Biotin sources

A considerable amount of biotin is synthesized by human intestinal bacteria, as evidenced by the fact that 3 to 6 times more biotin is excreted in the urine and feces than is ingested. But synthesis in the gut may occur too late in the intestinal passage to be absorbed well and play much of a direct role as a biotin source. Also, several variables affect the microbial synthesis in the intestines, including the carbohydrate source of the diet (starch, glucose, sucrose, etc.), the presence of other B vitamins, and the presence or absence of antimicrobial drugs and antibiotics. 

Fatty acids with odd numbers of carbon atoms are rare in mammals, but fairly common in plants and marine organisms. Humans and animals whose diets include these food sources metabolize odd-carbon fatty acids via the /3-oxida-tion pathway. The final product of /3-oxidation in this case is the 3-carbon pro-pionyl-CoA instead of acetyl-CoA. Three specialized enzymes then carry out the reactions that convert propionyl-CoA to succinyl-CoA, a TCA cycle intermediate. (Because propionyl-CoA is a degradation product of methionine, valine, and isoleucine, this sequence of reactions is also important in amino acid catabolism, as we shall see in Chapter 26.) The pathway involves an initial carboxylation at the a-carbon of propionyl-CoA to produce D-methylmalonyl-CoA (Figure 24.19). The reaction is catalyzed by a biotin-dependent enzyme, propionyl-CoA carboxylase. The mechanism involves ATP-driven carboxylation of biotin at Nj, followed by nucleophilic attack by the a-carbanion of propi-onyl-CoA in a stereo-specific manner. 

In terms of amino acids bacterial protein is similar to fish protein. The yeast s protein is almost identical to soya protein fungal protein is lower than yeast protein. In addition, SCP is deficient in amino acids with a sulphur bridge, such as cystine, cysteine and methionine. SCP as a food may require supplements of cysteine and methionine whereas they have high levels of lysine vitamins and other amino acids. The vitamins of microorganisms are primarily of the B type. Vitamin B12 occurs mostly hi bacteria, whereas algae are usually rich in vitamin A. The most common vitamins in SCP are thiamine, riboflavin, niacin, pyridoxine, pantothenic acid, choline, folic acid, inositol, biotin, B12 and P-aminobenzoic acid. Table 14.4 shows the essential amino acid analysis of SCP compared with several sources of protein. 

This system is present in many tissues, including hver, kidney, brain, lung, mammary gland, and adipose tissue. Its cofactor requirements include NADPfl, ATP, Mn, biotin, and HC03 (as a source of CO2). Acetyl-CoA is the immediate substrate, and free palmitate is the end product. 

Bicarbonate as a source of CO2 is required in the initial reaction for the carboxylation of acetyl-CoA to mal-onyl-CoA in the presence of ATP and acetyl-CoA carboxylase. Acetyl-CoA carboxylase has a requirement for the vitamin biotin (Figure 21-1). The enzyme is a multienzyme protein containing a variable number of identical subunits, each containing biotin, biotin carboxylase, biotin carboxyl carrier protein, and transcarboxylase, as well as a regulatory allosteric site. The reaction takes place in two steps (1) carboxylation of biotin involving ATP and (2) transfer of the carboxyl to acetyl-CoA to form malonyl-CoA. 

Vitamins such as thiamin, biotin, and vitamin Bj2 are often added. Once again, the requirements of anaerobes are somewhat greater, and a more extensive range of vitamins that includes pantothenate, folate, and nicotinate is generally employed. In some cases, additions of low concentrations of peptones, yeast extract, casamino acids or rumen fluid may be used, though in higher concentrations, metabolic ambiguities may be introduced since these compounds may serve as additional carbon sources. 

Recently, SETA BioMedicals has developed a new near-infrared squaraine-based label Seta-633, which can be used to study the interaction between low-molecular-weight analytes and proteins using fluorescence lifetime as the readout parameter [19]. This label exhibits lower quantum yields and shorter fluorescence lifetimes when free in solution, but these values substantially increase upon interaction with proteins, which is contrary to tracers like Cy5 or Alexa 647. It was demonstrated in a model assay that a biotinylated Seta-633 binds to anti-biotin with high specificity. Importantly, the lifetime of Seta-633-biotin increases about 2.76 fold upon binding to a specific antibody (anti-biotin, MW =160 kDa), while the titration with BSA or nonspecific antibody does not result in a noticeable change in lifetime (Fig. 13). The label is compatible with readily available light sources (635 nm or 640 nm lasers) and filter sets (as for Cy5 or Alexa 647) and its... 

The methods used for in vivo incorporation of azido-monomers and performing a labeling reaction with live cells are relatively simple. The following protocol is based on the methods of Saxon and Bertozzi (2000), which uses acetylated azidoacetylmannosamine as the azido-monomer source and a biotin-PEG-phosphine compound to biotinylate cell surface glycoproteins at the specific azide-sialic acid incorporation sites (Figure 17.19). 

