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Phosphoribosyl pyrophosphate synthetase and glutathione reductase in erythrocytes from hyperuricaemic and gout patients
217
C&&n Chit&a Acta, 126 (1982) 217-226 Elsevier Biomedical Press
CCA 2328
Phosphoribosyl pyrophosphate synthetase and glutathione reductase in erythrocytes from hyperuricaemic and gout patients T.R. Hardwell a, J. Braven b,* , S. Shaw b and Mary Whittaker’ a Department of Pathology, Torbay Hospital, Torquay, Devon (UK), h Department of Environmental Sciences, P&mouth Polytechnic, Drake Circus, Plymouth, Devon PL4 SAA (UK) and’ Depurtment Chemistry, Exeter lJnit;ersir_y, Exeter, Devon (UK) (Received
of
April 22nd, 1982)
Summary The enzymic activities of erythrocyte phosphoribosyl pyrophosphate synthetase (EC 2.7.6.1) and glutatbione reductase (EC 1.6.4.2) have been measured in 54 primary gout patients, 35 indi~duals having h~eruricae~a and 51 healthy controls. Statistical analyses have shown a significant increase ( p < 0.01) in the enzymic activity of erythrocyte PRPP synthetase in both the hyperuricaemic and gout groups compared with the controls. No correlation between activity and age was found in any of the three clinical groups. A significant decrease ( p = 0.01) was found in the enzymic activity of red cell glutathione reductase in the gout group compared with the other two groups. The biochemical significance of the changes in enzymic activities of the two enzymes in primary gout is discussed.
introduction The excessive production of uric acid, which occurs in patients having primary gout, has been attributed to defects that result in the formation of increased concentrations of phosphoribosyl pyrophosphate (PRPP), a substrate for the first stage of de novo purine biosynthesis [ 11. Although this reaction, catalysed by PRPP glutamine amidotransferase (EC 2.4.2.14), is subjected to feedback inhibition by purine nucleotides, the presence of excessive amounts of PRPP not only stimulates the purine biosynthesis but nullifies the allosteric inhibition by the purine nucleotides. Independent reports have shown elevation of erythrocyte PRPP synthetase activity of individuals with familial gout [2,3]. Further investigations have revealed distinct differences in the kinetic properties of red cell PRPP synthetase occurring in different families. It therefore seems likely that the clinical condition of gout could * To whom correspondence 0009-8981/82/0000-0000/$02.7$
should be addressed
Q 1982 Elsevier Biomedical
Press
218
be associated with a number of variants of PRPP synthetase. Investigations of any changes in the properties of this enzyme in primary gout patients should also be performed on a group of asymptomatic hyperuricaemic individuals with no family history of gout and a group of healthy control individuals. An increased enzymic activity of erythrocyte glutathione reductase has been reported in a group of Caucasians with untreated primary gout [4] and similar findings were reported in 23/28 Negro gout patients [5]. Control screening of a large negro population indicated that - 31% had an increased enzymic activity and a higher mean serum urate concentration than the 69% with a ‘normal’ enzymic activity [6]. Although one must have some reservations about the significance of changes in enzymic activity in any pathological state, it nonetheless seemed worthwhile to find out whether the reported elevation of enzymic activities of PRPP synthetase and glutathione reductase in erythrocytes from patients with gout was substantiated in a statistically valid group of subjects.
Materials and methods Measurements were carried out on 54 gout patients who had been diagnosed by their general practitioner on the basis of clinical criteria and a raised level of serum uric acid. All specimens received from GPs were processed on arrival at the laboratory within 4 h of the sample being taken. Control samples were obtained from 51 healthy volunteers. Any blood sample having a plasma uric acid level greater than 420 pmol .l-’ was automatically transferred to the hyperuricaemic group. The hyperuricaemic group of patients consisted of 35 individuals having a high metabolic plasma uric acid content as well as hospital patients with uremia. One individual with a long history of hyperuricaemia but whose plasma uric acid was currently controlled chemotherapeutically by allopurinol was also included in this group to ascertain whether chemotherapy resulted in any change of enzyme activity. Venepuncture samples (10 ml) were collected in lithium heparin. The samples were centrifuged at 1000 x g for 10 min and the plasma and buffy coat separated. The packed erythrocytes were washed three times with physiological saline and centrifuged at 1000 X g for 10 min. After the final wash, the original volume of blood was restored by adding physiological saline to the packed erythrocytes. An aliquot (1 ml) of the homogenous suspension of red cells in saline was added to ice-cold water (8 ml) and mixed for 10 min on a rotor mixer. Phosphate buffer (8 ml, 90 mmol . l- ‘, pH 7.4 containing 4 mmol . l- ’ EDTA) was added to the laked cells and mixed for 10 min on the rotor mixer prior to centrifugation at 1000 X g for 10 min. The supernatant was aliquoted into plastic tubes and stored frozen until required. The protein content of the haemolysate was measured in a Gilford Staser III using the biuret reagent and a standard reference serum. The assay of PRPP synthetase was a modification of that reported by Valentine and Kurschner [7] which monitors the formation of AMP, using the following linked
