Erythrocyte adenosine kinase activity in gout

15

Clinica Chimica Acta, 67 (1976) 15-20 0 Elsevier Scientific Publishing Company,

Amsterdam

- Printed

in The Netherlands

CGA 7505

ERYTHROCYTE

TSUNEO

ADENOSINE

NISHIZAWA,

YUTARO

KINASE ACTIVITY

NISHIDA

Department of Medicine and Physical Therapy, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113 (Japan)

(Received

August

IN GOUT

and IEO AKAOKA Faculty

of Medicine,

University

of Tokyo,

18, 1975)

Summary

1. Erythrocyte adenosine kinase (AK) (EC 2.7.1.20) and guanosine monophosphate (GMP) reductase (EC 1.6.6.8) were measured in healthy male controls and primary gouty subjects. Adenosine kinase activity in 19 controls and 26 gouty subjects was 0.717 f 0.176 and 0.615 * 0.128 nmol/mg protein/h, respectively. The difference was statistically significant (P < 0.05). GMP reductase activity in 39 controls and 46 gouty subjects was 30.90 f 6.28 and 33.43 f 7.97 pmol/mg protein/h, respectively, without statistically significant difference. 2. When gouty patients were divided into two groups according to gouty heredity and/or tophus formation, a group with such clinical traits had a lower adenosine kinase activity when compared with another group having none of these clinical features (P < 0.10). Adenosine kinase activity had negative correlations to serum urate level (r = -0.433, P < 0.05) and to erythrocyte adenosine deaminase (ADA) activity (r = -0.689, P< 0.05). The possible biochemical role of adenosine kinase activity in purine metabolism and in the pathogenesis of primary gout is discussed.

Introduction

The pathogenesis of hyperuricemia in primary gout can be attributed to a reduced ability of the kidney to excrete uric acid, an excessive production of uric acid, or a combination of these two factors. However, it is difficult to be certain if some patients with primary gout do have a specific renal abnormality in urate transport, because our understanding of the normal process involved in the renal handling of uric acid is relatively incomplete [1,2]. On the other hand, there have been substantial recent advances in understanding the pathogenesis of excessive uric acid production. The main causes of overproduction of uric acid in some gouty patients are a partial deficiency of hypoxanthineguanine phosphoribosyltransferase (HGPRT) [ 31, a defective phosphoribosyl-

16

pyrophosphate amidotransferase [ 41 and an increased phosphoribosyll)yrophosphate synthetase (PRPP synthetase) [ 5,6]. Gouty patients with abnormalities of these enzymes, however, are a small minority and the cause of hyperuricemia in the majority of primary gouty patients is still poorly understood. We reported that erythrocyte adenosine deaminase (ADA) activity was significantly elevated in primary gout [ 71 . In order to elucidate the cause of elevated ADA activity in primary gout, measurements of erythrocyte adenosine kinase (AK) and guanosine monophosphate (GMP) reductase were carried out. Adenosine kinase (ATP:adenosine-5’-phosphotransferase, EC 2.7.1.20) phosphorylates adenosine to adenosine monophosphate (AMP). GMP reductase (NADPH:GMP oxidoreductase (deaminating), EC 1.6.6.8), a NADPH-linked of GMP to inosine monophosenzyme, catalyzes the reductive deamination phate (IMP). Both enzymes are known to occur in human red blood cells and their characteristics have been studied in detail [S-+11] . Materials and methods Measurements of erythrocyte GMP reductase were carried out on 46 primary male gouty subjects, average age 46.8 years (range 23-84 years). The majority of gouty subjects were out-patients, including 22 non-treated subjects. The average age of 39 male healthy controls was 46.7 years (range 3048 years). Measurement of erythrocyte AK activity was made on 26 patients, randomly selected from 46 above-mentioned gouty patients. The mean age of 26 gouty subjects was 46.7 years (range, 27-72 years). The erythrocyte AK activity was determined in 19 healthy persons, randomly chosen from a control group. The age of the 19 controls averaged 46.3 years (range, 36-68 years). After an overnight fast, whole blood was obtained by venipuncture, and collected in a heparinized tube which was centrifuged at room temperature and 1000 X g for 5 min. The plasma, buffy coat and the upper $ layer of the red blood cell column, being rich in reticulocytes, were discarded. The packed erythrocytes were washed three times with 0.9% saline and centrifuged in the same way. The packed erythrocytes were diluted with 1 vol. of water and erythrocyte lysates were prepared by twice freezing-thawing the diluted erythrocytes. The supernatant of the hemolysate, after centrifugation at 15000 X g at 4°C for 10 min, was dialysed overnight against 200 vol. of 0.01 M phosphate buffer (pH 7.4) at 4°C. Erythrocyte GMP reductase activity was measured by the method of Mackenzie [ 111, using the NADPH system with minor modifications. 100 1.11of the 1:20 diluted hemolysate was employed and the final reaction mixture was 3.1 ml consisting of 33 mM potassium phosphate buffer (pH 7.5), 4.8 mM 2-mercaptoethanol, 165 FM GMP and 200 PM NADPH. The reaction was stopped by the addition of 396 PM xanthosine monophosphate (XMP). Erythrocyte AK activity was measured by the method of Meyskens and Williams [8] with minor modified experimental conditions, using 100 ~1 of the 1:2 diluted hemolysate in 1.1 ml of the final reaction mixture. The reaction was stopped by 0.1 mM p-chloromercuribenzoate [12]. The reaction products were separated by thin-layer chromatography, using Eastman cellulose chromatogram sheet (Eastman Kodak Company). The solvent system used consisted

