osteochondritis, three joints with traumatic arthritis and seven joints infected with bacteria. Serum and urine from individual horses were also examined for the ...
EQUINE VETERINARY JOURNAL
44
Equine ve!. J . (1991) 23 ( I )
44-47
Glycosaminoglycans in horses with osteoarthritis W. H. ALWAN, S. D. CARTER*, D. BENNETT and G. 6. EDWARDS Departments of Veterinary Pathology and Veterinary Clinical Science, University of liverpool, PO. Box 147, Liverpool 169 3BX, UK.
Summary
Materials and methods
Horse articular cartilage glycosaminoglycans (GAGs) were measured in synovial fluids from 48 joints affected with osteoarthritis (OA), 22 normal joints, four joints with osteochondritis, three joints with traumatic arthritis and seven joints infected with bacteria. Serum and urine from individual horses were also examined for the presence of GAGs. High levels of GAGs were found in synovial fluids (SF) from horses with OA. In each case, the level was higher in the synovial fluid than in the serum or urine from the same horse. Horses with OA showed high GAG levels in SF, serum and urine compared to horses with normal and infected joints. High levels were also found in horses with osteochondritis and traumatic arthritis. Levels of synovial fluid GAG reflect cartilage destruction in arthritis and may be useful for monitoring disease progression in the equine species.
Sample collection
Introduction DEGENERATIVE forms of arthritis are common in horses, constituting approximately 33 per cent of all equine lameness, and osteoarthritis is certainly the most important (Rose 1979; Rossdale, Hopes, Digby and Offord 1985). A major problem in measuring arthritis in any species is the absence of a reliable method of measuring disease activity which can be related to the degree of joint damage present. There is a definite need for laboratory markers of disease processes that are specific, sensitive, reproducible and related to the underlying pathological mechanisms. Over the last few years, several measurements have been described for human arthritic patients, but none is completely specific for osteoarthritis (OA) (Thompson 1987). Recent work has shown that, in human joint disease, breakdown of articular cartilage can be monitored by determining the levels of individual degraded proteoglycan molecules, ie glycosaminoglycans (GAGs), in both synovial fluids and sera (Thonar et a1 1985; Carroll 1989). Because degeneration of articular cartilage with loss and fragmentation of the proteo lycans is an important feature of OA (Perricone, Palmoski and Brandt 1977), and because OA is so common in horses, a study was performed to determine the concentration of GAGs in synovial fluid (SF),serum and urine of arthritic horses.
*Correspondence should be addressed to Dr S. D. Carter, Department of Veterinary Pathology, University of Liverpool. PO Box 147. Liverpool L69 3BX, UK.
Synovial fluids were obtained from horses under sterile conditions, centrifuged at 10,000 g for 20 mins to remove cells and debris and stored in aliquots at -70°C until assayed. Aliquots of SF were also collected for cell counts. Urine and serum from the same cases were also obtained, centrifuged and stored in 0.5 ml aliquots at -7O'C. Clinical cases of OA were referred to the Large Animal Hospital as lameness problems for second opinion evaluation. The diagnoses of OA, traumatic arthritis and osteochondritis were based on history, clinical examination and radiographic assessment. Most cases involved the carpal and fetlock joints. Septic arthritis (acute cases) was diagnosed by clinical and radiographical examination and by cytological examination of SF (high polymorphonuclear cell counts) and in some cases by a positive bacterial culture from SF. Horses with normal joints were selected from clinical cases presented at the Large Animal Hospital with no history of locomotor disease; most were colic cases. The absence of joint disease was confirmed by clinical examination, S F analysis and, in some cases, radiography. GAG assay