219
systems: PRPP
R-5-P + ATP
--j
PRPP + AMP
synch&m
adenylic
AMP + ATP f
kinase
2)
2ADP
phosphokinase
2PEP+2ADP
2 pyruvate
+ 2 ATP
(2, lactic acid dehydrogenase
2 pyruvate
+ 2 NADH
+ 2 Hi+
f&
2 lactate + 2 NAD+
All reagents were of analytical grade except for the enzymes, ATP (disodium salt) and ribose-5-phosphate (disodium salt), which were obtained from Boehringer Ltd. PEP (monocyclohexyl ammonium salt) and NADH were obtained from BDH Ltd. The assay solution contained the following concentrations (mmol . l- ‘) of reagents: MgCl, = 2.00, ATP = 1.33, PEP = 2.00, KF = 3.33, NADH = 0.23, AK 5 y1 (720 units - ml- I), PK 2 ~1 (2000 units. ml “-I), LDH 2 ~1 (2750 units. ml- ‘) in 2 ml phosphate buffer (67.5 mmol *l- ’ pH 7.4). 200 ~1 haemolysate were added to 2 ml assay reagents in a blank and a test cuvette and incubated at 37°C for 30 min. Ribose-S-phosphate (20 ~1, 66.6 mmol - 1-l) and water (20 ~1) were added to the test and blank cuvettes respectively. The change in absorbance at 340 nm at 37°C was monitored continuously on a Gilford 3500 rate analyser linked to a recorder. Enzymic activity, expressed as pmol substrate per mg . ml-’ protein AA/P, where AA = change in absorbance/h and P is the concentration mg . ml- ’ of haemolysate.
per h = 892.3 of protein in
Glutathione reductase was assayed by the method of Lee Kum-Tatt et al [8f. The mean enzymic activities of PRPP synthetase and glutathione reductase from the three clinical groups were compared statistically using a one-way analysis of variance followed by a multiple comparison by Students’ r-test. Statistical analysis was carried out using the MINITAB computer package. Results The mean plasma uric acid content of 140 individuals divided into three clinical groups is given in Table I. The data has been further divided according to sex and included in this table. Histograms of PRPP synthetase activity for normal and hyperuricaemic individuals and individuals suffering from gout are shown in Fig. 1, together with confidence limits of the mean activities for the three groups. The mean enzymic activities of these groups, subdivided into sex, are included in Table I.
220
20 -
10 .
1 0
. 100
I 200
-a 300
20
10
0
HVPERURICAEMIC
GOUT :fl?ERKJRICAEViC
Fig. 1. Histograms of the enzyme activity, and 95% confidence intervals for the mean enzyme activity, erythrocyte PRPP synthetase in three clinical groups. Ordinate = number of individuals; abscissae activity units in pmol substrate’mg-‘.ml protein.h-‘.
of =
The distribution of the activities of erythrocyte glutathione reductase and the confidence limits of the mean activities of the three groups is presented in Fig. 2. Analysis of the mean enzymic activities of each group when divided by sex is given in Table I.
221
20
NORMAL 10
0 20
GOUT
10
A-loo
200
300
HYPERURICAEMIC
l
100
2Co
300
NORVhI. GOUT HYPEWRICAEHIC
1
--
65
1
95
125
155
Fig. 2. Histograms of the enzyme activity, and 95% confidence intervals for the mean enzyme activity, erythrocyte giutathione reductase in three clinical groups. Ordinate = number of individuals; abscissae activity units in pmol substratesmg-‘*ml protein’h‘.
of =
Discussion It is apparent from the data of Table I that there is an uneven sex distribution in our two pathological groups. The selection of gout patients was made from the diagnosis of a GP or consultant. It is significant that 47 of 54 patients were male. This uneven distribution of the sexes in our sample indicates that more males than
222
TABLE
I
MEAN PLASMA URIC ACID, ERYTHROCYTE PRPP SYNTHETASE ACTIVITY AND ERYTHROCYTE GLUTATHIONE REDUCTASE ACTIVITY IN 140 INDIVIDUALS CLASSIFIED INTO THREE CLINICAL GROUPS Standard Clinical
deviations group
are given in brackets. Number of individuals
Mean plasma uric acid (pmol.ll’)
Mean erythrocyte PRPP synthetase (pm01 substrate. mg -‘.rnl protein’h-‘)
Mean erythrocyte glutathione reductase (am01 substrate. mg -‘.ml protein. h- ‘)
51 54 35
315 (76) 481 (138) 527 (153)
96 (31.2) 122 (44.2) 113 (34.7)
107 (62.0) 83 (42.7) 120 (74.7)
21 47 23
336 (67) 492 (137) 508 (146)
106 (29.6) 120 (37.6) 121 (35.6)
102 (69.9) 82 (43. I) 127 (76.3)
30 I 12
301 (80) 404 (135) 564 (166)
88 (30.6) 134 (79.1) 99 (29.2)
111 (56.7) 87 (42.6) 107 (72.9)
Total
normal gout hyperuricaemic M&
normal gout hyperuricaemic FCWUlle
normal gout hyperuricaemic
