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Liver xanthine oxidase in gouty patients.

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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 1.2.3.2.) 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. J., and Smellie, J. M.: Xan-
thinuria. Brit. Med. J. 2: 1217, 1959.
4. Ayvazian, J. H.; Xanthinuria and hemochromatosis. New Eng. J. Med. 2:18, 1964.
5. Engelman, K., Watts, R. W. E., Klinenberg,
J. R., Sjoerdsma, A., and Seegmiller, J. E.: Clinical,
physiological and biochemical studies of a patient
with xanthinuria and pheochromocytoma. Amer. J.
Med. 37:839, 1964.
6. Watts, R. W. E., Engelman, K., Klinenberg,
20
J. R., Seegmiller, J. E., and Sjoerdsma, A.: Enzyme
defect in a case of xanthinuria. Nature 201:395,
1964.
7. Shammaa, M. H., Nasrallah, S., Chaglassian,
T., Khachadurian, A. K., and Khalidi, A.: Serum
xanthine oxidase: a sensitive test of acute liver
injury. Gastroenterology 48:226, 1965.
8. Hall, A. P., Holloway, V.P., and Scott, J. T.:
4-Hydroxypyrazolo-( 3,4-d)-pyrimidine ( HPP) in
the treatment of gout. Ann. Rheum. Dis. 23:439,
1964.
9. Rundles, R. W., Silberman, H. R., Hitchings,
G. H., and Elion, G. B.: Effects of xanthineoxidase inhibitor on clinical manifestation and
purine metabolism in gout. Ann. Intern. Med. 60:
717, 1964.
10. Klinenberg, J. R., Goldfinger, S. E., and
Seegmiller, J. E.: The effectiveness of the xanthine-oxydase inhibitor allopurinol in the treatment of gout. Ann. Intern. Med. 62:639, 1965.
11. Krakoff, I. H., and Meyer, R. L.: Prevention
of hyperuricemia in leukemia and lymphoma:
use of allopurinol, a xanthine-oxidase inhibitor.
J.A.M.A. 193:1, 1965.
12. Marcolongo, R., Carcassi, A., Lunghetti, R.,
Bravi, A., Bianco, G., and Di Paolo, N.: L’allopurinolo nel trattamento delle iperuricemie primitive e secondarie. Metabolism0 3:593, 1967.
13. Marcolongo, R., Carcassi, A., and Lunghetti,
CARCASS1 ET AL.
R.: Modeme vedute sulla terapia della gotta. Med.
Clin. Sperim. 17:490, 1967.
14. Archibald, R. M.: Colorimetric measurement
of uric acid. Clin. Chem. 3: 102, 1957.
15. Marinello, E., Martelli, P., Verme, G., and
Gioannhi, P.: Determinazione di alcune attiviti
enzimatiche in frammenti bioptici di fegato umano.
Biochim. Appl. 9:11, 1962.
16. Burch, H. B., Lowry, 0. H., Padilla, A. M.,
and Combs, A. M.: Effects of riboflavin deficiency
and realimentation on flavin enzymes of tissues.
J. Biol. Chem. 223:29, 1956.
17. Lowry, 0. H., Rosebroug, N. J., Farr,
A. L., and Randall, R. J.: Protein measurement
with the folin phenol reagent. J. Biol. Chem.
193:265, 1951.
18. Stirpe, F., and Della Corte, E.: Regulation
of xanthine dehydrogenase in chick liver. Effect of
starvation and of administration of purine nucleotides. Biochem. J. 94:309, 1965.
19. Kelley, W. N., Rosenbloom, F. M., and
Seegmiller, J. E.: The effects of azathioprine
(Imuran) on purine synthesis in clinical disorders
of purine metabolism. J. Clin. Invest. 46:1518,
1967.
20. Kelley, W. N., Rosenbloom, F. M., Miller,
J., and Seegmiller, J. E.: An enzymatic basis for
variation in response to allopurinol. New Eng. J.
Med. 278:287, 1968.
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