The Dimethylmethylene blue (DMB) assay was adapted from the method of Farndale, Buttle and Barrett (1986). Briefly, samples of SF, serum or urine diluted 1:lO with 50mM phosphate buffered saline, pH 7.0, were prepared. Aliquots (100 111) were transferred, in duplicate, to polystyrene tubes. A further 100 p1 of the same buffer, containing N-acetylcysteine (final concentration 2 mmol/l) and papain (0.13 unit), were added and the tubes were mixed, capped and incubated at 65'C for 2 h to digest proteins. After digestion the papain was inactivated with iodoacetic acid (final concentration 4 mmol/litre) and the NaCl concentration of the digest was adjusted to 0.5 mmol/litre in a final volume of 250 PI. DMB reagent (2.5 ml) was then added and the absorbance at 525 nM was read after 15 secs in a spectrophotometer. The DMB reagent was made according to the method published by Tayler and Jeffree (1969) and prepared by adding 3.04 g of glycine, 2.37 g of NaCl and 16 mg of 1.9-dimethylemethylene blue to one litre of deionised water. The pH was adjusted to 0.3 with HCI. The assay was calibrated with chondroitin sulphate and keratan sulphate and suitable solvent blanks. Measurement of individual sulphated GAG was achieved by using polysaccharidases (Sigma), which included chondroitinase AC (from Arrhrobacter aurescens), chondroitinase ABc (from Proreus vulgaris) and keratanase
45
EQUINE VETERINARY JOURNAL
0.3
1
1
-z-.
Chondroitin
-+-
TABLE 1: Mean (It sd) levels of GAG in SF,serum and urine from horses with OA, horses with normal joints and those with infected and traumatic arthritis and osteochondritis (OCD)
Keratan
(Pg/ml)
OA
Urine
Serum
SF
256 f 153* 30.85 It 11.35 15.5 f 7.28 8.3 k 4.05 8.25 f 2.05
Normal 66 f 32 12.31 f 5.4 Infected 110 rt 38.3 16.25 1 2.27 Traumatic 118 f 45 NT
0.2
n
OCD
N
m
NT NT
NT
154 f 73
No. of cases 48 22 7 3 4
NT Not tested; *: sd
8 0 .I
70C
0 .o 0
10
20 30 40 50 CONCENTRATIONS pg/rnl
64
600
8
500
.
Fig I : Standard curves for sulphared GAG with dimethylmerhylene blue. The assay was made with somples ofwhale chondroitin sulphate (chondroitin) and keratan sulphate (keratan). The results are means of four experiments. each concentration was tested in duplicate
a
(from Pseudomonas spp). Some SF samples were treated with 100 iu/ml of sheep testis hyaluronidase to digest hyaluronic acid and GAGs. To assess the accuracy of the assay, 10 and 20 pg of standard chondroitin sulphate were added to a sample of SF which, when tested at 150 dilution, had a GAG concentration of 50 pg/ml.
\
Statistical analyses
0 L 0
The data were analysed by the Student t-test and linear regression analysis.
a
L.
E
! 400
L
m
-'E'
a Y Y
a a
300
Results The standard calibration curves for sulphate GAGs are shown in Figure 1. The curves for chondroitin sulphate and keratan sulphate were similar because the colour yield of each was linear with concentration, but the intensity of metachromasia was different; chondroitin sulphate showed a higher absorbance than keratan sulphate. From these standard curves the amount of individual sulphated GAG could be estimated in the samples by digestion with enzymes; ie to measure chondroitin sulphate the samples should be digested with keratanase to digest keratan sulphate, to measure keratan sulphate, the samples should be digested with chondroitinase ABC or hyaluronidase. For this study the levels of GAG were calculated by referring to the chondroitin sulphate standard curve.
a
:
W
.a
to
a. a
C
200
.. 4 a a
ISI 100
a
a m 0
a
a
GAG in equine synovial fluids
0Synovial fluids from joints with OA showed very high levels of GAG, ranging from 25 to 600 pg/ml, with a mean (k sd) of 256
(k 153) pg/ml. Serum and urine also showed increased levels of
OA
-
3 a a a a
1 NORMAL
I NFEC
GAG which were significantly different from normal (P
