BIOCHEMICAL
MEDICINE
AND
METABOLIC
BIOLOGY
43, 183-192 (1990)
y-Glutamyl Transpeptidase in Synovial Fluid, Serum, and Urine of Patients with Rheumatoid Arthritis IL RAMBABU,’ A. A. ANSARI, I. A. SHAAFIE, A. P. CHELVAM,*.’ Department
of Laboratory
Medicine and *Department of Orthopaedics, University, P.O. Box 13040. Benghazi, Libya
AND M. M. Zru Al-Arab
Medical
Received October 14, 1988, and in revised form December 13, 1989
y-Glutamyl transpeptidase (GGT), which catalyzes the transfer of glutamyl moiety from y-glutamyl peptides and their derivatives to suitable acceptors, is widely distributed in human tissues and fluids (1). GGT plays a prominent role in the transport of amino acids and small peptides across cell membrane (2). In inflammation metabolic processes are activated with subsequent migration of leukocytes to the damaged site. A greater turnover and transport of amino acids at the site of inflammation occurs in an attempt to repair the damaged tissue. Meister and Tate (3) have suggested that increased GGT activity at the site of inflammation may have a role in amino acid transport. Participation of GGT in the formation of leukotrienes has been reported (4). Hammarstrom (5) reported that GGT is responsible for the conversion of leukotriene C, to leukotriene D+ The present study was undertaken to evaluate the effect of inflammation on GGT activity in synovial fluid (SF-GGT), serum (SGGT), and urine (UGGT) in rheumatoid arthritis patients (RA patients). PATIENTS
AND METHODS
Patients. The subjects consisted of 59 patients (38 females and 21 males, age range 28-73) presenting with rheumatoid arthritis as defined by Ropes et ~11.(6). Radiographs were graded O-5, according to the method of Larsen et al. (7). A score of 5 denotes the most severe joint destruction. In addition, 11 patients were receiving o-penicillamine, 7 were receiving gold salts, and 6 were on azathioprine therapy. These drugs have no evident influence on SGGT (1,8). Control subjects. This group consisted of 18 patients (11 males and 7 females, age range 30-65) who were admitted to or attended the hospital with traumatic arthritis. I To whom correspondence should be addressed. ’ Present address: University Department of Orthopaedics. Street, Liverpool, England.
Royal Liverpool
Hospital, Prescot
183 0885-4505/90 $3 .OO Copyright 0 1990 by Academic Press. Inc. All rightr of reproduction m any form revsvzd.
184
RAMBABU
ET AL.
Specimen collection. Synovial fluid was aspirated from knee joints of patients at the orthopedics department of Al-Jallah Hospital, Benghazi. Complete aspiration of joint fluid was performed under aseptic conditions using a 1.2-mm-bore needle and total volume measured. No local anesthetic was used. The effusions were collected in sterile tubes and then stored at 4°C. Early morning urine specimens (10 PM to 6 AM) were collected without any preservative. Venous blood samples were collected from RA patients and from control subjects. Serum was separated from clot and stored at 4°C until use. Synovial fluid, serum, and urine were analyzed for enzyme activities within 12 hr after collection. Determination of GGT activity. SF-WBC was separated from enzyme activity by filtration through cotton, VSP according to the method of Fleming (9). The cotton was boiled in five changes of water and 1.0 g (dry weight) was packed into a 5-ml syringe clamped in a vertical position for 3 ml of the synovial fluid processed. The 50-~1 aliquot of filtrate was used for SF-GGT estimation. The UGGT activity was separated from its inhibitor(s) employing ion retardation resin as described earlier (10). The SGGT activity was assayed directly in 50 ~1 serum without employing ion retardation resin. The SF-GGT, UGGT, and SGGT activities were estimated according to the method of Szasz (11). The assay system of GGT consisted of buffered substrate containing 4.4 pmole of y-glutamyl-p-nitroanilide, 22 pmole of diglycine, 11 pmole of MgClz ,50 pmole of ammediol-HCl buffer, pH 8.6, and 50 pl of synovial fluid, serum, or urine. The liberated p-nitroaniline was determined by measuring the absorbance at 405 nm at 1-min intervals in a Beckman Model-42 spectrophotometer. The millimolar absorptivity for p-nitroaniline in cm*/mM is 9.87. One unit of GGT activity in synovial fluid, serum, or urine was defined as that amount of enzyme producing 1 pmole p-nitroanilinelmin under the assay conditions. Estimation ofSF-WBC count. The SF-WBC count was obtained with a ModelS Coulter counter, using the microsample mode. However, automated particle counting (Coulter) was found to be unsatisfactory for control subjects because of low SF-WBC counts which approached the background of the system. The manual method was employed for the SF-WBC count of control subjects. Determination of SF-protein. The SF-protein was determined by the biuret method (12). Estimation of urinary creatinine. Creatinine in the overnight urine specimens was estimated by the initial rate method (13). Determination of rheumatoid factor and erythrocyte sedimentation rate (ESR). The rheumatoid factor (serum) and ESR (blood) were determined by the latex slide technique (14) and the Westergren method (15), respectively. Statistical analysis. The correlation coefficients were determined by the Pearson product moment correlation method (16). All P values shown for the correlation coefficient are two-tailed. The regression line is fitted by the least-squares method.
