Rheumatology and Rehabilitation, 1975,14, 50 ORIGINAL PAPER
W-ACETYL-0-D-GLUCOSAMINIDASE
ACTIVITY IN
SYNOVIAL FLUID BY E. M. VEYS, P. GABRIEL, L. DECRANS, AND G. VERBRUGGEN
SUMMARY AT-acetyl-jS-D-glucosaminidase activity in the synovial fluid of different articular diseases was studied and statistical investigations were carried out after logarithmic transformation of the data. The enzyme activity in the synovial fluid of rheumatoid arthritis is increased when compared with osteoarthrosis and traumatic effusions. The enzyme activity in traumatic effusions is also increased in comparison with osteoarthrosis. A linear relation was found between the enzyme activity in cell-free fluid and the polymorphonuclear leucocyte (PMN) count in rheumatoid arthritis, osteoarthrosis and in miscellaneous synovitis. The activity per PMN cell was determined and found to be relatively constant in the synovial fluid of inflammatory diseases (rheumatoid arthritis, chondrocalcinosis, miscellaneous synovitis). The A^acetyl-j3-p-glucosaminidase activity per PMN cell in serum was found to be significantly lower than in synovial fluid. THE role of lysosomal enzymes in the pathogeny of inflammatory and degenerative joint diseases has been emphasized by many authors (Hollander et al., 1965; Hamerman, 1966; Hamerman and Barland, 1966). JV-acetyl-p-D-glucosaminidase and p-glucuronidase are particularly important in the pathology of joint diseases, as they are responsible for the enzymatic cleavage of hyaluronic acid. Depolymerized hyaluronic acid is split into N-acetyl-p-D-glucosamine and p-glucuronic acid by these two enzymes (Linker, Meyer and Weissmann, 1955). Greiling and his co-workers (1971) suggested that depolymerization of proteoglycans precedes the breakdown of collagen fibrils in joint diseases. Caygill and Pitkeathly (1966), Fallet, Micheli and Boussina (1971) and Eberhard et al. (1972) investigated JV-acetyl-p-D-glucosaminidase activity in synovial fluid in rheumatoid arthritis (RA) and in osteoarthrosis (OA); they found an increased enzyme activity in RA. Traumatic effusions were not included in these investigations. We have determined JV-acetyl-p-D-glucosaminidase activity in RA, in OA, and in traumatic effusions and the sources of the enzyme activity are approached. MATERIAL Two groups of patients were studied. In the first group (85 synovial fluids of 73 different patients), we determined only the N-acetyl-p-D-glucosaminidase activity in cell-free synovial fluid; the cellular content being discarded after separation in a centrifuge. In this group we examined 42 synovial fluids of 34 cases, in which RA definite or RA classic was diagnosed according to the A.R.A. criteria, as well as 15 traumatic effusions and 28 synovial fluids of 24 patients with osteoarthrosis. In the cases where two samples were analysed, synovial fluids were obtained from different joints. In the second group, which included 77 synovial fluids (29 cases with RA, 10 cases with OA, 8 traumatic effusions, 10 cases of chondrocalcinosis and 20 cases of miscellaneous Accepted for publication June 1974. 50
Downloaded from http://rheumatology.oxfordjournals.org/ at University of Iowa Libraries/Serials Acquisitions on July 14, 2015
Department of Physical Medicine and Orthopaedic Surgery Section of Rheumatology, University of Ghent, De Pintelaan 135, B-9000 GHENT (Belgium)
VEYS ET AL.: ENZYME ACTIVITY IN SYNOVIAL FLUID
51
synovitis with ill-defined aetiology), the enzyme activity was determined simultaneously in cell-free fluid and in the cellular material.
