Pyridoxine-5 -phosphate

Other compounds, not shown in Table VI, which are hydrolyzed by alkaline phosphatase are TPN (28), poly A (28), phosphocellulose (28), pyrophosphoserine (102, 103), phosphoserine (102-104), pyridoxine phosphate (104), pyridoxal phosphate (104), phosphothreonine (104), and phosphocholine (104) These compounds are all hydrolyzed at approximately the same rate. 

The six principal B6 vitamers are widely distributed in foods (102,103). They include pyridoxine (PN), pyridoxal (PL), pyridoxamine (PM), and their 5 -phosphate esters, pyridoxine phosphate (PNP), pyridoxal phosphate (PLP), and pyridoxamine phosphate (PMP) (Fig. 5). The predominate B6 vitamer in animal-based foods is PLP, whereas plant products generally contain PN and PM or their phosphorylated forms. Conjugated vitamers in the form of PN-glycosides have also been isolated from plant-based foods. Pyridoxal is readily converted to PM during cooking and food processing. Total vitamin B6 is the sum of the six principal vitamers inclusion of the conjugated forms depends on the extraction procedure. 

PN = pyridoxine PM = pyridoxamine PL = pyridoxal PNP = pyridoxine phosphate PMP = pyridoxamine phosphate PLP = pyridoxal phosphate PNG = 5 -0-(/8-D-glucopyranosyl)pyri-doxine. 

The assay contained in a volume of 1 mL 20 mM potassium phosphate buffer (pH 5.75), 0.08 mM ZnCl2,0.06 mM KG, 0.02 mM isopyridoxal (internal standard), 1.2 mM ATP, 0.1 mM pyridoxine, and liver extract as the source of enzyme. To assay the yeast enzyme, ZnQ2 was replaced by 0.1 mM MgG2 and KC1 was omitted. The reaction was started by adding enzyme, and incubations were continued in the dark at 37°C for 90 minutes. The reaction was stopped by heating the test tubes in a boiling water bath for 3 minutes. After centrifugation, an aliquot of the supernate was injected into the HPLC system. The reaction was linear for at least 90 minutes when the rate of pyridoxine phosphate formation was not more than 13 nmol/h. 

The phosphorylated vitamers are dephosphorylated by membrane-bound alkaline phosphatase in the intestinal mucosa pyridoxal, pyridoxamine, and pyridoxine are all absorbed rapidly by carrier-mediated diffusion. Intestinal mucosal cells have pyridoxine kinase and pyridoxine phosphate oxidase (see Figure 9.1), so that there is net accumulation of pyridoxal phosphate by metabolic trapping. Much of the ingested pyridoxine is released into the portal circulation as pyridoxal, after dephosphorylation at the serosal surface. 

Tissue uptake of vitamin Be is again by carrier-mediated diffusion of pyridoxal (and other unphosphorylated vitamers), followed by metabolic trapping by phosphorylation. Circulating pyridoxal and pyridoxamine phosphates are hydrolyzed by extracellular alkaline phosphatase. All tissues have pyridoxine kinase activity, but pyridoxine phosphate oxidase is found mainly in the liver, kidney, and brain. 

Pyridoxine phosphate oxidase is a flavoprotein, and activation of the erythrocyte apoenzyme by riboflavin 5 -phosphate in vitro can be used as an index of riboflavin nutritional status (Section 7.4.3). However, even in riboflavin deficiency, there is sufficient residual activity of pyridoxine phosphate oxidase to permit normal metabolism ofvitamin Be (Lakshmi and Bamji, 1974). Pyridoxine phosphate oxidase is inhibited by its product, pyridoxal phosphate, which binds a specific lysine residue in tbe enzyme. In tbe brain, tbe Ki of pyridoxal phosphate is of the order of 2 /xmol per L - the same as the brain concentration of free and loosely bound pyridoxal phosphate, suggesting that this inhibition may be a physiologically important mechanism in the control of tissue pyridoxal phosphate (Choi et al., 1987). 

Although pyridoxine is taken up and phosphorylated by muscle (and other tissues), the resultant pyridoxine phosphate is not oxidized to pyridoxal phosphate. It has been suggested that the neurotoxicity of high intakes of pyridoxine (Section 9.9.6.4) may be caused by the uptake and trapping of pyridoxine, and hence competition with pyridoxal, resulting in depletion of tissue pyridoxal phosphate and a deficiency of the metabolically active form of the vitamin. 

TLC of vitamin Be compounds, on various layers in different solvents, was studied. The Rf values of pyri-doxine, pyridoxal, pyridoxamine, pyridoxal ethyl acetate, 4-pyridoxic acid, 4-pyridoxic acid lactone, pyridoxine phosphate, pyridoxal phosphate, and pyridoxamine phosphate were 0.62, 0.68, 0.12, 0.54, 0.91, 0.91, 0.95, 0.95, and 0.86, respectively, by TLC on silica gel HF254 with... 