y-GLUTAMYL
TRANSPEF’TIDASE
IN SYNOVIAL
185
FLUID
60
I
.
27.62
I- = 0.381,
l
+ 0.6496X
C
P = < 0.025
140-
,
1
L
60
70
1
SF
80 GGT
90 ACTIVITY,
I
100 U/L
I
110
120
1
130
FIG. 1. Correlation between SF-GGT activity and SF-protein concentration (A), SGGT activity (B), and UGGT activity (C). Regression line displayed in corresponding correlation holds for 59 RA patients. Closed circles represent female and open circles represent male RA patients.
RESULTS Figure I represents correlation between SF-protein concentration, SGGT and UGGT activities, and SF-GGT activity. A statistically significant correlation between SF-protein concentration and SF-GGT activity was observed (r = -0.543, P < 0.001) in a total of 59 RA patients investigated (Fig. IA). A
186
RAMBABU
ET AL.
significant correlation between SGGT activity and SF-GGT (r = 0.399, P < 0.05) was noticed only in male patients (Fig. 1B). Table 1 shows that the mean ratio of SF-GGT to SGGT in control subjects (0.46 + 0.23) was lower than that in RA patients (3.65 + 1.50). The excretion of GGT was markedly increased in all RA patients screened. In 9 patients, the UGGT activity increased fivefold compared to the upper limit of normal value (range 15.5-31.1 U/g creatinine). The increase was only twoto threefold in the other 50 cases (Fig. 1C). The correlation between UGGT and SF-GGT was significant only in female RA patients (r = 0.580, P < 0.001). A small but statistically significant correlation between SF-WBC Count and SF-GGT activity was observed only in female patients (r = 0.332, P < 0.01). The data on individual samples are presented in Fig. 2D. Likewise, there was a correlation between ESR and SF-GGT activity in females (r = 0.416, P < 0.025) but not in males (r = 0.293, P < 0.10) (Fig. 2E). The data on correlation between UGGT and SF-protein, SGGT, and SF-GGT are presented in Fig. 3. A strong positive correlation between SGGT and UGGT activities was noticed in all RA patients (r = 0.743, P < 0.001) (in females, r = 0.567, P < 0.01, and in males, r = 0.435, P < 0.02). Although the relation may be more complex than linear, the mean values of the ratio of UGGT to SGGT were 3.46 ? 1.46 and 1.52 2 1.07 in RA patients and control subjects, respectively. SF-GGT levels in 9 patients were 8-fold higher than the upper limit of normal value (range 5.50-15.0 U/liter). In 50 of the remaining patients the levels were 4- to 6-fold higher. The mean increase in specific activity of SF-GGT was found to be IO-fold higher in RA patients compared to control subjects (Table 1). Conversely, the SF-WBC Count correlated strongly with UGGT more in males
Laboratory
TABLE I Data in Control Subjects and Rheumatoid Arthritis Patients RA patients
Parameter SF-protein (g/liter) SGGT (U/liter) SF-GGT (U/liter) UGGT (U/g creatinine) SF-WBC count (X 103) ESR (mm/hr)
Total n = 59 means f SD 47.60 (SO.50 26.60 (23.70 85.00 ( 9.34 82.80 (23.90 31.40 ( 2.77 82.30 (10.30
k -t 2 k k 2 k 2 2 f t k
7.70 8.20) 9.72 10.80) 13.6 3.06) 23.10 3.48) 10.10 1.64) 35.30 4.76)
n = 38 means k SD 47.40 (51.30 27.20 (27.42 84.60 ( 9.30 91.30 (24.75 31.10 ( 2.61 78.1 (10.00
Nore. Control subjects values are given in parentheses.