of 0.1 M phosphate buffer (pH 6.8), saturated ammonium sulfate and n-propanol (100:60:2, v/v) 1133. E~throcyte ADA activity was measured by a modified Hopkinson’s method as described previously [ 71, Erythrocyte protein was determined by a modified Lowry’s method [ 143 and serum uric acid was measured by an automated calorimetric method. Results Mean erythrocyte GMP reduetase and AK activities in healthy males and gouty subjects are listed in Table I. Mean AK activity in gouty subjects was significantly lower than in controls (P < 0.05). Mean GMP reductase activities in the two groups were not significantly different. A mixture of hemolysate from a gouty patient with normal hemolysate resulted in the expected intermediate level of AK and GMP reductase activity, respectively. Relations of AK activity to percentage overweight, renal impa.irment, gouty heredity and tophus formation in gouty subjects are shown in Table II. In two groups of gouty subjects divided according to percentage overweight, the difference in mean AK activities was not statistically significant. In 8 controls having a percentage overweight greater than llO.O%, AK activity averaged 0.758 + 0.162 nmol/mg protein/h, while in 11 healthy persons having a percentage overweight less than 109.9%, mean AK activity was 0.657 +_0.203 nmol/mg protein/h, without significant difference in the two control groups. In the group, having impairment of renal function (proteinuria, abnormal urine sediment, and/or below 25% at 15 min of PSP retention), the average AK activity was slightly lower than in the group having no renal disturbance, with no significant difference. In the group with gouty heredity and/or tophi, the mean AK activity was significantly lower than in the group having neither go&y heredity nor tophi (P < 0.10). In 15 treated gouty subjects AK activity averaged 0.629 + 0.125 nmol/mg protein/h, while in 11 non-treated gouty subjects the mean AK activity was 0.617 + 0.093 nmol/mg protein/h, with no significant difference in the two gouty groups. With respect to the factors of overweight, renal impa~ment, gouty heredity and tophus formation, no definite differences were seen in the GMP reductase activity of gouty subjects. A relationship between serum urate level and erythrocyte AK activity, with a significant negative correlation coefficient (r = -0.433, P < 0.05), in 19 controls and 11 non-treated gouty subjects is shown in Fig. 1. The relation of AK activity to ADA activity in 9 gouty subjects also shows a significant negative correlation (r = -0.689, P < 0.05) (Fig. 2). Erythrocyte GMP reductase shows

TABLE1 ERYTH~~YTEGMPREDUCTASEANDADENOSINEKINASEACTIVITY(MEAN~S.D.) -GMP reductase Contro1 Gout

*v <

0.05.

n = 39 n = 46

(pmol/mg 30.90 33.43

protein/h)

t 6.28 f 7.97

Adenosine n = 19 n = 26

kinase (nmol/mg 0.717 0.615

protein/h)

+ 0.176 f 0.128

*

18

TABLE:

II

RELATIONS AGE

OF

ERYTHROCYTE

OVERWEIGHT,

GOlJTY

RENAL

ADENOSINE

GOUTY

ACTIVITY HEREDITY

(MEANS AND

’ S.D.)

