Michio Suda

Purification and immunohistochemical tissue localization of human xanthine oxidase

Xanthine oxidase was purified 1600-fold from human liver cytosol. The purified enzyme was shown as a single band of 300 kDa on polyacrylamide gel electrophoresis and 150 kDa on SDS-PAGE. Using this purified enzyme, polyclonal antibody against xanthine oxidase was raised in a rabbit. On Ouchterlonys double immunodiffusion method, the raised antibody and the human liver cytosol made a precipitation line stained by activity stain and protein stain, respectively. With the raised anti-xanthine oxidase sera, the immunohistochemical localization of xanthine oxidase in human tissues was examined. Immunostaining of frozen hepatic tissue section showed that the cytoplasm of hepatocytes and endothelial lining cells were stained. In a number of other tissues, the xanthine oxidase antigen was detected only in the endothelial lining cells from heart, kidney, brain, aorta, lung and mesentery, except for the duodenal mucosa cells. A possible role for xanthine oxidase in the endothelial cells from various human tissues in the pathogenesis of reperfusion injury was suggested.

In vitro oxidation of pyrazinamide and allopurinol by rat liver aldehyde oxidase

Aldehyde oxidase was purified about 120-fold from rat liver cytosol by sequential column chromatography using diethylaminoethyl (DEAE) cellulose, Benzamidine-Sepharose 6B and gel filtration. The purified enzyme was shown as a single band with M(r) of 2.7 x 10(5) on polyacrylamide gel electrophoresis (PAGE) and M(r) of 1.35 x 10(5) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Using this purified enzyme, in vitro conversion of allopurinol, pyrazinamide and pyrazinoic acid was investigated. Allopurinol and pyrazinamide were oxidized to oxypurinol and 5-hydroxy-pyrazinamide, respectively, while pyrazinoic acid, the microsomal deamidation product of pyrazinamide, was not oxidized to 5-hydroxypyrazinoic acid. The apparent Km value of the enzyme for pyrazinamide was 160 microM and that for allopurinol was 1.1 mM. On PAGE, allopurinol- or pyrazinamide-stained band was coincident with Coomassie Brilliant Blue R 250-stained band, respectively. These results suggest that aldehyde oxidase may play a role in the oxidation of allopurinol to oxypurinol and that of pyrazinamide to 5-hydroxypyrazinamide with xanthine dehydrogenase which can oxidize both allopurinol and pyrazinamide in vivo. The aldehyde oxidase may also play a major role in the oxidation of allopurinol and pyrazinamide in the subgroup of xanthinuria patients (xanthine oxidase deficiency) who can oxidize both allopurinol and pyrazinamide.

Effect of BOF-4272 on the oxidation of allopurinol and pyrazinamide in vivo: Is xanthine dehydrogenase or aldehyde oxidase more important in oxidizing both allopurinol and pyrazinamide?

Allopurinol or pyrazinamide was administered to rats treated with BOF-4272 (a potent xanthine oxidase inhibitor) to investigate to what degree xanthine dehydrogenase participates in the oxidation of these agents. BOF-4272 markedly decreased the plasma concentration and the urinary excretion of both oxypurinol and 5-hydroxypyrazinamide. It also decreased the sum of the urinary excretion of allopurinol and oxypurinol and that of pyrazinamide and its metabolites, although it did not affect the sum of the plasma concentrations of allopurinol and oxypurinol at 105 min after administration of allopurinol or the plasma concentration of pyrazinamide during the period after the administration of pyrazinamide. These results suggested that BOF-4272 almost completely inhibited the oxidation of allopurinol and pyrazinamide and had some effect on the excretion and/or the tissue incorporation of these two compounds. Since the in vitro study demonstrated that BOF-4272 did not inhibit the activity of aldehyde oxidase, which oxidized both allopurinol to oxypurinol and pyrazinamide to 5-hydroxypyrazinamide, the results suggested that xanthine dehydrogenase was the more important enzyme in converting allopurinol to oxypurinol and pyrazinamide to 5-hydroxypyrazinamide.

Metabolism of pyrazinamide and allopurinol in hereditary xanthine oxidase deficiency

The metabolism of pyrazinamide and allopurinol was studied in three xanthinuric patients from two families with hereditary xanthinuria to determine whether both substrates were oxidized only by xanthine oxidase or by other oxidases as well. One xanthinuric patient could neither metabolize pyrazinamide into 5-hydroxypyrazinamide nor allopurinol into oxypurinol. Two xanthinuric patients could metabolize both pyrazinamide into 5-hydroxypyrazinamide and allopurinol into oxypurinol but could not oxidize pyrazinoic acid to 5-hydroxypyrazinoic acid. These findings suggest that xanthinuria comprises at least two subgroups.

Effect of ethanol ingestion on nucleotides and glycolytic intermediates in erythrocytes and purine bases in plasma and urine: Acetaldehyde-induced erythrocyte purine degradation

The effect of ethanol on nucleotides and glycolytic intermediates in erythrocytes and purine bases in plasma and urine was investigated. Ethanol ingestion (0.45 mL/kg body weight) increased plasma concentrations and urinary excretion of oxypurines (hypoxanthine and xanthine) and concentrations of adenosine monophosphate (AMP), adenosine diphosphate (ADP), and glyceraldehyde 3-phosphate+dihydroxyacetonephosphate in erythrocytes. In an in vitro incubation study using erythrocytes, acetaldehyde increased the concentrations of AMP, ADP, and glyceraldehyde 3-phosphate+dihydroxyacetonephosphate in erythrocytes as well as the concentration of hypoxanthine in the incubation medium. These results suggest that acetaldehyde (a metabolite of ethanol) induces an increase in purine degradation by erythrocytes and then contributes to the ethanol-induced enhanced purine degradation in vivo.

