Liver Xanthine Oxidase in Gouty Patients By ALDOCARCASSI, M.D., ROBERTO MARCOLONGO, JR., M.D., ENRIC~ MARINELLO,M.D., RIAFUO-SFORZA, M.D., AND CARLOBOGGIANO, M.D. GJXJSEPPE The activity of xanthine oxidase was studied in liver biopsy material from normal subjects and patients with primary gout. Increased enzyme activity was present in all patients with gout. The possibility that increased xanthine oxidase activity may represent a primary biochemical lesion in gout is discussed. X OXIDASE (xanthine: 02 oxpreviously reported subjects with xanthine idoreductase, EC 126.96.36.199.) is an en- stones may have had xanthinuria, but in zyme which in man has special importance none were studies of xanthine excretion in the final steps of purine catabolism, performed. Recent works have further stimulated where it catalyzes the aerobic dehydrogenation of hypoxanthine to xanthine and of interest in xanthine oxidase. Since the enxanthine to uric acid. zyme in man is found mainly in the liver, The literature contains little data on the its increase in the plasma should be an activity of xanthine oxidase in man, prob- index of hepatocellular lesion; its imporably because of technical daculties in the tance in the enzymatic diagnosis of hepatic assay of the enzyme in small quantities of diseases, and specifically of acute hepatitis, The rebiological material. Watts et a1.l have dem- has recently been demon~trated.~ onstrated that in man only liver and the cent introduction of allopurinol in the treatsmall intestine mucosa contain more than a ment of primary and secondary hyperuricetrace of xanthine oxidase activity. Investi- mias-13 has also focused the attention on gation of this enzyme assumed a special xanthine oxidase. Allopurinol ( 4-hydroimportance in human pathology following xypyrazolo [3,4-d] pyrimidine ) , an isomer reports of 3 well-documented cases of xan- of xanthine, competitively inhibits the forthinuria,2-6characterized by the excretion mation of uric acid by xanthine oxidase. of xanthine as the major urinary end prod- Allopurinol has elucidated some pathogeuct of purine metabolism. It has been netic aspects of hyperuricemia in gouty postulated that this disease is due to a con- patients and specihally the role of xanthine genital deficiency of xanthine oxidase.4 One oxidase. The purpose of this paper is to reof the cases was associated with pheochro- port on the activity of xanthine oxidase in m o ~ y t o m a It . ~ is likely that some of the 32 the liver of patients suffering from gout, ANTHINE From the Departments of Medicine, Infectious Diseases, and Biochemistry, University of Siena, Siena, Italy. ALDO CARCASSI,M.D.: Assistant Professor, Department of Medicine (Libero Docente in Semeiotica Medica). ROBERTOMARCOLONGO, JR., M.D.: Assktant Professor, Department of lnfectious Diseases (Libero Docente in Patologia Speciale Med- ica). ENRICOMARINELLO, M.D.: Assistant Professor, Department of Biochemistry (Libero Docente in Chimica Bidogica). GNSEPPE RIARIOSFORZA,M.D.: Assistant Professor, Department of Biochemistry. CARLOBOGGMNO,M.D.: Assistant Professor, Department of Infectious Diseases. Reprint requests should be addressed to Dr. Marcolongo. ARTHRITIS AND RHEUMATISM, VOL.12, No. 1 ( FEBRC rmy 1969) 17 18 CARCASS1 ET AL. Table l.-Xanthine Oxidase Activity in Normal Subjects and Gouty Patients * Normal subjects Xanthine oridase Serumuric acid, mg. % Gouty patients u* unc aud, mg./24 hr. 0.030 0.042 0.058 3.10 4.20 2.90 0.042 3.00 0.023 2.20 0.031 3.90 0.032 4.00 0.048 2.50 0.022 3.30 0.024 1.90 0.042 3.80 0.058 2.70 Mean = 0.037 f0.0045 * Enzyme activity is expressed 460 390 500 440 380 520 280 360 470 300 290 520 Xanthine oxidase Serumuric acid,mg.% u n c aud mg.124 h;. 0.100 0.490 0.105 0.062 0.160 0.090 0.114 0.123 6.80 10.00 12.20 11.30 12.10 5.60 10.00 8.10 890 1150 1080 1200 890 920 880 980 urinarly Mean = 0.155 f0.29 in pmoles of substrate transformed in 60 min./mg. protein. and to compare it with that of control material. MATERIALS AND METHODS Experiments were carried out on 8 male patients, aged 35-73, affected by primary gout; the controls were 12 subjects of both sexes, aged 20-55, who were surely not affected by gout or by any other metabolic disease. All the patients were submitted to the usual routine tests necessary for the diagnosis. Serum and urinary uric acid were determined by the modified method of Archibald.14 The gouty patients had a net overproduction of