iV-ACETYL-P-D-GLUCOSAMINIDASE ACTIVITY
A modification of the technique described by Caygill and Jevons (1964) was used. The reaction mixture contained 1 ml of a solution of 4-nitrophenyl-N-acetyl-p-Dglucosamine (0.6 mM) (B.D.H. Chemicals) in citrate buffer (0.05 M, pH 4.5) and 0.1 ml of the sample. After incubation at 37 °C for 30 min, the reaction was stopped by adding 0.1 ml sodium carbonate (2 M). The pH of the final solution was was 9.8. The released p-nitrophenol was estimated by measuring the absorption at 402 m/t. Enzyme activity was calculated from the molar extinction coefficient of />-nitrophenol and is reported in milli-units/millilitre (mU/ml). RESULTS A^ACETYL-P-D-GLUCOSAMINIDASE ACTIVITY IN SYNOVIAL FLUID
The frequency distribution of the enzyme activity in the three groups we investigated (RA, OA, traumatic effusions) was found to be not of the normal type. A Gaussian distribution curve was obtained by plotting the log of enzyme activity (Fig. 1). Statistical investigations were made after logarithmic transformation of the data. The results are reported in Table I. jWe see that the enzyme activity of synovial TABLE I iV-ACETYL-/?-D-GLUCOSAMINIDASE ACTIVITY IN SYNOVIAL FLUID
Diagnosis RA OA Traumatic effusions
n
m mU/ml
min mU/ml
42 28 15
17.86 9.38 11.79
6.97 2.99 5.13
max mU/ml
m log
s log
sm log
42.57 23.84 46.44
1.202 1.000 0.920
0. 213 0. 221 0. 202
0.033 0.057 0.038
n = number of samples, m = mean of the enzyme activities, m log = mean of the logs of the enzyme activity, s log = standard deviation of the logs, sm log = standard error of the mean.
Downloaded from http://rheumatology.oxfordjournals.org/ at University of Iowa Libraries/Serials Acquisitions on July 14, 2015
METHODS Synovial fluid (1 ml) was collected in dried EDTA tubes for leucocyte count and differentiation. To the remaining synovial fluid, 40 i.u./ml testicular hyaluronidase (Evans Medical Ltd., Liverpool) were added immediately to prevent enclosure of leucocytes in the mucine clot. Investigation revealed that hyaluronidase interferes with the substrate used in the AT-acetyl-p-D-glucosaminidase dosage at a ratio of 1%, so the interference can be ignored. After incubation for 1 hour at 37 °C, a constant amount of synovial fluid (10 ml) was centrifuged at 2 500 g and 4 °C for 10 min. In this way a clear cell-free supernatant without any particulate contaminants and a cellular sediment were obtained. The supernatant was used as such. The sediment was washed three times with saline and finally resuspended in saline to a final volume of exactly 10 ml. This suspension was submitted to ultrasonic disintegration (MSE, type 5.65; power set at 100 W) for 15 min, with cooling in an ice-bath. N-acetyl-p-D-glucosaminidase activity was then determined for both fractions, designated cell-free fluid and cellular material.
52
RHEUMATOLOGY AND REHABILITATION VOL. XIV NO. 1
in \\j
6 n A
1
2
0
1
1
1
0.30
0.50
0.70
|
RA{n>42)
"1 0.90 1.10 Log enz.act.
1.30
1.50
1.70
10
8-
1
6 -
H
n 4 -•
f
2-
_T
—I
i n
i
0.30
1
I
0.50
i
0.70
0A(n=28)
"1
i
i
0.90 . 1.10 Log enz.act.
1 1
1.30
I
1.50
1
1.70
10 -
8 -
6n
L.
2 1
1
0.30
0.50
r , ,1 l
0.70
0.90 1.10 Log enz.act.
1.30
Traumatic effusions 1
1.50
I
1.70
Fio. 1.—Frequency distribution of the logs of the enzyme activity in synovial fluid (n = number of the samples).
Downloaded from http://rheumatology.oxfordjournals.org/ at University of Iowa Libraries/Serials Acquisitions on July 14, 2015
n
6-
VEYS ET AL.: ENZYME ACTIVITY IN SYNOVIAL FLUID
53
1.5 1.4
i.