females are diagnosed clinically as having gout, in the Torbay area. The selection of the control group and hyperuricaemic individuals was essentially identical and therefore any variation in results is not due to the distribution of sexes and should be of pathological origin. As indicated above there is a higher incidence of hyperuricaemia in males than in females, and this is confirmed by an analysis of hospital records (Table II). Van Maris et al [9] have measured PRPP and some enzymes of purine metabolism in erythrocytes from 43 young hyperuricaemia males. They report that the mean erythrocyte PRPP synthetase activity in their hyperuricaemic individuals was slightly but significantly ( p < 0.01) higher than the means of their small control group (10 males). Meyskens and Williams [lo] have assayed the concentration and synthesis of PRPP in erythrocytes from normal, hyperuricaemic and gout-suffering subjects and found no difference in the mean PRPP synthetase activity. In the case of our gout patients, the large standard deviation found overall, and for females (Table I), is mainly due to an exceptionally large value recorded for one individual. Statistical analysis, shown in Table III, indicates a significant increase in enzymic activity for the gout and hyperuricaemic groups compared with the healthy controls ( p < 0.05) and is at variance with the results of Meyskens and Williams [lo]. The increase in enzymic activity of PRPP synthetase is inadequate for use in the diagnosis of gout. The confidence limits in Fig. 1 clearly illustrate these differences in the mean PRPP synthetase activities of the three groups. The reliability of the
223
TABLE
II
PERCENTAGE LABORATORY
OF HYPERURICAEMIC SAMPLES FOUND DURING SCREENING FROM JANUARY 1976 TO APRIL 1978
Year
Percentage of hyperuricaemic
Number of individuals investigated
1976 1977 1978
A ROUTINE
CLINICAL
samples
male
female
total
male
female
total
1876 1946 709
1278 1327 479
3 154 3273 1188
46.5 42.8 42.5
23.2 21.9 24.0
37.1 34.3 35.0
4531
3084
7615
44.2
22.8
35.6
(Jan-April) Jan 1976 to April
1978
different assay methods of PRPP synthetase has long been suspect; the origin of this is the apparent instability of the enzyme. Our assay has not only been compared with other spectrophotometric systems [7,11,12] but has been subjected to the analysis of day-to-day variation and to the optimal conditions for maintaining enzymic activity following storage of haemolysates at -20°C. Details of these investigations will be published elsewhere. We have as yet no evidence for a genetic variant of PRPP synthetase characterised by high enzymic activity as reported by Becker et al [3]. It is, however, proposed to investigate in depth repeat samples from the female patient mentioned above and to study her family should any unusual properties of the enzyme emerge. A significant decrease (p < 0.05) in erythrocyte glutathione reductase, compared to normal and hyperuricaemics, was found in gout patients (Table III). There is no significant difference in this mean enzymic activity in the normal and hyperuricaemic groups (Table III). The difference between the control group and the gout patients may be reduced by an age effect. The two pathological groups were on average older than the control group. A positive correlation (0.53) with age has been found for erythrocyte glutathione reductase in the controls, but there is no such correlation in either pathological group. It can be assumed that there is something swamping the age correlation in the gout and hyperuricaemia groups, but it is not necessary to consider the age factor in either group because the difference in the glutathione reductase activities would be magnified by taking age into account. There is no correlation of PRPP synthetase with age in any of the three groups (Table IV). We can, therefore, reject any age effect. There is a good correlation between erythrocyte PRPP synthetase activity and erythrocyte glutathione reductase activity in both the hyperuricaemic and gout groups, but no correlation of these two enzymes in the control group (Table V). The mechanism causing the reduction of the mean erythrocyte glutathione reductase activity in the gout group might not be responsible for the increase in the mean PRPP synthetase activity in this group of patients since a positive correlation between the two enzymes is observed for all three clinical groups. Plasma uric acid
224 TABLE
III
STANDARD ERROR ERYTHROCYTE PRPP CAL GROUPS Group comparison
Normal gout
OF DIFFERENCE BETWEEN MEAN ENZYMIC SYNTHETASE AND GLUTATHIONE REDUCTASE
PRPP synthetase
Glutathione
Standard error of difference between mean
1
Significance *
Standard error of difference between means
ACTIVITIES OF IN THREE CLINI-
reductase
I
Significance *
vs. 1.34
- 3.60
xxx
11.54
2.12
Normal vs. hyperuricaemic
8.25
-2.11
x
12.97
~ 1.00
NS
Gout vs. hyperuricaemic
8.15
NS
12.82
-2.93
xx
* Significant
TABLE
1.10
IV OF AGE WITH ENZYMIC
Enzyme
Clinical
PRPP synthetase
Glutathione
reductase
xxx, Significant
ACTIVITIES
type
IN 3 CLINICAL
GROUPS
OF INDIVID-
Number of individuals
Correlation coefficient
Significance
normal gout hyperuricaemic
51 54 35
0.10 -0.21 -0.21
NS NS NS
normal gout hyperuricaemic
51 54 35
0.53 0.04 0.003
xxx NS NS
at 1 in 1000; NS, not significant.