Notes. When using biotin-labeled secondary antibodies instead of enzyme-labeled antibodies, you have first to detect biotin with enzyme-labeled (strept) avidin and proceed further with the Substrate Step (9). Do not add normal serum, non-fat dried milk, culture media or other potential sources of biotin to (strept)avidin-containing reagents. This may result in reduced sensitivity. Solutions containing sodium azide or other inhibitors of peroxidase activity should not be used in diluting the peroxidase substrate. 

Fig. s.n On-line continuous-flow monitoring of biochemical interaction with (a) fluorescence and (b) MS SIM (m/z 390) detection. Fluorescein-biotin (96 nM), streptavidin (32 nM), 20-pL loop injections of 1000 nM biotin (n = 3). MS instrument Q-ToF2 (Waters) equipped with a Waters Z-spray electrospray (ESI) source. Point 1 Carrier pump, protein and reporter ligand pumps... 

Fixation of carbon dioxide by biotin-enzyme complexes is not unique to acetyl-CoA, and another important example occurs in the generation of oxaloacetate from pyravate in the synthesis of glucose from non-carbohydrate sources (gluconeogene-sis). This reaction also allows replenishment of Krebs... 

In the ruminant mammary tissue, it appears that acetate and /3-hydroxybutyrate contribute almost equally as primers for fatty acid synthesis (Palmquist et al. 1969 Smith and McCarthy 1969 Luick and Kameoka 1966). In nonruminant mammary tissue there is a preference for butyryl-CoA over acetyl-CoA as a primer. This preference increases with the length of the fatty acid being synthesized (Lin and Kumar 1972 Smith and Abraham 1971). The primary source of carbons for elongation is malonyl-CoA synthesized from acetate. The acetate is derived from blood acetate or from catabolism of glucose and is activated to acetyl-CoA by the action of acetyl-CoA synthetase and then converted to malonyl-CoA via the action of acetyl-CoA carboxylase (Moore and Christie, 1978). Acetyl-CoA carboxylase requires biotin to function. While this pathway is the primary source of carbons for synthesis of fatty acids, there also appears to be a nonbiotin pathway for synthesis of fatty acids C4, C6, and C8 in ruminant mammary-tissue (Kumar et al. 1965 McCarthy and Smith 1972). This nonmalonyl pathway for short chain fatty acid synthesis may be a reversal of the /3-oxidation pathway (Lin and Kumar 1972). 

Biotin is involved in many carboxylation and decarboxylation reactions in carbohydrate, fatty acid, protein, and nucleic acid metabolism. Milk is a fairly good source of this vitamin, generally providing about 3/xg/100 g. Pasteurization has a minimal effect on the biotin content of milk. 

Vitamins and Minerals. Milk is a rich source of vitamins and other organic substances that stimulate microbial growth. Niacin, biotin, and pantothenic acid are required for growth by lactic streptococci (Reiter and Oram 1962). Thus the presence of an ample quantity of B-complex vitamins makes milk an excellent growth medium for these and other lactic acid bacteria. Milk is also a good source of orotic acid, a metabolic precursor of the pyrimidines required for nucleic acid synthesis. Fermentation can either increase or decrease the vitamin content of milk products (Deeth and Tamime 1981 Reddy et al. 1976). The folic acid and vitamin Bi2 content of cultured milk depends on the species and strain of culture used and the incubation conditions (Rao et al. 1984). When mixed cultures are used, excretion of B-complex vita-... 

To replace losses, oxaloacetate can be synthesized from pyruvate and C02 in a reaction that uses ATP as an energy source. This is indicated by the heavy gray line leading downward to the right from pyruvate in Fig. 10-1 and at the top center of Fig. 10-6. This reaction depends upon yet another coenzyme, a bound form of the vitamin biotin. Pyruvate is formed from breakdown of carbohydrates such as glucose, and the need for oxaloacetate in the citric acid cycle makes the oxidation of fats in the human body dependent on the concurrent metabolism of carbohydrates. 

Nucleic acids can also be biotinylated by nonenzymatic methods with photobiotin, a photoactivatable biotin analog (6), which can be commercially obtained from BRL, Sigma, and other commercial sources I have not compared the suitability of this method of biotin incorporation with that reported here, but expect that the method would be fully acceptable FMC (Rockland, ME) markets an alternate nonradioactive sequence detection kit known as Chemiprobe. The basis of this system is a chemical modification of cytosine residues m the probe DNA. After hybridization, the probe is detected by means of a monoclonal antibody that specifically recognizes the sulfonated DNA. Detection of the bound monoclonal antibody is achieved by means of an alkaline phosphatase-conjugated second antibody. 

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