uric acid, evaluated through 24 hr. urinary excretion, as indicated in Table 1. Hepatic function tests were performed on all patients to exclude the possibility of hepatic disease. The gouty patients had previously received neither uricosuric drugs nor allopurinol, but only antiphlogistic drugs (colchicine, indomethacin). All drugs were suspended for one week, after which they were submitted to hepatic biopsy, using Menghini’s needle, following the procedure described by Marinello et al.15 The hepatic specimen thus obtained was washed with saline solution, blotted with filter paper, and immediately homogenized at O°C for 1 min. in phosphate buffer, 0.2 M, p H 7.6, using a Potter-Elvehjem glass homogenizer. With this procedure 3 per cent homogenates in phosphate buffer were prepared. Xanthine oxidase activity was evaluated by the fluorimetric method of Burch et al.16 with minor modifications, Incubation mixtures contained 2amino-4-hydroxypteridine, 1 X 10-5 M, and liver in a concentration of 2 mg./ml. of substrate. The incubation was carried out at 37OC for 30 min. Readings were taken at zero time and every 5 min. in a fluorimeter (Turner model 110) at 395 mp (filter 7-60) and an emission wavelength of 460 mp (filter 2 A ) ; sensitivity was reduced 100 times with a filter Kodak N. 96. A blank consisting of 4 ml. of phosphate buffer, 0.2 M, p H 7.6, was run in parallel, with a quantity of tissue equivalent to that of the samples. Enzymatic activity was expressed in pmoles of substrate transformed in 60 min. per milligram of protein, the latter being determined by the method of Lowry et al.17 RESULTS Results are reported in Table 1 and Figure 1. In 12 normal subjects, activity of hepatic xanthine oxidase was on the aver0.0045 pmoles/60 min./mg. age 0.037 protein, while in the gouty patients the average was 0.155 k 0.29 pmoles/60 min./ mg. protein. The difference between the two averages was statistically significant ( p < 0.05). * DISCUSSION From our results it is evident that xanthine oxidase is increased in the liver of the patients affected by primary gout, in comparison with the controls. The increase of 19 LIVER XANTHINE OXIDASE AND GOUT 0 0 Ck 0.100 0 0 0 <*I, 0 < -2 a0601 2 + 0 .,I (1020 I NORMAL PATI E NTS GOUTY PATIENTS Fig. 1.-Levels of xanthine oxidase activity in liver of normal subjects and gouty patients. xanthine oxidase activity in the gouty patients, which was evident in all the cases we have tested, is difficult to understand. Since xanthine oxidase is an inducible enzyme,ls its increase could be primary, genetically determined, or secondary to possible modifications of phosphoribosylpyrophosphate-amidotransferase activity related to factors governing the feedback mechanisms, as suggested by various aut h o r ~ , *or~ to other factors still unknown. We are planning to evaluate xanthine oxidase activity in secondary gout associated with overproduction of uric acid (e.g., polycythemia Vera, leukemia) to elucidate this point. No definite conclusion regarding the causal role of xanthine oxidase in the overproduction of uric acid in primary gout is possible at the present time. Other enzymatic activities have been shown to have peculiar behavior in gouty patients and their role has to be studied. Recently, e.g., Kelley et a1.20 have demonstrated a deficiency of the enzyme hypoxanthineguanine phosphoribosyl transferase in some patients with gout. In any case, the study of xanthine oxidase and other enzymes of purine metabolism, e.g., the hepatic phosphoribosyl-pyrophosphate-amidotransferase activity, in gouty patients, seems to be useful in understanding the pathogenesis of gout on a biochemical basis. SUMMAFUO IN INTERLINGUA Le activitate de oxydase de xanthina esseva studiate in material de biopsia hepatic ab subjectos normal e ab patientes con gutta primari. Augmentate activitate del enzyma esseva presente in omne le patientes con gutta. Es commentate le possibilitate que augmentos del activitate de oxydase de xanthina representa un primari lesion biochimic in gutta. REFERENCES 1. Watts, R. W. E., Watts, J. E. M., and Seegmiller, J. E.: Xanthine oxidase activity in human tissues and its inhibition by allopurinol (4-hydroxypyrazolo[3,4-d]pyrimidine). J. Lab. Clin. Med. 66:688, 1965. 2. Dent, C. E., and Philpot, G. R.: Xanthinuria: an inborn error (or deviation) of metabolism. Lancet 1:182, 1954. 3. Dickinson, C. 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