V
CORRELATION ERYTHROCYTES Clinical
I in 100; x, 1 in 20; NS, not significant
at: xxx, 1 in 1000; xx,
CORRELATION UALS
TABLE
x
group
Normal Gout Hyperuricaemic xx, significant
OF PRPP SYNTHETASE AND GLUTATHIONE REDUCTASE FROM THREE CLINICAL GROUPS OF INDIVIDUALS Correlation 0.245 0.445 0.426
1 in 100; x, significant
coefficient
Significance NS xx x
1 in 20; NS, not significant.
ACTIVITIES
IN
225
showed negligible correlation with either enzyme in any of the three clinical groups. Erythrocyte glutathione reductase activity is increased in patients with neoplastic disease, hepatitis and obstructive jaundice [13] and in most patients with anaemia [ 141. During protein-calorie malnutrition, however, this enzymic activity is reduced [15]; Beutler [16] has reported that erythrocyte glutathione reductase activity is dependent on diet and it is now accepted that not only disease, e.g. diabetes mellitus. but the vitamins nicotinamide and riboflavin increase the enzymic activity. The gout group is older than the control group and one must query whether ill-balanced diet and alcoholism are more frequent in the gout patients. If this is so, some vitamin difficiencies could partially explain the reduced erythrocyte glutathione reductase activity. On another track, one should consider whether PRPP is a feedback inhibitor of erythrocyte glutathione reductase. Glutathione reductase increases NADP+ production from the coenzyme, and an increased NADP+/NADPH ratio would accelerate the pentose monophosphate shunt pathway with increased production of ribose-5phosphate, which in turn is converted by PRPP synthetase into PRPP. The latter may be an allosteric regulator of glutathione reductase when erythrocyte PRPP synthetase activity is increased in some pathological conditions, such as gout. This hypothesis has not to our knowledge been investigated and indeed some doubt has been expressed about a rate-determining role for NADP+ in the phosphogluconate oxidative pathway [ 171. No anomalous result was apparent for the lady with the long history of hyperuricaemia in the hyperuricaemic clinical group. Acknowledgement Financial support from the MRC (MW) is gratefully recorded. Valuable discussions with Dr. G. Manley (Torbay Hospital) and Dr. P. Hickline (Plymouth Hospital) are acknowledged. References SeegmillerJE. In: Duncan’s diseases of metabolism,
edn. 6. Bond PK. Rosenberg L, eds. Philadelphia: W.B. Saunders, 1969: 516. Sperling 0, Boer P, Persky-Brosh S, Kanarek E, DeVries A. Altered kinetic property of erythrocyte phosphoribosylpyrophosphate synthetase in excessive purine production. Rev Em Etud Clin Biol 1972; 17: 703. Becker MA, Meyer LJ, Wood AW, Seegmiller JE. Purine overproduction in man associated with increased phosphoribosylpyrophosphate synthetase activity. Science 1973; 179: 1123. Long WK. Red blood cell glutathione reductase in gout. Science 1962; 138: 991. Long WK. Glutathione reductase in red blood cells: variant associated with gout. Science 1967; 155: 712. Long WK. Association between glutathione reductase variants and plasma uric acid concentration in a Negro population. Program abstracts. Am J Hum Genet 1970; 22: 14a-15a. Valentine WN, Kurschner KK. Studies on human erythrocyte nucleotide metabolism. I. Nonisotopic methodologies. Blood 1972; 39: 666. Lee Kum-Tatt, It-Koon Tan, Ai-Mee Seet. A new colourimetric method for the determination of NADH-NADPH-dependent glutathione reductase in erythrocytes and in plasma. Clin Chim Acta 1975; 58: 101.
226
9 Van Maris AGCCM, Tax WJM, Oei TL et al. Phosphoribosylpyrophosphate and enzymes of purine metabolism in erythrocytes from young hyperuricaemic males. Biochem Med 1980; 23: 263. 10 Meyskens FL, Williams HE. Concentration and synthesis of phosphoribosylpyrophosphate in erythrocytes from normal, hyperuricemic and gouty subjects. Metabolism 1971; 20: 737. 11 Ferrari M, Giacomello A, Salerno A, Messina A. A spectrophotometric assay for phosphoribosylpyrophosphate synthetase. Anal Biochem 1978; 89: 355. 12 Switzer RL. Regulation and mechanism of phosphoribosylpyrophosphate synthetase: 1. Purification and properties of the enzyme from Salmonella fyphimurium. J Biol Chem 1969; 244: 2854. 13 Kerpola W, Nikkila EA, Pitkanen E. Serum TPN-linked enzymes glucose 6-phosphate dehydrogenase, isocitric dehydrogenase and glutathione reductase activities in health and in various disease states. Acta Med Stand 1959; 164: 357. 14 Ramachandran M, Iyer GYN. Erythrocyte glutathione reductase in iron deficiency anaemia. Clin Chim Acta 1974; 52: 225. 15 Verjee ZH, Behal R. Protein-calorie malnutrition: a study of red blood cell and serum enzymes during and after crisis. Clin Chim Acta 1976; 70: 139. 16 Beutler E. Effect of flavin compounds on glutathione reductase activity in in viva and in vitro studies. J Clin Invest 1969; 48: 1957. 17 Paniker NV, Srivastava SK, Beutler E. Glutathione metabolism of the red cells. Effects of glutathione reductase deficiency on the stimulation of hexose monophosphate shunt under oxidative stress. Biochem Biophys Acta 1970; 215: 456.
