Subject pyridoxal

W2. Wachstein, M., Kellner, J. D., and Oritz, J. M., Pyridoxal phosphate in plasma and leukocytes of normal and pregnant subjects following Be load tests. Froc. Soc. Exptl. Biol. Med. 103, 350-353 (1960). 

As indicated in Section 6.3.3 and Table 6.2 the key control step is mediated by glycogen phosphorylase, a homodimeric enzyme which requires vitamin B6 (pyridoxal phosphate) for maximum activity, and like glycogen synthase (Section 6.2) is subject to both allosteric modulation and covalent modification. 

Structures of vitamin B6 derivatives and the bonds cleaved or formed by the action of pyridoxal phosphate (a). The reactive part of the coenzyme is shown in red in (a). The bonds shown in red in (d) are the types of bonds in substrates that are subject to cleavage. 

Although, owing to the wide distribution of vitamin Bg in nature, clinical deficiency symptoms are seldom observed, there is little doubt that pyridoxine is essential in human nutrition. Pyridoxine is absorbed from the gastrointestinal tract and is converted to the active form pyri-doxal phosphate. Absorption is decreased in gastrointestinal diseases and also in subjects taking isoniazid (3). It is excreted in the urine as 4-pyridoxic acid (2). The metabolism of vitamin Bg in human beings has been investigated (56). 

This reaction is catalyzed by the enzyme ALA synthase (Fig. 2d) which requires the coenzyme pyridoxal phosphate (see Topic M2) and is located in the mitochondria of eukaryotes. This committed step in the pathway is subject to regulation. The synthesis of ALA synthase is feedback-inhibited by heme. 

In subjects with hypophosphatasia, the rare genetic lack of extracellular alkaline phosphatase, plasma concentrations of pyridoxal phosphate are very much higher than normal (up to 4 /rmol per L, compared with a normal range of about 100 nmol per L), and intracellular concentrations of pyridoxal phosphate are lower than normal (Narisawa et al., 2001). 

Free pyridoxal either leaves the cells or is oxidized to 4-pyridoxic acid by aldehyde dehydrogenase (which is present in all tissues) and also by hepatic and renal aldehyde oxidases. 4-Pyridoxic acid is actively secreted by the renal tubules, so measurement of the plasma concentration provides an index of renal function (Coburn et al., 2002). There is some evidence that oxidation to 4-pyridoxic acid increases with increasing age in elderly people, the plasma concentration of pyridoxal phosphate is lower, and that of 4-pyridoxic acid higher, than in younger subjects even when there is no evidence of impaired renal function (Bates et al., 1999b). Small amounts of pyridoxal and pyridox-amine are also excreted in the urine, although much of the active vitamin Be that is filtered in the glomerulus is reabsorbed in the kidney tubules. 

Gershoff and Prien (G6) found that normal subjects excrete significantly less xanthurenic acid and 4-pyridoxic acid and more citric acid than patients with chronic formation of calcium oxalate. A marked rise in excretion of calcium oxalate followed administration of tryptophan in these patients, whereas ingestion of pyridoxine was followed by a decrease in urinary oxalate. 

Urinary excretions of nicotinic acid metabolites and 2-pyridone, as well as of 4-pyridoxic and xanthurenic acids were determined in 15 South African Bantu pellagrins before and after tryptophan administration (P13). Red blood cell riboflavine levels and serum glutamic-oxalacetic transaminase levels were also measured. The authors discussed the apparent inability of the pellagra patients to convert tryptophan to nicotinic acid as indicated by their low excretion of nicotinic acid metabolites before and after tryptophan load. The possibility that the subjects were also suffering from a riboflavine deficiency was also discussed. 

Most of the vitamin in the body is eventually degraded to pyridoxic acid (PX) and excreted in the urine. Vitamin deficiency can result in a decrease in the amount of PX excreted, as illustrated by the data tn Table 9.4. Human subjects who had consumed a B i-sufficient diet were fed a B(,-deficient diet for 45 days. The results... 

