Yuji Moriwaki

Effects of combination treatment using anti-hyperuricaemic agents with fenofibrate and/or losartan on uric acid metabolism

Objective: To assess the effect of a combination treatment using anti-hyperuricaemic agents with fenofibrate and/or losartan on uric acid metabolism in hypertriglyceridaemic and/or hypertensive patients with gout. Methods: Twenty seven patients with gout were included in a fenofibrate plus anti-hyperuricaemic agents combination study, and 25 in a losartan plus anti-hyperuricaemic agents combination study. Serum uric acid concentration, uric acid clearance, and 24 hour urinary uric acid excretion were measured before and two months after the addition of fenofibrate (300 mg once daily) or losartan (50 mg once daily) to anti-hyperuricaemic agents. Results: Combination therapy of fenofibrate or losartan with anti-hyperuricaemic agents, which included benzbromarone (50 mg once daily) or allopurinol (200 mg twice a day), significantly reduced serum uric acid concentrations in accordance with increased uric acid excretion. Conclusion: A combination of fenofibrate or losartan with anti-hyperuricaemic agents is a good option for the treatment of gout patients with hypertriglyceridaemia and/or hypertension, though the additional hypouricaemic effect may be modest.

Close correlation between visceral fat accumulation and uric acid metabolism in healthy men

We evaluated the effect of accumulation of intraabdominal visceral fat on the metabolism of uric acid in 50 healthy male subjects to elucidate any relationship between such obesity and hyperuricemia. The area of abdominal fat (visceral fat and subcutaneous fat) was measured at the level of the umbilicus by abdominal computed tomographic scanning. Serum and urinary concentrations of uric acid and creatinine were determined with an autoanalyzer. Uric acid clearance and the ratio of urinary uric acid to creatinine excreted in urine were calculated. Univariate and multivariate analyses were used to evaluate the relationship between uric acid metabolism and body fat. The size of the area of visceral fat was significantly correlated with the serum concentration of uric acid (r = .37, P < .01), uric acid clearance (r = -.34, P < .05), and the urinary uric acid to creatinine ratio (r = .65, P < .0001). The size of the area of subcutaneous fat was significantly correlated only with the urinary uric acid to creatinine ratio (r = .38, P < .01). Multivariate analyses, including body mass index (BMI), showed that the size of the visceral fat area was the strongest contributor to an elevated serum concentration of uric acid, a decrease in uric acid clearance, and an increase in the urinary uric acid to creatinine ratio. These results suggest that accumulation of visceral fat may have a greater adverse effect on the metabolism of uric acid than BMI or accumulation of subcutaneous fat. Clearly, patients with hyperuricemia should lose weight to reduce excessive visceral fat stores, to help avoid attacks of gout.

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.

Tumor markers in pleural effusion diagnosis.

In order to discriminate between malignant and benign effusions, the values of carcinoembryonic antigen (CEA), ferritin, beta2‐microglobulin (BMG), acid‐soluble glycoprotein (ASP), tissue polypeptide antigen (TPA), adenosine deaminase (ADA), and immunosuppressive acidic protein (IAP) were measured in the pleural fluid of 54 patients with lung cancer, 20 with malignancies other than lung cancer, 18 with tuberculous pleurisy, and 22 with benign diseases other than tuberculosis. CEA levels in malignant effusions were significantly higher than those in benign effusions. At a cutoff level of 5 ng/ml, 68% of the patients with lung cancer and 44% of the patients with other malignancies showed elevated pleural fluid CEA levels. In 13 lung cancer cases with negative pleural fluid cytology, nine cases had elevated pleural fluid CEA levels. The mean pleural fluid BMG level of patients with benign diseases was significantly higher than that of patients with malignant diseases, but there was a marked overlap between those with malignant and benign diseases. No significant differences were found in the pleural fluid ferritin, ASP, TPA, and IAP levels between malignant and benign conditions. ASP and IAP pleural fluid levels showed significant correlations with the pleural fluid C‐reactive protein (CRP) concentrations suggesting that they also reflect inflammatory activity. The mean ADA activity in tuberculous effusion was significantly higher than that resulting from other causes of pleural effusion.

Immunohistochemical localization of aldehyde and xanthine oxidase in rat tissues using polyclonal antibodies

Tissues from male Wistar rats, fixed with 4% paraformaldehyde and embedded in paraffin, were studied with immunoperoxidase techniques using polyclonal antibodies raised against aldehyde oxidase or xanthine oxidase purified from rat liver. Immunohistochemical studies demonstrated that aldehyde oxidase-bearing cells were strongly stained in renal tubules, esophageal, gastric, intestinal and bronchial epithelium as well as liver cytoplasm. Weak but positive immunoreactivity was observed on the pulmonary alveolar epithelial cells, gastric glands and intestinal goblet cells. In contrast, it was demonstrated that cells with xanthine oxidase were strongly stained in renal tubules, esophageal, gastric, and small and large intestinal and bronchial epithelia etc. Positive immunostaining was also found in adrenal gland, skeletal muscle, spleen and cerebral hippocampus. Immunoreactivity againt aldehyde oxidase was not found in adrenal gland, spleen, mesentery or aorta, while immunoreactivity against xanthine oxidase was not found in mesentery or aorta. Although the significance of this ubiquitous and similar localization of aldehyde and xanthine oxidase seems unclear at present, these results may provide a clue as to the full understanding of the pathophysiological role of these oxidases in tissues.