C&&n Chit&a Acta, 126 (1982) 217-226 Elsevier Biomedical Press
CCA 2328
Phosphoribosyl pyrophosphate synthetase and glutathione reductase in erythrocytes from hyperuricaemic and gout patients T.R. Hardwell a, J. Braven b,* , S. Shaw b and Mary Whittaker’ a Department of Pathology, Torbay Hospital, Torquay, Devon (UK), h Department of Environmental Sciences, P&mouth Polytechnic, Drake Circus, Plymouth, Devon PL4 SAA (UK) and’ Depurtment Chemistry, Exeter lJnit;ersir_y, Exeter, Devon (UK) (Received
of
April 22nd, 1982)
Summary The enzymic activities of erythrocyte phosphoribosyl pyrophosphate synthetase (EC 2.7.6.1) and glutatbione reductase (EC 1.6.4.2) have been measured in 54 primary gout patients, 35 indi~duals having h~eruricae~a and 51 healthy controls. Statistical analyses have shown a significant increase ( p < 0.01) in the enzymic activity of erythrocyte PRPP synthetase in both the hyperuricaemic and gout groups compared with the controls. No correlation between activity and age was found in any of the three clinical groups. A significant decrease ( p = 0.01) was found in the enzymic activity of red cell glutathione reductase in the gout group compared with the other two groups. The biochemical significance of the changes in enzymic activities of the two enzymes in primary gout is discussed.
introduction The excessive production of uric acid, which occurs in patients having primary gout, has been attributed to defects that result in the formation of increased concentrations of phosphoribosyl pyrophosphate (PRPP), a substrate for the first stage of de novo purine biosynthesis [ 11. Although this reaction, catalysed by PRPP glutamine amidotransferase (EC 2.4.2.14), is subjected to feedback inhibition by purine nucleotides, the presence of excessive amounts of PRPP not only stimulates the purine biosynthesis but nullifies the allosteric inhibition by the purine nucleotides. Independent reports have shown elevation of erythrocyte PRPP synthetase activity of individuals with familial gout [2,3]. Further investigations have revealed distinct differences in the kinetic properties of red cell PRPP synthetase occurring in different families. It therefore seems likely that the clinical condition of gout could * To whom correspondence 0009-8981/82/0000-0000/$02.7$
should be addressed
Q 1982 Elsevier Biomedical
Press
218
be associated with a number of variants of PRPP synthetase. Investigations of any changes in the properties of this enzyme in primary gout patients should also be performed on a group of asymptomatic hyperuricaemic individuals with no family history of gout and a group of healthy control individuals. An increased enzymic activity of erythrocyte glutathione reductase has been reported in a group of Caucasians with untreated primary gout [4] and similar findings were reported in 23/28 Negro gout patients [5]. Control screening of a large negro population indicated that - 31% had an increased enzymic activity and a higher mean serum urate concentration than the 69% with a ‘normal’ enzymic activity [6]. Although one must have some reservations about the significance of changes in enzymic activity in any pathological state, it nonetheless seemed worthwhile to find out whether the reported elevation of enzymic activities of PRPP synthetase and glutathione reductase in erythrocytes from patients with gout was substantiated in a statistically valid group of subjects.
Materials and methods Measurements were carried out on 54 gout patients who had been diagnosed by their general practitioner on the basis of clinical criteria and a raised level of serum uric acid. All specimens received from GPs were processed on arrival at the laboratory within 4 h of the sample being taken. Control samples were obtained from 51 healthy volunteers. Any blood sample having a plasma uric acid level greater than 420 pmol .l-’ was automatically transferred to the hyperuricaemic group. The hyperuricaemic group of patients consisted of 35 individuals having a high metabolic plasma uric acid content as well as hospital patients with uremia. One individual with a long history of hyperuricaemia but whose plasma uric acid was currently controlled chemotherapeutically by allopurinol was also included in this group to ascertain whether chemotherapy resulted in any change of enzyme activity. Venepuncture samples (10 ml) were collected in lithium heparin. The samples were centrifuged at 1000 x g for 10 min and the plasma and buffy coat separated. The packed erythrocytes were washed three times with physiological saline and centrifuged at 1000 X g for 10 min. After the final wash, the original volume of blood was restored by adding physiological saline to the packed erythrocytes. An aliquot (1 ml) of the homogenous suspension of red cells in saline was added to ice-cold water (8 ml) and mixed for 10 min on a rotor mixer. Phosphate buffer (8 ml, 90 mmol . l- ‘, pH 7.4 containing 4 mmol . l- ’ EDTA) was added to the laked cells and mixed for 10 min on the rotor mixer prior to centrifugation at 1000 X g for 10 min. The supernatant was aliquoted into plastic tubes and stored frozen until required. The protein content of the haemolysate was measured in a Gilford Staser III using the biuret reagent and a standard reference serum. The assay of PRPP synthetase was a modification of that reported by Valentine and Kurschner [7] which monitors the formation of AMP, using the following linked
219
systems: PRPP
R-5-P + ATP
--j
PRPP + AMP
synch&m
adenylic
AMP + ATP f
kinase
2)
2ADP
phosphokinase
2PEP+2ADP
2 pyruvate
+ 2 ATP
(2, lactic acid dehydrogenase
2 pyruvate
+ 2 NADH
+ 2 Hi+
f&
2 lactate + 2 NAD+
All reagents were of analytical grade except for the enzymes, ATP (disodium salt) and ribose-5-phosphate (disodium salt), which were obtained from Boehringer Ltd. PEP (monocyclohexyl ammonium salt) and NADH were obtained from BDH Ltd. The assay solution contained the following concentrations (mmol . l- ‘) of reagents: MgCl, = 2.00, ATP = 1.33, PEP = 2.00, KF = 3.33, NADH = 0.23, AK 5 y1 (720 units - ml- I), PK 2 ~1 (2000 units. ml “-I), LDH 2 ~1 (2750 units. ml- ‘) in 2 ml phosphate buffer (67.5 mmol *l- ’ pH 7.4). 200 ~1 haemolysate were added to 2 ml assay reagents in a blank and a test cuvette and incubated at 37°C for 30 min. Ribose-S-phosphate (20 ~1, 66.6 mmol - 1-l) and water (20 ~1) were added to the test and blank cuvettes respectively. The change in absorbance at 340 nm at 37°C was monitored continuously on a Gilford 3500 rate analyser linked to a recorder. Enzymic activity, expressed as pmol substrate per mg . ml-’ protein AA/P, where AA = change in absorbance/h and P is the concentration mg . ml- ’ of haemolysate.