Uptake and Metabolism. The vitamin Bg family consists of pyridoxine, pyridoxal, pyridoxamine, pyridoxine phosphate, pyridoxal phosphate (PLP), and pyridoxamine phosphate (Fig. 8.33). The commercial form is pyridoxine. Pyridoxal phosphate is the coenzyme form. It and pyridoxamine phosphate are from animal tissues. Pyridoxine is from plant tissues. All phosphorylated forms are hydrolyzed in the intestinal tract by phosphatases before being absorbed passively. Conversion to the phosphorylated forms occurs in the liver. Notice that niacin (NAD) and riboflavin (FMN, FAD) are required for interconversion among the vitamin Bq family. The phosphorylated forms are transported to the cells where needed. The major excretory product is 4-pyr-idoxic acid. 

Figure 8.35. Pyridoxine phosphate-catalyzed transamination and decarho lation.

Vitamin Bg and Related Compounds.—Analogues of pyridoxol and pyridoxal phosphates in which the 5 -methylene (35) or the 5 -phosphate group (36) have been modified have been used to study the substrate specificity of pyridoxine phosphate oxidase. The methylene analogues acted as substrates whereas (36) or the 2-cyanoethyl ester of pyridoxol... 

Vitamin B6 has been shown to be essential in many biochemical reactions that occur in plants and animals. Although it may occur in any one of the three forms listed above, the compound usually acts as the phosphate ester, pyridoxine phosphate. Pyridoxine phosphate functions as a coenzyme in the transformation of amino acids, the building blocks from which proteins are made. A coenzyme is a chemical compound that works with an enzyme to catalyze some essential chemical reaction in the body. Pyridoxine phosphate appears to be necessary for the synthesis of proteins from amino acids as well as the metabolism of amino acids to produce energy needed for normal body functioning. 

Tumour cells (ascites hepatoma) of the rat liver can take up (i) pyridoxine phosphate and (ii) pyridoxal phosphate without eliminating the phosphate... 

Vitamin B Vitamin Bg is the generic name for six naturally occurring vitamers which are derivatives of 2-methyl-3-hydroxy pyridone. All forms of vitamin Bg have been detected in human plasma with the exception of pyridoxine phosphate. Both free and protein bound forms can be extracted with trichloroacetic acid or perchloric acid, the excess of which should be removed prior to chromatography. Cation-exchange chromatography with linear gradients of hydrochloric acid and phosphate buffers produces a profile of the vitamin Bg compounds in 50 min. 

Studies based on the use of an antivitamin, deoxypyri-doxine, have established that the daily requirement of the vitamin ranges between 1 and 2 mg in the human adult. A normal diet has been reported to provide 1-1.5 mg daily of the vitamin. Food appears to be the only source of the vitamin because most of the vitamin produced by the bacterial flora of the intestine is excreted in the feces, possibly after oxidation to 4-pyridoxic acid. The ingested vitamin is rapidly and completely absorbed, but the exact site of the absorption is not known. Although both pyridoxine and pyridoxamine can be excreted as such and are therefore normal constituents of human urine, part of the vitamin is oxidized to the 4-pyridoxic acid before excretion in the urine. Mammalian tissues contain at least two enzymes capable of oxidizing pyridoxine. Both enzymes seem to be flavoproteins. One attacks pyridoxine, the other attacks pyridoxine phosphate. The pyridoxine phosphate oxidase of liver has been purified 65 times. Although the enzyme was shown to act on pyridoxamine phosphate, pyridoxamine phosphate was oxidized only when the pH of the incubation mixture was raised to 10. Pyridoxine phosphate oxidase has no effect on pyridoxamine phosphate at physiological pH. 

Pyridoxine phosphate may either be used unchanged in enzymic reactions, or it may be split by nonspecific phosphatases to yield pyridoxine and inorganic phosphate. Pyridoxine is then further oxidized to yield 4-pyridoxic acid in the presence of a purified preparation of liver pyridoxic oxidase and aldehyde oxidase. 

The role of pyridoxal in the transamination reaction has been extensively investigated by comparing the mechanism of action of nonenzymic transamination (see Fig. 4-28) in the presence of metal (iron) acting as a catalyst to the transamination catalyzed by the specific proteins. The mechanism of the nonenzymic transamination was deduced from a series of rather simple observations (1) when an amino acid is added to a dilute solution of pyridoxal or pyridoxine phosphate, the absorption maximum of the solution shifts from 345 mp to 430 mp. This change in absorption... 

Although it is possible to describe the clinicopatho-logical manifestations of pyridoxine deficiency and the metabolic role of pyridoxal phosphate, each pathological alteration cannot be explained by a specific metabolic alteration. Deficiency of a vitamin involved in several steps of the intermediary metabolism of amino acids is bound to be associated with severe clinicopath-ological changes, but the specific metabolic alterations responsible for the anemia and convulsions in pyridoxine deficiency have not been identified. y-Amino butyric acid, cystathione, sphingosine, and 5-hydroxy-tryptamine are compounds abundant in the brain. Pyridoxal phosphate is involved in their metabolic formation. Is there any correlation between the role of pyridoxal phosphate in the metabolism of these compounds and the development of convulsions and ataxia in pyridoxine deficiency Is the role of pyridoxine phosphate in the intermediary metabolism of sulfur amino acid related to the development of seborrheic dermatitis ... 

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