k k 2 2 + ” f k ? k k 2
7.50 9.97) 9.60 8.48) 14.10 3.08) 22.10 3.20) 9.74 1.60) 34.70 4.39)
Male n = 21 means k SD 47.90 (51.40 25.60 (21.45 85.70 ( 9.37 67.40 (23.46 32.00 ( 2.88 87.30 (10.63
2 zi k r k + k ” 2 ” + 2
8.30 3.20) 10.10 11.92) 12.8 3.21) 16.2 3.71) Il.00 1.73) 34.00 5.18)
+3LUTAMYL
I
= 15.34
TRANSPEPTIDASE
+ 0.1aw
I- = 0.254,
IN SYNOVIAL
187
FLUID D
x
P = < 0.025
D-
D/ o-
I
= 2.76
r
= 0.345.
+ 0.8748
i0 -
0
. .
E
x
P = < 0.08
.
0
.
0
l0 8
. o
l
i0
.
.
0
IO
. 1
. .
0 e
1
60
I
70 SF
a0 GGT
90 ACTIVITY,
1
100
110
120
I
130
UIL
FIG. 2. Correlation between SF-GGT activity and SF-WBC Count (D) and ESR (E). Regression line displayed in corresponding correlation holds for 59 RA patients. Closed circles represent female and open circles represent male RA patients.
(r = 4D). (v = (Fig.
0.987, P < 0.001) than in female RA patients (Y = 0.422, P < 0.01) (Fig. Likewise, UGGT activity correlated strongly with ESR in male patients 0.482, P < 0.001) but not in female RA patients (Y = 0.097, P > 0.10) 4E and Table 1). DISCUSSION
Because of the paucity of interpretable long-term clinical data and difficulty in performing prospective studies, attention has not been centered on GGT activity in various body fluids. In the present study, we selected a consecutive population of adult RA patients without confounding influences. Our study comprises an investigation of the changes in GGT levels in various body fluids, SF-
188
RAMBABU
Y = r=-
50.60
+ 0.0360
x,
0.109
P
Oo*O
0 a
> 0.20
n
0
a
” a 0 “.
ET AL.
a8
0
i
00
8
-i
a l
0’
0
Y = 26.66 r
140
=
0.143,
Y = 66.51 r
=
40
+ 0.2997 P =
+
0.381,
MEDICINE
AND
METABOLIC
BIOLOGY
43, 183-192 (1990)
y-Glutamyl Transpeptidase in Synovial Fluid, Serum, and Urine of Patients with Rheumatoid Arthritis IL RAMBABU,’ A. A. ANSARI, I. A. SHAAFIE, A. P. CHELVAM,*.’ Department
of Laboratory
Medicine and *Department of Orthopaedics, University, P.O. Box 13040. Benghazi, Libya
AND M. M. Zru Al-Arab
Medical
Received October 14, 1988, and in revised form December 13, 1989
y-Glutamyl transpeptidase (GGT), which catalyzes the transfer of glutamyl moiety from y-glutamyl peptides and their derivatives to suitable acceptors, is widely distributed in human tissues and fluids (1). GGT plays a prominent role in the transport of amino acids and small peptides across cell membrane (2). In inflammation metabolic processes are activated with subsequent migration of leukocytes to the damaged site. A greater turnover and transport of amino acids at the site of inflammation occurs in an attempt to repair the damaged tissue. Meister and Tate (3) have suggested that increased GGT activity at the site of inflammation may have a role in amino acid transport. Participation of GGT in the formation of leukotrienes has been reported (4). Hammarstrom (5) reported that GGT is responsible for the conversion of leukotriene C, to leukotriene D+ The present study was undertaken to evaluate the effect of inflammation on GGT activity in synovial fluid (SF-GGT), serum (SGGT), and urine (UGGT) in rheumatoid arthritis patients (RA patients). PATIENTS
AND METHODS
Patients. The subjects consisted of 59 patients (38 females and 21 males, age range 28-73) presenting with rheumatoid arthritis as defined by Ropes et ~11.(6). Radiographs were graded O-5, according to the method of Larsen et al. (7). A score of 5 denotes the most severe joint destruction. In addition, 11 patients were receiving o-penicillamine, 7 were receiving gold salts, and 6 were on azathioprine therapy. These drugs have no evident influence on SGGT (1,8). Control subjects. This group consisted of 18 patients (11 males and 7 females, age range 30-65) who were admitted to or attended the hospital with traumatic arthritis. I To whom correspondence should be addressed. ’ Present address: University Department of Orthopaedics. Street, Liverpool, England.