I.I
w
B •i
10
0.8 0.7 X
0.6
x
0.50
I
2
3
^
4
5
Log PMN
Fio. 2.—Log-log plot of enzyme activity in cell-free fluid and of PMN count {x — osteoaithrosis, O = traumatic effusions, O = rheumatoid arthritis, A = chondrocalcinosis and • = miscellaneous synovitis). fluid in RA is increased when compared with osteoarthrosis (t = 7.46, P
W-ACETYL-0-D-GLUCOSAMINIDASE
ACTIVITY IN
SYNOVIAL FLUID BY E. M. VEYS, P. GABRIEL, L. DECRANS, AND G. VERBRUGGEN
SUMMARY AT-acetyl-jS-D-glucosaminidase activity in the synovial fluid of different articular diseases was studied and statistical investigations were carried out after logarithmic transformation of the data. The enzyme activity in the synovial fluid of rheumatoid arthritis is increased when compared with osteoarthrosis and traumatic effusions. The enzyme activity in traumatic effusions is also increased in comparison with osteoarthrosis. A linear relation was found between the enzyme activity in cell-free fluid and the polymorphonuclear leucocyte (PMN) count in rheumatoid arthritis, osteoarthrosis and in miscellaneous synovitis. The activity per PMN cell was determined and found to be relatively constant in the synovial fluid of inflammatory diseases (rheumatoid arthritis, chondrocalcinosis, miscellaneous synovitis). The A^acetyl-j3-p-glucosaminidase activity per PMN cell in serum was found to be significantly lower than in synovial fluid. THE role of lysosomal enzymes in the pathogeny of inflammatory and degenerative joint diseases has been emphasized by many authors (Hollander et al., 1965; Hamerman, 1966; Hamerman and Barland, 1966). JV-acetyl-p-D-glucosaminidase and p-glucuronidase are particularly important in the pathology of joint diseases, as they are responsible for the enzymatic cleavage of hyaluronic acid. Depolymerized hyaluronic acid is split into N-acetyl-p-D-glucosamine and p-glucuronic acid by these two enzymes (Linker, Meyer and Weissmann, 1955). Greiling and his co-workers (1971) suggested that depolymerization of proteoglycans precedes the breakdown of collagen fibrils in joint diseases. Caygill and Pitkeathly (1966), Fallet, Micheli and Boussina (1971) and Eberhard et al. (1972) investigated JV-acetyl-p-D-glucosaminidase activity in synovial fluid in rheumatoid arthritis (RA) and in osteoarthrosis (OA); they found an increased enzyme activity in RA. Traumatic effusions were not included in these investigations. We have determined JV-acetyl-p-D-glucosaminidase activity in RA, in OA, and in traumatic effusions and the sources of the enzyme activity are approached. MATERIAL Two groups of patients were studied. In the first group (85 synovial fluids of 73 different patients), we determined only the N-acetyl-p-D-glucosaminidase activity in cell-free synovial fluid; the cellular content being discarded after separation in a centrifuge. In this group we examined 42 synovial fluids of 34 cases, in which RA definite or RA classic was diagnosed according to the A.R.A. criteria, as well as 15 traumatic effusions and 28 synovial fluids of 24 patients with osteoarthrosis. In the cases where two samples were analysed, synovial fluids were obtained from different joints. In the second group, which included 77 synovial fluids (29 cases with RA, 10 cases with OA, 8 traumatic effusions, 10 cases of chondrocalcinosis and 20 cases of miscellaneous Accepted for publication June 1974. 50
Downloaded from http://rheumatology.oxfordjournals.org/ at University of Iowa Libraries/Serials Acquisitions on July 14, 2015
Department of Physical Medicine and Orthopaedic Surgery Section of Rheumatology, University of Ghent, De Pintelaan 135, B-9000 GHENT (Belgium)
VEYS ET AL.: ENZYME ACTIVITY IN SYNOVIAL FLUID
51
synovitis with ill-defined aetiology), the enzyme activity was determined simultaneously in cell-free fluid and in the cellular material.