Metabolism of pyridoxine-related compounds in mammals. Enzymes 1, pyridoxal kinase (present in all mammalian tissues) 2, nonspecific (probably alkaline) phosphatases 3, pyridoxine oxidase (cofactor is FMN O2 is required subject to product inhibition) 4, aldehyde oxidase or aldehyde dehydrogenase 5, aminotransferase,... 

These results show that the phosphate and aldehyde moieties of pyridoxal-5-phosphate and its analogues are required for antagonist activity at P2X Punnoceptors in rabbit vas deferens. Among this series of compounds, PPADS was found to be the most interesting P2-antagonist, and it was therefore the subject of a more detailed pharmacological investigation. 

The catabolic pathway of vitamin Bg (1) is probably the best studied. In animals (including humans) 4-pyridoxic acid (4) is the primary catabolic product of vitamin Bg, found in urine. It is formed by the oxidation of pyridoxal (2) by a nonspecific flavin adenine dinucleotide (FAD)-dependent aldehyde oxidase. The catalytically active form of vitamin Bg, pyridoxal-5 -phosphate, PLP (17), does not undergo similar oxidation to form 4. The various forms of vitamin Bg (1, 2, 15) and their respective phosphate esters (16, 17) are readily enzymatically interconvertible. Further degradation of 4 is unlikely in humans as the subjects administered with doses of 4 were found to excrete it quantitatively. Estimation of 4 in urine serves as a nutritional marker, as lower than normal amounts of 4 is indicative of vitamin Bg deficiency. ... 

It is possible for a tissue to have a low or impaired histamine-forming capacity (HFG) and yet to have a high histamine content by virtue of its ability to store the amine. Thus in rats subjected to prolonged inhibition of histamine formation by means of a pyridoxal-deficient diet , the HFC in abdominal skin, tongue and lung was reduced to less than 10 per cent of normal without diminishing the histamine content. In the gastric mucosa,... 

Further reactions may occur after the formation of a Schiff base. Thus aldehydes react with substituted phenylethylamines under mild experimental conditions to form ultimately tetrahydroisoquinolines i . A particular case of this is the reaction between pyridoxal and DOPA [Figure 4.5). A similar reaction occurs between pyridoxal and histidine [Figure 4.5). The observation that DOPA decarboxylase is subject to substrate inhibition aroused... 

The histidine decarboxylase activity of tissues can be raised or lowered by changing the hormonal state of the animal or by subjecting it to certain stressful stimuli. Administration of thyroid hormones to rats produces a marked increase in the specific histidine decarboxylase activity of the glandular mucosa of the stomach " , while the activity of the nonspecific enzyme in the liver is lowered . Studies of the action of thyroid hormones on other pyridoxal-dependent enzymes in rat liver suggest that, in this organ at least, these changes arise from corresponding alterations in both pyridoxal phosphate and apo-enzyme synthesis . 

An unusual variant of a /8 elimination reaction is shown by kyneurinase which catalyses the production of L-alanine and 3-hydroxyanthranilate from 3-hydroxy kyneurine (Fig. 33), an intermediate in tryptophan biosynthesis. Although the reaction has not been subjected to mechanistic investigation a pathway consistent with the previously demonstrated properties of pyridoxal-P to stabilise a j8-carban-ion is illustrated in Fig. 34. 

There is a rapid decrease in urinary vitamin Bg when normal subjects are placed on a diet low in Be content (K3). Recently Miller et(d. M14) reported a study of five women receiving a constant diet of known Bo content. Three subjects taking OCAs showed most of the expected changes in tryptophan metabolites in urine and some evidence for decreased urinary excretion of Bo. This was in contrast to the earlier findings by Aly et al. (A5) that Be excretion in urine was unchanged by OCAs, and could have been due to a number of factors including differences in the organisms used to assay urine Bo. Urinary 4-pyridoxic add output in urine is also decreased quickly on a Ba-deficient diet, and has been reported to be normal in OCA-users (P8). 