Effect of ethanol and fructose on plasma uridine and purine bases

To determine whether both ethanol and fructose increase the plasma concentration of uridine, we administered ethanol (0.6 g/kg) or fructose (1.0 g/kg) to seven normal subjects. Both ethanol and fructose increased the plasma concentration of uridine together with an increase in the plasma concentration of oxypurines, whereas fructose also increased the plasma concentration of uric acid, but ethanol did not. In ethanol ingestion and fructose infusion, an increase in the plasma concentration of purine bases correlated with that of uridine. These results strongly suggest that an increase in the plasma concentration of uridine is ascribable to increased pyrimidine degradation following purine degradation increased by ethanol and fructose.

Determination of human plasma xanthine oxidase activity by high-performance liquid chromatography

An assay for human plasma xanthine oxidase activity was developed with pterin as the substrate and the separation of product (isoxanthopterin) by high-performance liquid chromatography with a fluorescence detector. The reaction mixture consists of 60 microliters of plasma and 240 microliters of 0.2 M Tris-HCl buffer (pH 9.0) containing 113 microM pterin. With this assay, the activity of plasma xanthine oxidase could be easily determined despite its low activity. As a result, it could be demonstrated that the intravenous administration of heparin or the oral administration of ethanol did not increase plasma xanthine oxidase activity in normal subjects, and also that plasma xanthine oxidase activity was higher in patients with hepatitis C virus infection than in healthy subjects or patients with gout. In addition, a single patient with von Gierkes disease showed a marked increase in the plasma activity of this enzyme, relative to that apparent in normal subjects.

Apolipoprotein E phenotypes in patients with gout: relation with hypertriglyceridaemia.

OBJECTIVE--To elucidate the relationship, if any, between lipid abnormalities and apolipoprotein E (apo E) polymorphism, by investigating apo E phenotype and allele frequency. METHODS--Fasting blood samples were taken for determination of apo E phenotype and serum lipids in 221 male patients with gout and 141 control male subjects. Apo E phenotype was determined by one dimensional flat gel isoelectric focusing. RESULTS--Frequencies of apo E phenotypes in gout were apo E3/3 67.9%, E4/3 18.1%, E4/4 2.3%, E4/2 1.8%, E3/2 9.5%, and E2/2 0.5%; those in control male subjects were 74.5%, 15.6%, 0%, 1.4%, 7.1%, and 1.4%, respectively. Frequencies of the e2, e3, and e4 alleles in gout were 0.061, 0.817 and 0.122, compared with the corresponding control frequencies of 0.057, 0.858 and 0.085. These differences in apo E phenotype and allele frequencies between gout and control subjects were not significant. The frequency of apo e4 allele in hyperlipidaemic gout subjects was significantly greater than that in normolipidaemic gout subjects; in contrast, its frequency was not different between hyperlipidaemic and normolipidaemic control subjects. Serum triglyceride, total cholesterol, apo B and E concentrations were significantly greater in gouty patients with the apo E4/3 phenotype than in those with gout having the apo E3/3 phenotype. CONCLUSIONS--These data suggest that gout subjects with hyperlipidaemia (hypertriglyceridaemia, hypercholesterolaemia or both) possess the apo e4 allele with higher frequency than those with normolipidaemia. They also suggest that apo e4 may induce some susceptibility to the development of hyperlipidaemia in gout in addition to that induced by obesity or excessive alcohol consumption, and may contribute to the high prevalence of atherosclerotic diseases in gout patients.

Urinary excretion of pseudouridine in patients with hepatocellular carcinoma

The urinary concentration of pseudouridine, primarily a degradation product of transfer ribonucleic acid, was determined by high‐performance liquid chromatography in 23 patients with primary hepatocellular carcinoma, 13 patients with liver cirrhosis, and 24 healthy controls. The urinary concentration of pseudouridine in the patients with hepatocellular carcinoma was significantly higher than that in the patients with liver cirrhosis or the healthy controls. Sixteen (70%) of the 23 patients with hepatocellular carcinoma had urinary pseudouridine levels higher than the mean value for the healthy controls plus 2 standard deviations. Nine of the 13 patients (69%) who had a serum alpha‐fetoprotein level below 400 ng/ml, had elevated urinary pseudouridine levels. Thus, the two markers, urinary pseudouridine and serum alpha‐fetoprotein in combination, which were detected in a total of 19 of the 23 patients (83%) with hepatocellular carcinoma, are considered to serve as complementary markers for the diagnosis of this disease. Cancer 57:1571–1575, 1986.

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.