Effect of Glucose Infusion on the Renal Transport of Purine Bases and Oxypurinol

The effect of glucose infusion on renal handling of purine bases and oxypurinol was examined in 6 normal subjects. Five hundred milliliters of 1.1 M glucose solution were administered intravenously in 1 h. Fractional clearances of uric acid, xanthine and oxypurinol were significantly increased during glucose infusion, but that of hypoxanthine was not changed, while a 1-hour infusion of 500 ml of 1.1 M mannitol had no effect on the fractional clearances of purine bases and oxypurinol. These data indicate that the effect of glucose infusion on the renal clearances of uric acid, xanthine and oxypurinol was not related to osmotic diuresis but induced by glycosuria and/or hyperglycemia. Accordingly, the glycosuria- and/or hyperglycemia-induced decrease in the biological half-life of oxypurinol must be considered in the administration of allopurinol to gouty patients with uncontrolled diabetes mellitus.

Ethanol as a xanthine dehydrogenase inhibitor

In the present study, we investigated whether ethanol inhibits the activity of xanthine dehydrogenase. Ethanol and/or inosine were administered to normal subjects, and plasma concentration and urinary excretion of purine bases were measured together with blood concentrations of lactic acid and pyruvic acid. In addition, ethanol and pyrazinamide were administered to these subjects, and plasma concentration and urinary excretion of pyrazinamide and its major metabolites were measured. Increases in plasma concentration and urinary excretion of xanthine induced by a combination of ethanol and inosine were greater than the sums of increases induced separately by ethanol and inosine, although increases in plasma concentration and urinary excretion of uric acid induced by the combination of ethanol and inosine were not different from the sums of increases induced separately by ethanol and inosine. Ethanol increased the ratio of blood lactic acid to blood pyruvic acid and decreased plasma concentration and urinary excretion of 5-hydroxypyrazinamide and 5-hydroxypyrazinoic acid. These results suggest that ethanol inhibits xanthine dehydrogenase presumably by an ethanol-induced increase in the cytosolic concentration of NADH in the liver.

Novel apoprotein A-I-containing lipoprotein produced by a human hepatoma-derived cell line HuH-7

The apoprotein A-I (apo A-I)-containing lipoprotein (LPHuH-7apoA-I) was isolated from the concentrated conditioned medium of human hepatoma-derived cell line HuH-7 by immunoaffinity chromatography. LpHuH-7apoA-I consists of two kinds of lipoproteins. One is a lipoprotein of large particle size (LpL) with broad electrophoretic mobility on agarose gel ranging from the origin to the position of prebeta-lipoprotein. LpL is protein-rich in composition (protein, 75.4% by weight) and is heterogeneous in size (34-17 nm in diameter) electron microscopically. However, the most intriguing properties of LpL are its partial electrophoretic mobility towards the cathode on agar gel. The other lipoprotein is of small particle size (LpS). It demonstrates prebeta-electrophoretic mobility on agarose gel. LpS is also protein-rich in composition (protein, 95.4% by weight) and is heterogeneous in size (16.5-8.4 nm in diameter) electron microscopically. LpL is obviously different from LP-X and LP-Y in property, although LP-X, LP-Y and a part of LpL migrate towards the cathode on agar gel electrophoresis. LpS is also different from human apo A-I-containing lipoprotein without apo A-II in property, although these two lipoproteins possess the same mobility on agarose gel electrophoresis. These results indicate that both LpL and LpS are novel lipoproteins which have not yet been reported. The major isoproteins of the apo A-I of LpHuH-7apoA-I are apo A-I isoprotein 2 (apo A-I2), apo A-I isoprotein 4 (apo A-I4) and apo A-I isoprotein 5 (apo A-I5), and are different from those of apo A-I in human plasma and in the conditioned medium of hepatoma-derived cell line HepG2. This result suggests the presence of a proteinase which converts proapoprotein A-I (apo A-I2) to apoprotein A-I (apo A-I4) in the conditioned medium of HuH-7.

Studies on abnormal lipid metabolism in experimental nephrotic syndrome.

The specific lipid alterations in plasma and four different tissues, as well as the activities of plasma lecithin-cholesterol acyltransferase (LCAT) and tissue lipolytic enzymes were determined in experimental nephrotic and control rats. In nephrotic rats, the cholesterol level in the heart was significantly increased, while the kidney level was decreased. When expressed per unit protein, the cholesterol level in the adrenal was also increased. The respective triglyceride and phospholipid levels were similar in both groups, except for the significant increase in the adrenal triglyceride of nephrotic rats when expressed per unit protein. The tissue lipolytic activities were significantly reduced in the heart and adrenals of the nephrotic rats. The plasma LCAT was increased, and electron-microscopically heterogenous lipoproteins (very-low density and high-density lipoproteins) were demonstrated in the nephrotic rats. These results suggest that an excess of abnormal lipoproteins in the circulation may contribute to an increased uptake of cholesterol by circulatory organs like the heart and that an accumulation of cholesterol in the circulatory organs may accelerate atherosclerosis in nephrotic syndrome.

A Xanthinuric Family - The Proposita Having Immunologically Reactive Xanthine Oxidase but no Xanthine Oxidase Activity

The antibody was raised against purified human liver xanthine oxidase in a rabbit. In a xanthinuric patient, the double immunodiffusion method demonstrated the existence of an immunologically reactive duodenal mucosa xanthine oxidase which did not possess xanthine oxidase activity. These results indicated that xanthine oxidase protein is abnormal in structure and/or amino acid sequence.