iV-ACETYL-P-D-GLUCOSAMINIDASE ACTIVITY
A modification of the technique described by Caygill and Jevons (1964) was used. The reaction mixture contained 1 ml of a solution of 4-nitrophenyl-N-acetyl-p-Dglucosamine (0.6 mM) (B.D.H. Chemicals) in citrate buffer (0.05 M, pH 4.5) and 0.1 ml of the sample. After incubation at 37 °C for 30 min, the reaction was stopped by adding 0.1 ml sodium carbonate (2 M). The pH of the final solution was was 9.8. The released p-nitrophenol was estimated by measuring the absorption at 402 m/t. Enzyme activity was calculated from the molar extinction coefficient of />-nitrophenol and is reported in milli-units/millilitre (mU/ml). RESULTS A^ACETYL-P-D-GLUCOSAMINIDASE ACTIVITY IN SYNOVIAL FLUID
The frequency distribution of the enzyme activity in the three groups we investigated (RA, OA, traumatic effusions) was found to be not of the normal type. A Gaussian distribution curve was obtained by plotting the log of enzyme activity (Fig. 1). Statistical investigations were made after logarithmic transformation of the data. The results are reported in Table I. jWe see that the enzyme activity of synovial TABLE I iV-ACETYL-/?-D-GLUCOSAMINIDASE ACTIVITY IN SYNOVIAL FLUID
Diagnosis RA OA Traumatic effusions
n
m mU/ml
min mU/ml
42 28 15
17.86 9.38 11.79
6.97 2.99 5.13
max mU/ml
m log
s log
sm log
42.57 23.84 46.44
1.202 1.000 0.920
0. 213 0. 221 0. 202
0.033 0.057 0.038
n = number of samples, m = mean of the enzyme activities, m log = mean of the logs of the enzyme activity, s log = standard deviation of the logs, sm log = standard error of the mean.
Downloaded from http://rheumatology.oxfordjournals.org/ at University of Iowa Libraries/Serials Acquisitions on July 14, 2015
METHODS Synovial fluid (1 ml) was collected in dried EDTA tubes for leucocyte count and differentiation. To the remaining synovial fluid, 40 i.u./ml testicular hyaluronidase (Evans Medical Ltd., Liverpool) were added immediately to prevent enclosure of leucocytes in the mucine clot. Investigation revealed that hyaluronidase interferes with the substrate used in the AT-acetyl-p-D-glucosaminidase dosage at a ratio of 1%, so the interference can be ignored. After incubation for 1 hour at 37 °C, a constant amount of synovial fluid (10 ml) was centrifuged at 2 500 g and 4 °C for 10 min. In this way a clear cell-free supernatant without any particulate contaminants and a cellular sediment were obtained. The supernatant was used as such. The sediment was washed three times with saline and finally resuspended in saline to a final volume of exactly 10 ml. This suspension was submitted to ultrasonic disintegration (MSE, type 5.65; power set at 100 W) for 15 min, with cooling in an ice-bath. N-acetyl-p-D-glucosaminidase activity was then determined for both fractions, designated cell-free fluid and cellular material.
52
RHEUMATOLOGY AND REHABILITATION VOL. XIV NO. 1
in \\j
6 n A
1
2
0
1
1
1
0.30
0.50
0.70
|
RA{n>42)
"1 0.90 1.10 Log enz.act.
1.30
1.50
1.70
10
8-
1
6 -
H
n 4 -•
f
2-
_T
—I
i n
i
0.30
1
I
0.50
i
0.70
0A(n=28)
"1
i
i
0.90 . 1.10 Log enz.act.
1 1
1.30
I
1.50
1
1.70
10 -
8 -
6n
L.
2 1
1
0.30
0.50
r , ,1 l
0.70
0.90 1.10 Log enz.act.
1.30
Traumatic effusions 1
1.50
I
1.70
Fio. 1.—Frequency distribution of the logs of the enzyme activity in synovial fluid (n = number of the samples).
Downloaded from http://rheumatology.oxfordjournals.org/ at University of Iowa Libraries/Serials Acquisitions on July 14, 2015
n
6-
VEYS ET AL.: ENZYME ACTIVITY IN SYNOVIAL FLUID
53
1.5 1.4
i.
I.I
w
B •i
10
0.8 0.7 X
0.6
x
0.50
I
2
3
^
4
5
Log PMN
Fio. 2.—Log-log plot of enzyme activity in cell-free fluid and of PMN count {x — osteoaithrosis, O = traumatic effusions, O = rheumatoid arthritis, A = chondrocalcinosis and • = miscellaneous synovitis). fluid in RA is increased when compared with osteoarthrosis (t = 7.46, P