Royal Liverpool
Hospital, Prescot
183 0885-4505/90 $3 .OO Copyright 0 1990 by Academic Press. Inc. All rightr of reproduction m any form revsvzd.
184
RAMBABU
ET AL.
Specimen collection. Synovial fluid was aspirated from knee joints of patients at the orthopedics department of Al-Jallah Hospital, Benghazi. Complete aspiration of joint fluid was performed under aseptic conditions using a 1.2-mm-bore needle and total volume measured. No local anesthetic was used. The effusions were collected in sterile tubes and then stored at 4°C. Early morning urine specimens (10 PM to 6 AM) were collected without any preservative. Venous blood samples were collected from RA patients and from control subjects. Serum was separated from clot and stored at 4°C until use. Synovial fluid, serum, and urine were analyzed for enzyme activities within 12 hr after collection. Determination of GGT activity. SF-WBC was separated from enzyme activity by filtration through cotton, VSP according to the method of Fleming (9). The cotton was boiled in five changes of water and 1.0 g (dry weight) was packed into a 5-ml syringe clamped in a vertical position for 3 ml of the synovial fluid processed. The 50-~1 aliquot of filtrate was used for SF-GGT estimation. The UGGT activity was separated from its inhibitor(s) employing ion retardation resin as described earlier (10). The SGGT activity was assayed directly in 50 ~1 serum without employing ion retardation resin. The SF-GGT, UGGT, and SGGT activities were estimated according to the method of Szasz (11). The assay system of GGT consisted of buffered substrate containing 4.4 pmole of y-glutamyl-p-nitroanilide, 22 pmole of diglycine, 11 pmole of MgClz ,50 pmole of ammediol-HCl buffer, pH 8.6, and 50 pl of synovial fluid, serum, or urine. The liberated p-nitroaniline was determined by measuring the absorbance at 405 nm at 1-min intervals in a Beckman Model-42 spectrophotometer. The millimolar absorptivity for p-nitroaniline in cm*/mM is 9.87. One unit of GGT activity in synovial fluid, serum, or urine was defined as that amount of enzyme producing 1 pmole p-nitroanilinelmin under the assay conditions. Estimation ofSF-WBC count. The SF-WBC count was obtained with a ModelS Coulter counter, using the microsample mode. However, automated particle counting (Coulter) was found to be unsatisfactory for control subjects because of low SF-WBC counts which approached the background of the system. The manual method was employed for the SF-WBC count of control subjects. Determination of SF-protein. The SF-protein was determined by the biuret method (12). Estimation of urinary creatinine. Creatinine in the overnight urine specimens was estimated by the initial rate method (13). Determination of rheumatoid factor and erythrocyte sedimentation rate (ESR). The rheumatoid factor (serum) and ESR (blood) were determined by the latex slide technique (14) and the Westergren method (15), respectively. Statistical analysis. The correlation coefficients were determined by the Pearson product moment correlation method (16). All P values shown for the correlation coefficient are two-tailed. The regression line is fitted by the least-squares method.
y-GLUTAMYL
TRANSPEF’TIDASE
IN SYNOVIAL
185
FLUID
60
I
.
27.62
I- = 0.381,
l
+ 0.6496X
C
P = < 0.025
140-
,
1
L
60
70
1
SF
80 GGT
90 ACTIVITY,
I
100 U/L
I
110
120
1
130
FIG. 1. Correlation between SF-GGT activity and SF-protein concentration (A), SGGT activity (B), and UGGT activity (C). Regression line displayed in corresponding correlation holds for 59 RA patients. Closed circles represent female and open circles represent male RA patients.
RESULTS Figure I represents correlation between SF-protein concentration, SGGT and UGGT activities, and SF-GGT activity. A statistically significant correlation between SF-protein concentration and SF-GGT activity was observed (r = -0.543, P < 0.001) in a total of 59 RA patients investigated (Fig. IA). A
186
RAMBABU
ET AL.