Figure 11.13 Reactions at a-carbon of a-amino acids catalyzed by pyridoxal enzymes All three substituents at C are subject to labilization in the three types of a-carbon reactions. The hydrogen is labilized in recemization reactions, the amino group is labUized in the transamination and the carboxyl group is labilized in decarboxylation. a-Amino acid condenses with pyridoxal phosphate to yield pyridoxylidene imino acid (an aldimine). The common intermediate, aldimine and distinct ketimines leading to the production of oxo-acid (in transamination), amino acid (in racemization) and amine (in decarboxylation) are shown. The catalytic acid (H-A-) and base (-B ) are symbolic both can be from the same residue such as Lys258 in aspartate aminotransferase.

An investigation has been conducted by Mpller (1953) in which measurements were made of the contents of pyridoxal, pyridoxamine, and phos-phorylated vitamin Be derivatives in tissue samples (liver, heart, kidney, spleen) obtained from 10 subjects ranging in age from 0 to 76 years. No significant variations with age in the concentrations of these compounds were encountered essentially similar values were observed for samples from newborn children and from adult individuals. 

Each monomer contains one pyridoxal phosphate and a highly conserved primary structure in the vicinity of the cofactor binding site. The enzymes follow the same catalytic mechanism, a rapid equilibrium random Bi Bi mechanism. However, unlike animal phosphorylases, the microbial and plant enzymes are not subjected to covalent or allosteric control. Within the group of higher plant phosphorylases two types of enzyme have been distinguished which differ in monomer size, peptide pattern, glucan specificity, and intracellular location (1-3). Based on immunochemical studies on leaf tissues (4-5), one enzyme form has been localized in the cytosol whereas the other one resides in the chloroplast. Thus, the two plant phosphorylase types represent non-interconvertible proteins which presumably have entirely different metabolic functions. 

A more specific type of chemical assay is based on enzymatic measurement of vitamin co-enzyme activity. This approach is designed to detect a vitamin deficiency in tissues, and is only feasible for those vitamins that serve as co-enzymes. For instance, thiamin depletion in a subject can be diagnosed by measuring the transketolase activity in red blood cells with and without the addition of thiamin pyrophosphate (TPP) in vitro. If TPP increases the activity by more than a given amount, thiamin deficiency is indicated. Similarly, a subnormal level of riboflavin is indicated in tissues if the activity of erythrocyte glutathione reductase is increased after the addition of flavin adenine dinucleotide (FAD). Erythrocyte transaminase activation by pyridoxal-5 -phosphate (PLP) can be measured to establish a deficiency of vitamin B . 

The chemistry of pyridoxal and some of its phosphorylated derivatives has been the subject of continuous research directed toward the elucidation of the relationship between structure and biological activity. A variety of different derivatives of vitamin Bg have been described. For many of them the precise function is not understood, yet these derivatives have novel functions and could be crucial to fully understanding the biological relevance of vitamin Bg. 

The reactions of amino acids, catalysed by enzymes requiring a derivative of vitamin Bg (Figure 5a) as cofactor, have been the subject of a number of mechanistic studies. Enzymes catalysing decarboxylation, racemisation, dehydration (of serine or threonine) or desulphy-dration (of cysteine) require pyridoxal-5 -phosphate as cofactor (Figure 5b), and are dealt with in later sections. Enzymes catalysing transamination, on the other hand, require either pyridoxal-5 -phosphate or pyridoxamine-5 -phosphate (Figure 5c). Snell and coworkers showed that the reactions of amino acids normally... 

Previous investigations of this subject led to the misleading results that oxygen was consumed and CO2 produced in the reaction also that riboflavin and DPN, as well as pyridoxal phosphate, were required for maximal activation. The confusion resulted from the fact that oxidation of pyruvate was being measured in the crude bacterial preparations as well as the decomposition of tryptophan to indole. For literature, see . 

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