per h = 892.3 of protein in
Glutathione reductase was assayed by the method of Lee Kum-Tatt et al [8f. The mean enzymic activities of PRPP synthetase and glutathione reductase from the three clinical groups were compared statistically using a one-way analysis of variance followed by a multiple comparison by Students’ r-test. Statistical analysis was carried out using the MINITAB computer package. Results The mean plasma uric acid content of 140 individuals divided into three clinical groups is given in Table I. The data has been further divided according to sex and included in this table. Histograms of PRPP synthetase activity for normal and hyperuricaemic individuals and individuals suffering from gout are shown in Fig. 1, together with confidence limits of the mean activities for the three groups. The mean enzymic activities of these groups, subdivided into sex, are included in Table I.
220
20 -
10 .
1 0
. 100
I 200
-a 300
20
10
0
HVPERURICAEMIC
GOUT :fl?ERKJRICAEViC
Fig. 1. Histograms of the enzyme activity, and 95% confidence intervals for the mean enzyme activity, erythrocyte PRPP synthetase in three clinical groups. Ordinate = number of individuals; abscissae activity units in pmol substrate’mg-‘.ml protein.h-‘.
of =
The distribution of the activities of erythrocyte glutathione reductase and the confidence limits of the mean activities of the three groups is presented in Fig. 2. Analysis of the mean enzymic activities of each group when divided by sex is given in Table I.
221
20
NORMAL 10
0 20
GOUT
10
A-loo
200
300
HYPERURICAEMIC
l
100
2Co
300
NORVhI. GOUT HYPEWRICAEHIC
1
--
65
1
95
125
155
Fig. 2. Histograms of the enzyme activity, and 95% confidence intervals for the mean enzyme activity, erythrocyte giutathione reductase in three clinical groups. Ordinate = number of individuals; abscissae activity units in pmol substratesmg-‘*ml protein’h‘.
of =
Discussion It is apparent from the data of Table I that there is an uneven sex distribution in our two pathological groups. The selection of gout patients was made from the diagnosis of a GP or consultant. It is significant that 47 of 54 patients were male. This uneven distribution of the sexes in our sample indicates that more males than
222
TABLE
I
MEAN PLASMA URIC ACID, ERYTHROCYTE PRPP SYNTHETASE ACTIVITY AND ERYTHROCYTE GLUTATHIONE REDUCTASE ACTIVITY IN 140 INDIVIDUALS CLASSIFIED INTO THREE CLINICAL GROUPS Standard Clinical
deviations group
are given in brackets. Number of individuals
Mean plasma uric acid (pmol.ll’)
Mean erythrocyte PRPP synthetase (pm01 substrate. mg -‘.rnl protein’h-‘)
Mean erythrocyte glutathione reductase (am01 substrate. mg -‘.ml protein. h- ‘)
51 54 35
315 (76) 481 (138) 527 (153)
96 (31.2) 122 (44.2) 113 (34.7)
107 (62.0) 83 (42.7) 120 (74.7)
21 47 23
336 (67) 492 (137) 508 (146)
106 (29.6) 120 (37.6) 121 (35.6)
102 (69.9) 82 (43. I) 127 (76.3)
30 I 12
301 (80) 404 (135) 564 (166)
88 (30.6) 134 (79.1) 99 (29.2)
111 (56.7) 87 (42.6) 107 (72.9)
Total
normal gout hyperuricaemic M&
normal gout hyperuricaemic FCWUlle
normal gout hyperuricaemic
females are diagnosed clinically as having gout, in the Torbay area. The selection of the control group and hyperuricaemic individuals was essentially identical and therefore any variation in results is not due to the distribution of sexes and should be of pathological origin. As indicated above there is a higher incidence of hyperuricaemia in males than in females, and this is confirmed by an analysis of hospital records (Table II). Van Maris et al [9] have measured PRPP and some enzymes of purine metabolism in erythrocytes from 43 young hyperuricaemia males. They report that the mean erythrocyte PRPP synthetase activity in their hyperuricaemic individuals was slightly but significantly ( p < 0.01) higher than the means of their small control group (10 males). Meyskens and Williams [lo] have assayed the concentration and synthesis of PRPP in erythrocytes from normal, hyperuricaemic and gout-suffering subjects and found no difference in the mean PRPP synthetase activity. In the case of our gout patients, the large standard deviation found overall, and for females (Table I), is mainly due to an exceptionally large value recorded for one individual. Statistical analysis, shown in Table III, indicates a significant increase in enzymic activity for the gout and hyperuricaemic groups compared with the healthy controls ( p < 0.05) and is at variance with the results of Meyskens and Williams [lo]. The increase in enzymic activity of PRPP synthetase is inadequate for use in the diagnosis of gout. The confidence limits in Fig. 1 clearly illustrate these differences in the mean PRPP synthetase activities of the three groups. The reliability of the
