Serum Inhibitors of Elastase, Trypsin, and Plasmin in Chronic Rheumatic Diseases
Authors: W. Ardelt, S. Ksiezny, Izabella Niedzwiecka-Namyslowska, A.Z. Budzynski
Journal: Annals of the Rheumatic Diseases (1966)

There have been few studies of the serum level of proteolytic inhibitors in chronic rheumatic diseases.Patients with rheumatoid arthritis (RA) were found to have an increased level of serum trypsin inhibitor (Coke, 1949; Ragan, Grokoest, and Boots, 1949) as well as of plasmin inhibitor (Thomas and Dingle, 1955; Kope6 and Amatuni, 1961).Pancreatic elastase is an enzyme with high affinity for some of the protein components of connective tissue, particularly elastin.Disturbances of connect- ive tissue metabolism in chronic rheumatic diseases are of special interest with respect to this specific lytic enzyme system and its inhibitors present in serum.Serum inhibitor of elastase was found in normal human subjects by Balo and Banga (1949).Its level is said to be increased in pregnancy (Walford and Schneider, 1959; Schneider, Walford, and Dig- nam, 1960; Gilfillan, Sbarra, and Bardwil, 1961) and in cases of the Ehler-Danlos syndrome (Hall, Keech, Reed, Saxl, Tunbridge, and Wood, 1955), but the latter observation was not confirmed by Carter and Walford (1963).Contradictory reports on inhibition of elastolysis by serum of patients suffering from atherosclerosis have also been pub- lished (Balo and Banga, 1953; Walford and Schneider, 1959; Balo, 1960; Banga, 1963; Gilfillan and others, 1961).According to Gilfillan and others, (1961) the inhibitor of elastase was increased in certain cases of RA and tuberculosis.Our previous preliminary communication (Ardelt, Nied- zwiecka-Namyslowska, Loegler, Ksiezny, and Bud- zynski, 1966) indicated an increase in the level of inhibitor of the elastolytic function of elastase in both RA and ankylosing spondylitis (AS).No further detailed studies have been made, however, of the inhibiting activity against all three enzymes.Our present aim was to study the inhibition of the elastolytic and proteolytic functions of elastase by serum of patients suffering from RA and AS, and by serum of normal human subjects.Furthermore, in some of the patients, the serum inhibitors of trypsin, plasmin, and both of the elastase activities were determined in order to study the mutual relationship between the inhibitors of the various en- zymes.Material The studies were conducted on patients suffering from RA and AS, and control sera were obtained from healthy blood donors.Stained elastin was used as a substrate for the deter- mination of the elastolytic function of elastase.Elastin was obtained from ox ligamentum nuchae by the method of Partridge, Davis, and Adair (1955).This preparation was stained with orcein (G.T. Gurr, London) according to the method described by Banga (1963).Additional elution of weakly-bound orcein was performed in 0-08 M TRIS-HCL buffer atpH 9*0.Stained elastin was dried at room temperature.Casein solution for proteolytic determinations was obtained from light white casein (BDH, England) according to the method of Kunitz as described by Laskowski (1955).This solution was used for determining the proteolytic activity of elastase and trypsin but it appeared unsatisfactory for determining that of plasmin.For this purpose another casein solution was prepared as follows: about 5 per cent.casein solution in 0 1 M sodium phosphate bufferpH 7 -6 was exhaustively dialyzed against this buffer.Thereafter the solution was diluted with the same buffer to 3 per cent.casein concentration and heated in boiling water bath for 15 minutes.Elastase was obtained from commercial pancreatin (Fluka, Buchs) according to the method of Bagdy and Banga (1957).Trypsin was a crystalline preparation (Spofa, Prague).Plasmin, obtained by streptokinase activation of human plasminogen prepared according to the method of Kline (1953), was kindly supplied by Mr. Z.Wegrzynowicz, Institute of Nuclear Research, Warsaw.All the other reagents were Polish commercial products of analytical grades of purity.