TOPHIJS

TO

PF:KCF:NT-

FORMATION

IN

SURJECTS

Percentage

overweight

< 109.9% No. km&r protein/h)

* Calculated

from

the

*

~110.0%

15

Adenosme (nmol/mg

KINASE

IMPAIRMENT.

10

Renal

impairment

Goutv

hereditv

tophus

formation

and/or

+

+ 10

16

10

15

0.562

0.646

0.586

0.648

0.577

0.658

0.192

0.092

0.152

0.105

0.123

0.095

formula:

Percentage

overweight

= 100

X Weight/[(Height

100)

no significant relationship to serum urate level and erythrocyte for both gouty patients and healthy persons.

X 0.91

ADA activity

Discussion As mentioned in the introductory remarks, the specific abnormalities leading to defective uric acid excretion have not been welldefined. The majority of gouty subjects were out-patients, so urinary uric acid excretion or uric acid clearance under purine free diet was not studied. It was therefore very difficult to divide gouty patients into overproducers or underexcretors of uric acid. The mean AK activity in gouty patients was statistically lower than in controls (P < 0.05). The average AK activity in the group with gouty heredity and/or tophus formation, including only 5 patients with gouty heredity alone, tended

Fig.

1.

Relationship

between

serum

urate

Fig.

2.

Relationship

betwern

adenosine

level

kinase

and

erythrocyte

and

adenosine

adenosinr

deaminase

kinase

in gout

activity.

19

to be lower than in another gouty group having none of these two clinical traits (P < 0.10). Therefore, gouty subjects with abnormalities of HGPRT and PRPP synthetase, which were not measured in the present study, were possibly very few in number, if any, because patients with abnormalities of these two enzymes form a very small minority of the gouty population. The low value of erythrocyte AK activity in gouty patients could not be attributed to the presence of an inhibitor or to the absence of an activator of the enzyme in erythrocytes. Patients from four different families who are heterozygous for a deficiency of adenine phosphoribosyltransferase (APRT) have been described [ 15-181 . Although some of these patients have been noted to be hyperuricemic, there does not appear to be a direct relationship between heterozygosity for APRT and an aberration in purine metabolism [16,17,18]. Recently a case with complete APRT deficiency, with a serum urate level of only 6 mg/lOO ml, has been reported [ 191. The inference drawn from these findings is that APRT relatively lacks importance in uric acid metabolism and in maintaining a constant AMP level, which has the feedback inhibition upon PRPP amidotransferase, a ratelimiting step of de novo purine synthesis. Early results with purified purine nucleoside phosphorylase from human erythrocytes indicated that it did not react with adenosine [20,21]. The utilization of adenine in human tissues occurs mainly through the reaction catalyzed by APRT, because alternate pathways of utilization are either nonexistant or insignificant [ 221. An AK deficient mutant of the mouse fibroblast has recently been obtained in cell culture. In the mouse fibroblast the activity of AK is essential to retard the metabolic cycle of adenosine and to prevent cellular loss of purines [23]. According to the observations of Meyskens and Williams, AK activity seems to be important in the regulation of adenine nucleotide levels in the erythrocytes [8] . Our previous and present studies show that erythrocyte ADA activities in gouty patients are elevated significantly [7] and have a significant negative correlation to their erythrocyte AK activities (Fig. 2). Further, erythrocyte AK activity has a significantly negative correlation to human serum urate level (Fig. 1). Therefore, it is reasonable to presume that AK is more important than APRT in salvage pathways which maintain the level of AMP. If an AK deficient state was to occur in the human, an excessive deamination of adenosine, namely an elevation of ADA activity would occur and a condition of clinical gout would probably result. It is, therefore, logical to believe that an overproduction of uric acid resulting from AK deficiency may exist in some gouty patients. References 1

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