionine. 

The structure of biotin was determined in the early 1940s by Kogl in Europe and by dn Vigneand and coworkers in the United States. Interestingly, the biotin molecule contains three asymmetric carbon atoms, and biotin could thus exist as eight different stereoisomers. Only one of these shows biological activity. 

The product of the reaction in Entry 8 was used in the synthesis of the alkaloid pseudotropine. The proper stereochemical orientation of the hydroxy group is determined by the structure of the oxazoline ring formed in the cycloaddition. Entry 9 portrays the early stages of synthesis of the biologically important molecule biotin. The reaction in Entry 10 was used to establish the carbocyclic skeleton and stereochemistry of a group of toxic indolizidine alkaloids found in dart poisons from frogs. Entry 11 involves generation of a nitrile oxide. Three other stereoisomers are possible. The observed isomer corresponds to approach from the less hindered convex face of the molecule. 

The requirements of dairy cattle for B-vitamins, determined almost half a century ago, concluded that a ruminant animal does not require an exogenous supply of B-vitamins because its rumen microflora should synthesise enough of these compounds to avoid deficiency. Since then, dairy cows have greatly increased their average milk and milk component yields. More recent studies have shown that B-vitamin supply in dairy cows is increased by supplementation, although losses in the rumen are extensive (Santschi et al., 2005). Whilst there are few reports of B-vitamin supplementation affecting milk quality, supplemental biotin has been shown to directly improve milk yield (Majee et al., 2003). 

Iodoacetyl-LC-biotin has been used to localize the SH thiol of myosin by use of an avidin-biotin complex visualized by electron microscopy (Sutoh et al., 1984) and to determine the spatial relationship between SHj and the actin binding site on the myosin subfragment-1 surface (Yamamoto et al., 1984). 

The Derivative, 5-(biotinamido)pentylamine, contains a 5-carbon cadaverine spacer group attached to the valeric acid side chain of biotin (Thermo Fisher). The compound can be used in a carbodi-imide reaction process to label carboxylate groups in proteins and other molecules, forming amide bond linkages (Chapter 3, Section 1). However, the main use of this biotinylation reagent is in the determination of factor XHIa or transglutaminase enzymes in plasma, cell, or tissue extracts. 

Reactions done with NHS-PEG -biotin compounds typically are done with the reagent in molar excess over the amount of protein being modified. The efficiency of the reaction is dependent on the concentrations of reactants and the solvent exposed area of the amine groups on the protein. Reactions done with a 10-fold molar excess of NHS-PEG -biotin usually will result in at least 2-3 biotin labels per protein, while doubling the molar excess should provide 4-6 biotinylations. The optimal number of biotin groups added to a particular protein should be determined experimentally to provide the best performance in the intended application. 

Proteins biotinylated with this reagent will have a characteristic absorbance band at 354 nm, which can be used to determine accurately the number of biotin groups per molecule. No other biotinylation compound has such built-in quantification capability. This feature eliminates the need to consume conjugate by doing a HABA assay to test for the level of biotin incorporation (Chapter 23, Section 7). 

Measure the absorbance of the biotinylated protein solution at 354 nm. Use the molar extinction coefficient for the chromogenic group (e = 29,000 M-1cm-1) to determine the concentration of biotin present. To determine the molar ratio of biotin-to-protein, divide the molar concentration of biotin by the molar concentration of protein present (which may be determined by using the Coomassie assay or the BCA assay methods). 

It is often important to determine the extent of biotin modification after a biotinylation reaction is complete. Measuring biotin incorporation into macromolecules can aid in optimizing a particular (strept)avidin-biotin assay system. It also can be used to assure reproducibility in... 

Since a biotinylated molecule potentially is able to interact with (strept)avidin at its biotin binding sites just as strongly as biotin in solution, the degree of biotinylation may be determined using the HABA method as well. Comparison of the response of a biotinylated protein, for example, with a standard curve of various biotin concentrations allows calculation of the molar ratio of biotin incorporation. 

Dissolve (strept)avidin in 0.05 M sodium phosphate, 0.15 M NaCl, pH 6.0, at a concentration of 0.5 mg/ml. A total of 3 ml of the (strept)avidin solution is required to create a standard curve using known concentrations of biotin and an additional 3 ml is needed for each sample determination. 

To measure the response of the biotinylated protein sample, add 3 ml of the (strept)avidin solution plus 75 pi of the HABA dye to a cuvette. Mix well and measure the absorbance of the solution at 500 nm. Next, add a small amount of sample to this solution and mix. Record the absorbance at 500 nm. If the change in absorbance due to sample addition was not sufficient to obtain a significant difference from the initial (strept)avidin-HABA solution, add another portion of sample and measure again. Determine the amount of biotin present in the protein sample by using the standard curve. The number of moles of biotin divided by the moles of protein present gives the number of biotin modifications on each protein molecule. 

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