223
TABLE
II
PERCENTAGE LABORATORY
OF HYPERURICAEMIC SAMPLES FOUND DURING SCREENING FROM JANUARY 1976 TO APRIL 1978
Year
Percentage of hyperuricaemic
Number of individuals investigated
1976 1977 1978
A ROUTINE
CLINICAL
samples
male
female
total
male
female
total
1876 1946 709
1278 1327 479
3 154 3273 1188
46.5 42.8 42.5
23.2 21.9 24.0
37.1 34.3 35.0
4531
3084
7615
44.2
22.8
35.6
(Jan-April) Jan 1976 to April
1978
different assay methods of PRPP synthetase has long been suspect; the origin of this is the apparent instability of the enzyme. Our assay has not only been compared with other spectrophotometric systems [7,11,12] but has been subjected to the analysis of day-to-day variation and to the optimal conditions for maintaining enzymic activity following storage of haemolysates at -20°C. Details of these investigations will be published elsewhere. We have as yet no evidence for a genetic variant of PRPP synthetase characterised by high enzymic activity as reported by Becker et al [3]. It is, however, proposed to investigate in depth repeat samples from the female patient mentioned above and to study her family should any unusual properties of the enzyme emerge. A significant decrease (p < 0.05) in erythrocyte glutathione reductase, compared to normal and hyperuricaemics, was found in gout patients (Table III). There is no significant difference in this mean enzymic activity in the normal and hyperuricaemic groups (Table III). The difference between the control group and the gout patients may be reduced by an age effect. The two pathological groups were on average older than the control group. A positive correlation (0.53) with age has been found for erythrocyte glutathione reductase in the controls, but there is no such correlation in either pathological group. It can be assumed that there is something swamping the age correlation in the gout and hyperuricaemia groups, but it is not necessary to consider the age factor in either group because the difference in the glutathione reductase activities would be magnified by taking age into account. There is no correlation of PRPP synthetase with age in any of the three groups (Table IV). We can, therefore, reject any age effect. There is a good correlation between erythrocyte PRPP synthetase activity and erythrocyte glutathione reductase activity in both the hyperuricaemic and gout groups, but no correlation of these two enzymes in the control group (Table V). The mechanism causing the reduction of the mean erythrocyte glutathione reductase activity in the gout group might not be responsible for the increase in the mean PRPP synthetase activity in this group of patients since a positive correlation between the two enzymes is observed for all three clinical groups. Plasma uric acid
224 TABLE
III
STANDARD ERROR ERYTHROCYTE PRPP CAL GROUPS Group comparison
Normal gout
OF DIFFERENCE BETWEEN MEAN ENZYMIC SYNTHETASE AND GLUTATHIONE REDUCTASE
PRPP synthetase
Glutathione
Standard error of difference between mean
1
Significance *
Standard error of difference between means
ACTIVITIES OF IN THREE CLINI-
reductase
I
Significance *
vs. 1.34
- 3.60
xxx
11.54
2.12
Normal vs. hyperuricaemic
8.25
-2.11
x
12.97
~ 1.00
NS
Gout vs. hyperuricaemic
8.15
NS
12.82
-2.93
xx
* Significant
TABLE
1.10
IV OF AGE WITH ENZYMIC
Enzyme
Clinical
PRPP synthetase
Glutathione
reductase
xxx, Significant
ACTIVITIES
type
IN 3 CLINICAL
GROUPS
OF INDIVID-
Number of individuals
Correlation coefficient
Significance
normal gout hyperuricaemic
51 54 35
0.10 -0.21 -0.21
NS NS NS
normal gout hyperuricaemic
51 54 35
0.53 0.04 0.003
xxx NS NS
at 1 in 1000; NS, not significant.
V
CORRELATION ERYTHROCYTES Clinical
I in 100; x, 1 in 20; NS, not significant
at: xxx, 1 in 1000; xx,
CORRELATION UALS
TABLE
x
group
Normal Gout Hyperuricaemic xx, significant
OF PRPP SYNTHETASE AND GLUTATHIONE REDUCTASE FROM THREE CLINICAL GROUPS OF INDIVIDUALS Correlation 0.245 0.445 0.426
1 in 100; x, significant
coefficient
Significance NS xx x
1 in 20; NS, not significant.