significant correlation between SGGT activity and SF-GGT (r = 0.399, P < 0.05) was noticed only in male patients (Fig. 1B). Table 1 shows that the mean ratio of SF-GGT to SGGT in control subjects (0.46 + 0.23) was lower than that in RA patients (3.65 + 1.50). The excretion of GGT was markedly increased in all RA patients screened. In 9 patients, the UGGT activity increased fivefold compared to the upper limit of normal value (range 15.5-31.1 U/g creatinine). The increase was only twoto threefold in the other 50 cases (Fig. 1C). The correlation between UGGT and SF-GGT was significant only in female RA patients (r = 0.580, P < 0.001). A small but statistically significant correlation between SF-WBC Count and SF-GGT activity was observed only in female patients (r = 0.332, P < 0.01). The data on individual samples are presented in Fig. 2D. Likewise, there was a correlation between ESR and SF-GGT activity in females (r = 0.416, P < 0.025) but not in males (r = 0.293, P < 0.10) (Fig. 2E). The data on correlation between UGGT and SF-protein, SGGT, and SF-GGT are presented in Fig. 3. A strong positive correlation between SGGT and UGGT activities was noticed in all RA patients (r = 0.743, P < 0.001) (in females, r = 0.567, P < 0.01, and in males, r = 0.435, P < 0.02). Although the relation may be more complex than linear, the mean values of the ratio of UGGT to SGGT were 3.46 ? 1.46 and 1.52 2 1.07 in RA patients and control subjects, respectively. SF-GGT levels in 9 patients were 8-fold higher than the upper limit of normal value (range 5.50-15.0 U/liter). In 50 of the remaining patients the levels were 4- to 6-fold higher. The mean increase in specific activity of SF-GGT was found to be IO-fold higher in RA patients compared to control subjects (Table 1). Conversely, the SF-WBC Count correlated strongly with UGGT more in males
Laboratory
TABLE I Data in Control Subjects and Rheumatoid Arthritis Patients RA patients
Parameter SF-protein (g/liter) SGGT (U/liter) SF-GGT (U/liter) UGGT (U/g creatinine) SF-WBC count (X 103) ESR (mm/hr)
Total n = 59 means f SD 47.60 (SO.50 26.60 (23.70 85.00 ( 9.34 82.80 (23.90 31.40 ( 2.77 82.30 (10.30
k -t 2 k k 2 k 2 2 f t k
7.70 8.20) 9.72 10.80) 13.6 3.06) 23.10 3.48) 10.10 1.64) 35.30 4.76)
n = 38 means k SD 47.40 (51.30 27.20 (27.42 84.60 ( 9.30 91.30 (24.75 31.10 ( 2.61 78.1 (10.00
Nore. Control subjects values are given in parentheses.
k k 2 2 + ” f k ? k k 2
7.50 9.97) 9.60 8.48) 14.10 3.08) 22.10 3.20) 9.74 1.60) 34.70 4.39)
Male n = 21 means k SD 47.90 (51.40 25.60 (21.45 85.70 ( 9.37 67.40 (23.46 32.00 ( 2.88 87.30 (10.63
2 zi k r k + k ” 2 ” + 2
8.30 3.20) 10.10 11.92) 12.8 3.21) 16.2 3.71) Il.00 1.73) 34.00 5.18)
+3LUTAMYL
I
= 15.34
TRANSPEPTIDASE
+ 0.1aw
I- = 0.254,
IN SYNOVIAL
187
FLUID D
x
P = < 0.025
D-
D/ o-
I
= 2.76
r
= 0.345.
+ 0.8748
i0 -
0
. .
E
x
P = < 0.08
.
0
.
0
l0 8
. o
l
i0
.
.
0
IO
. 1
. .
0 e
1
60
I
70 SF
a0 GGT
90 ACTIVITY,
1
100
110
120
I
130
UIL
FIG. 2. Correlation between SF-GGT activity and SF-WBC Count (D) and ESR (E). Regression line displayed in corresponding correlation holds for 59 RA patients. Closed circles represent female and open circles represent male RA patients.
(r = 4D). (v = (Fig.
0.987, P < 0.001) than in female RA patients (Y = 0.422, P < 0.01) (Fig. Likewise, UGGT activity correlated strongly with ESR in male patients 0.482, P < 0.001) but not in female RA patients (Y = 0.097, P > 0.10) 4E and Table 1). DISCUSSION
Because of the paucity of interpretable long-term clinical data and difficulty in performing prospective studies, attention has not been centered on GGT activity in various body fluids. In the present study, we selected a consecutive population of adult RA patients without confounding influences. Our study comprises an investigation of the changes in GGT levels in various body fluids, SF-
188
RAMBABU
Y = r=-
50.60
+ 0.0360
x,
0.109
P
Oo*O
0 a
> 0.20
n
0
a
” a 0 “.
ET AL.
a8
0
i
00
8
-i
a l
0’
0
Y = 26.66 r
140
=
0.143,
Y = 66.51 r
=
40
+ 0.2997 P =
+
0.381,
