Pyridoxal-5 -phosphate plasma levels

Homocystinuria can be treated in some cases by the administration of pyridoxine (vitamin Bs), which is a cofactor for the cystathionine synthase reaction. Some patients respond to the administration of pharmacological doses of pyridoxine (25-100 mg daily) with a reduction of plasma homocysteine and methionine. Pyridoxine responsiveness appears to be hereditary, with sibs tending to show a concordant pattern and a milder clinical syndrome. Pyridoxine sensitivity can be documented by enzyme assay in skin fibroblasts. The precise biochemical mechanism of the pyridoxine effect is not well understood but it may not reflect a mutation resulting in diminished affinity of the enzyme for cofactor, because even high concentrations of pyridoxal phosphate do not restore mutant enzyme activity to a control level. 

In humans, peripheral neuropathy due to isoniazid is influenced by the acetylator phenotype (see chap. 5), being predominantly found in slow acetylators. This is probably due to the higher plasma level of isoniazid in this phenotype. In this case, therefore, acetylation is a detoxication reaction, removing the isoniazid and rendering it unreactive toward pyridoxal phosphate. 

Amino groups are tunneled to glutamate from all amino acids except lysine and threonine. The enzymes are aminotransferases, and they are reversible. The two most important of these enzymes are alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Aminotransferases require pyridoxal phosphate as a coenzyme. The presence of elevated levels of aminotransferases in the plasma can be used to diagnose liver disease. 

In 1998, the reference intake in the United States and Canada was reduced from the previous Recommended Daily Allowance of 2 mg per day for men and 1.6 mg per day for women (National Research Council, 1989) to 1.3 mg per day for both (Institute of Medicine, 1998). The report cites six smdies that demonstrated that this level of intake would maintain a plasma concentration of pyridoxal phosphate at least 20 nmol per L although, as shown in Table 9.5, the more generally accepted criterion of adequacy is 30 nmol per L. 

In rodents, preanalytical factors such as food intake and restraint may alter plasma ALT (see Chapter 12 also). A 50% food restriction over 210 days in rats resulted in elevated ALT values compared to controls (Schwartz, Tornaben, and Boxhill 1973), while reduced levels of ALT were observed in a study of fasting effects on the oral toxicity of several xenobiotics (Kast and Nishikawa 1981). Changes of ALT related to diet may reflect perturbations of gluconeogenesis (Toropila et al. 1996). There are now several examples where plasma ALT falls after the administration of xenobiotics due to effects on pyridoxal phosphate, which is a cofactor necessary for action of the aminotransferases AST and ALT (Dhami et al. 1979 Rhodes et al. 1987 Waner et al. 1990 Waner and Nyska 1991). Such effects may confuse the interpretation of data when hepatotoxicity occurs and tends to increase ALT, but where there is an opposing effect due to reductions in pyridoxal phosphate. Further complications with ALT have been described by Wells and To (1986) in covalent binding studies with acetaminophen. 

Plasma AST measurements are a useful adjunct to ALT, and increased AST can be an indication of mitochondrial and cytoplasmic injury, although this enzyme is less specific for hepatotoxicity compared to ALT. Sustained increases of the plasma aminotransferase levels can indicate a progressive injury, but the aminotransferases may not be elevated during acute necrosis if the timing of the sample collection has allowed the increased circulating plasma enzymes to be cleared. Plasma ALT and AST may be decreased when the enzyme cofactor pyridoxal phosphate is reduced in vivo (Dhami et al. 1979 also see Chapter 2). 

As the name implies, renal clearance of abnormal levels of homocystine in the plasma causes excessive excretion of the amino acid in the urine. In cystathionine P-synthase deficiency, plasma methionine concentrations are elevated as well -this serves as a point of distinction from the remethylation defects. At present, it appears that the pyridoxal phosphate response may be explained by the fact that this vitamin increases the steady-state concentration of the active enzymes by decreasing the rate of apoenzyme degradation and possibly by increasing the rate of holoenzyme formation. The explanation is not entirely satisfactory, however, since in vitro studies have shown detectable levels of enzyme activity in mutant fibroblasts that have no response, while in other mutant lines without detectable enzyme activity, response has occurred. Once again, a distressing lack of correspondence between in vivo observations and in vitro experiments forces investigators to probe the secrets of these diseases more deeply. 