ACTIVITIES
IN
225
showed negligible correlation with either enzyme in any of the three clinical groups. Erythrocyte glutathione reductase activity is increased in patients with neoplastic disease, hepatitis and obstructive jaundice [13] and in most patients with anaemia [ 141. During protein-calorie malnutrition, however, this enzymic activity is reduced [15]; Beutler [16] has reported that erythrocyte glutathione reductase activity is dependent on diet and it is now accepted that not only disease, e.g. diabetes mellitus. but the vitamins nicotinamide and riboflavin increase the enzymic activity. The gout group is older than the control group and one must query whether ill-balanced diet and alcoholism are more frequent in the gout patients. If this is so, some vitamin difficiencies could partially explain the reduced erythrocyte glutathione reductase activity. On another track, one should consider whether PRPP is a feedback inhibitor of erythrocyte glutathione reductase. Glutathione reductase increases NADP+ production from the coenzyme, and an increased NADP+/NADPH ratio would accelerate the pentose monophosphate shunt pathway with increased production of ribose-5phosphate, which in turn is converted by PRPP synthetase into PRPP. The latter may be an allosteric regulator of glutathione reductase when erythrocyte PRPP synthetase activity is increased in some pathological conditions, such as gout. This hypothesis has not to our knowledge been investigated and indeed some doubt has been expressed about a rate-determining role for NADP+ in the phosphogluconate oxidative pathway [ 171. No anomalous result was apparent for the lady with the long history of hyperuricaemia in the hyperuricaemic clinical group. Acknowledgement Financial support from the MRC (MW) is gratefully recorded. Valuable discussions with Dr. G. Manley (Torbay Hospital) and Dr. P. Hickline (Plymouth Hospital) are acknowledged. References SeegmillerJE. In: Duncan’s diseases of metabolism,
edn. 6. Bond PK. Rosenberg L, eds. Philadelphia: W.B. Saunders, 1969: 516. Sperling 0, Boer P, Persky-Brosh S, Kanarek E, DeVries A. Altered kinetic property of erythrocyte phosphoribosylpyrophosphate synthetase in excessive purine production. Rev Em Etud Clin Biol 1972; 17: 703. Becker MA, Meyer LJ, Wood AW, Seegmiller JE. Purine overproduction in man associated with increased phosphoribosylpyrophosphate synthetase activity. Science 1973; 179: 1123. Long WK. Red blood cell glutathione reductase in gout. Science 1962; 138: 991. Long WK. Glutathione reductase in red blood cells: variant associated with gout. Science 1967; 155: 712. Long WK. Association between glutathione reductase variants and plasma uric acid concentration in a Negro population. Program abstracts. Am J Hum Genet 1970; 22: 14a-15a. Valentine WN, Kurschner KK. Studies on human erythrocyte nucleotide metabolism. I. Nonisotopic methodologies. Blood 1972; 39: 666. Lee Kum-Tatt, It-Koon Tan, Ai-Mee Seet. A new colourimetric method for the determination of NADH-NADPH-dependent glutathione reductase in erythrocytes and in plasma. Clin Chim Acta 1975; 58: 101.
226
9 Van Maris AGCCM, Tax WJM, Oei TL et al. Phosphoribosylpyrophosphate and enzymes of purine metabolism in erythrocytes from young hyperuricaemic males. Biochem Med 1980; 23: 263. 10 Meyskens FL, Williams HE. Concentration and synthesis of phosphoribosylpyrophosphate in erythrocytes from normal, hyperuricemic and gouty subjects. Metabolism 1971; 20: 737. 11 Ferrari M, Giacomello A, Salerno A, Messina A. A spectrophotometric assay for phosphoribosylpyrophosphate synthetase. Anal Biochem 1978; 89: 355. 12 Switzer RL. Regulation and mechanism of phosphoribosylpyrophosphate synthetase: 1. Purification and properties of the enzyme from Salmonella fyphimurium. J Biol Chem 1969; 244: 2854. 13 Kerpola W, Nikkila EA, Pitkanen E. Serum TPN-linked enzymes glucose 6-phosphate dehydrogenase, isocitric dehydrogenase and glutathione reductase activities in health and in various disease states. Acta Med Stand 1959; 164: 357. 14 Ramachandran M, Iyer GYN. Erythrocyte glutathione reductase in iron deficiency anaemia. Clin Chim Acta 1974; 52: 225. 15 Verjee ZH, Behal R. Protein-calorie malnutrition: a study of red blood cell and serum enzymes during and after crisis. Clin Chim Acta 1976; 70: 139. 16 Beutler E. Effect of flavin compounds on glutathione reductase activity in in viva and in vitro studies. J Clin Invest 1969; 48: 1957. 17 Paniker NV, Srivastava SK, Beutler E. Glutathione metabolism of the red cells. Effects of glutathione reductase deficiency on the stimulation of hexose monophosphate shunt under oxidative stress. Biochem Biophys Acta 1970; 215: 456.