| Budget Amount *help |
¥1,900,000 (Direct Cost: ¥1,900,000)
Fiscal Year 1997: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1996: ¥1,100,000 (Direct Cost: ¥1,100,000)
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| Research Abstract |
Oxalate is synthesized mainly in the liver from glyoxylate which is formed from glycolate in liver peroxisomes. Usually, the conversion to glycine catalyzed by serine : pyruvate/alanine : glyoxylate aminotransferase (SPT/AGT) is the main rout of glyoxylate metabolism, but when SPT/AGT is missing in liver peroxisomes as in the case of primary hyperoxaluria type 1 more glyoxylate is utilized for oxalate production resulting in hyperoxaluria and finally systemic oxalosis. SPT/AGT locates in either mitochondria, peroxisomes or both depending on the food habit of animal species, and this dual organelle localization is caused by an alternative transcription initiation and point mutations in the translation initiation AUG codon, suggesting that only those animal species survive who gained, by mutations of the AGT gene, the proper organelle localization of SPT/AGT to meet the metabolic needs arisen from their food habit. The major source of glycolate is still elusive, but if it is the dietary glycolate itself it is rich in plant as an intermediate of photorespiration. The present study was undertaken to evaluate the contribution of dietary glycolate to oxalogenesis, and for this purpose enzymic methods for determination of glycolate were developed. In one of the methods, glycolate is oxidized by glycolate oxidase (GO) in the presence of excess cysteamine and resulting thiazolidine 2-carboxylate is quantitated by pico tag chromatography. In the other, the reaction of glycolate with GO is performed in Tris buffer (pH 8.3) and pyruvate formed from L-lactate is eliminated by the combined actions of GPT,GlDH and G6P-DH.Glyoxylate protected from both GO and GPT as Tris-glyoxylate adduct is then converted to 1,5-diphenylformazan and determined at 515-520 nm. Both methods reliably detect 1 nmol of glycolate in the sample, but the latter is simpler and pertinent to routine use while the former may be used for confirmatory purposes.
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