The alkaline phosphatase activity in plasma is usually reduced to values below the age-related normal range. Overall, there is a tendency to observe the lowest values in the more severely affected individuals, but individual cases may display activity just below the normal range. Heterozygotes may have reduced values. For that reason, the diagnosis should be confirmed by the demonstration of substrate accumulation ex vivo. As a consequence of reduced phosphatase activity, three compounds are present at increased concentrations pyridoxal-phosphate (PLP vitamin B6) in plasma, inorganic pyrophosphate (PPi) in plasma and urine, and phosphoethanolamine (PET) in urine. Increased PLP is a sensitive marker for hypophosphatasia (provided no oral supplement has been ingested) and is probably quite specific. Its elevation in plasma does not appear to have clinical consequences, as intracellular levels are not increased. PPi is equally sensitive but not routinely determined. Urinary PET is moderately sensitive (it may be normal in mild cases) but potentially nonspecific. 

Despite normal plasma pyridoxal 5-phosphate values, the urinary excretion of xanthurenic acid was found abnormally elevated in 3 epileptic children with disturbed tryptophan metabolism (HO). Administration of pyridoxine restored xanthurenuria to normal and raised plasma pyridoxal 5-phosphate levels. 

Release of free vitamin, mainly pyridoxal, occurs when physiological nonsaturating levels of vitamin are absorbed. Here the phosphates are hydrolyzed by nonspecific alkaline phosphatase located on the plasma membrane of cells. Some PLP is also released into the circulation by the liver. Because the reactive aldehyde is capable of forming Schiff bases with amino groups, PLP in plasma is more tightly com-plexed to proteins—mostly albumin—than is pyridoxal, which forms an intramolecular hemiacetal between the... 

Ubbink, J. B., Serfontein, W. J., Becker, P. J., and dc Villiers, L. S. (1987). Effect of different levels of oral pyridoxine supplementation on plasma pyridoxal-S -phosphate and pyridoxal levels and urinary vitamin B-6 excretion. Am. J. Clin. Nutr. 46,78-85. 

Potassium cyanide catalyzed oxidation of PLP with subsequent quantification of the highly fluorescent oxidation product, 4-pyridoxic acid-5 -phosphate, has been used to determine PLP levels in human plasma, brain tissue and cell cultures (74,126,127). The sensitivity of this method has been reported to be even better than methods based on PLP semicarbazone formation (127). PL is not determined by these methods this vitamer is oxidized to 4-pyridoxic acid lactone, which shows little fluorescence at pH 2.5 to 3.8 of the mobile phases used (Table 2). Hess and Vuilleumier (128) overcame this problem by alkaline delactonization of pyridoxic acid lactone to yield the highly fluorescent 4-PA. The determination of PL as 4-PA suffers from the disadvantage that each sample has to be analyzed twice. In the first analysis, cyanide oxidation is omitted and the basal 4-PA concentration is measured. In the second analysis, PL is converted to 4-PA as described. The concentration of PL is determined from the difference between the two assays for 4-PA. 

WJH Vermaak, HC Barnard, EMSP van Dalen, GM Potgieter. Correlation between pyridoxal-5 -phosphate levels and the percentage activation of aspartate aminotransferase enzyme in haemolysate and plasma during in vitro incubation studies with different vitamin B vitamers. Enzyme 35 215-224, 1986. 

R Delport, JB Ubbink, WJ Serfontein, PJ Becker, L Walters. Vitamin B-6 nutritional status in asthma the effect of theophylline therapy on plasma pyridoxal-5 -phosphate and pyridoxal levels. Int J Vitam Nutr Res 58 67-72, 1988. 

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