Effect of diacetylrhein on the phagocytosis of polymorphonuclear leucocytes and its influence on the biosynthesis of hyaluronate in synovial cells.

1. The influence of diacetylrhein on the luminol-induced chemiluminescence of zymosan-activated polymorphonuclear leucocytes (PMNL) was investigated. At a concentration of 4 x 10(-5) mol/l diacetylrhein an inhibition of about 40% was found. 2. A model for the degradation of hyaline cartilage by frustrated phagocytosis was developed, in which human polymorphonuclear leucocytes cause a release of glycosaminoglycan peptides from hyaline cartilage slices (bovine nasal septum). We observed a 20% inhibition of this release at a concentration of 10(-4) mol/l diacetylrhein. 3. Human synovial fibroblasts synthesize the glycosaminoglycan hyaluronate. As a parameter of the rate of hyaluronate synthesis we measured the incorporation of 14C-glucosamine into hyaluronate. At a concentration of 2 x 10(-4) mol/l diacetylrhein a 4-fold increase of 14C-glucosamine incorporation in the membrane fraction of the synovial cells (tryptic fraction) and a 1.6-fold elevation of glucosamine release into the medium was measured. The synovial fibroblasts show a higher (1.5-fold) glucose consumption and lactate production in the presence of diacetylrhein (2 x 10(-4) mol/l).

UDP-D-xylose: proteoglycan core protein beta-D-xylosyltransferase: a new marker of cartilage destruction in chronic joint diseases.

We investigated the diagnostic significance of UDP-D-xylose : proteoglycan core protein beta-D-xylosyltransferase (EC 2.4.2.26) in different chronic joint diseases. This enzyme is located almost exclusively within chondrocytes, where it initiates the formation of chondroitin sulphate during the biosynthesis of proteoglycans and from which it is easily released after damage of articular cartilage. Xylosyltransferase activity was determined in synovial fluid and serum by a radiochemical method, based on the incorporation of [14C]xylose from UDP-[14C]xylose into an exogenous acceptor protein. Serum has been shown to be the appropriate material for the determination of xylosyltransferase activity in blood, since in plasma fibrinogen causes an inhibition of enzyme activity of about 50%. The catalytic concentrations of xylosyltransferase in synovial fluids and sera of patients with chronic joint diseases (n = 131) ranged from 0.5 to 22.0 mU/l and from 0.8 to 5.6 mU/l, respectively. On most cases we found higher xylosyltransferase activities in synovial fluids than in the corresponding sera. The highest catalytic concentrations of the enzyme were observed in the synovial fluids of patients suffering from rheumatoid arthritis (median value: 5.56 mU/l, 90%-range: 3.2-22.0 mU/l). Synovial fluids of patients with arthritis urica, however, showing a comparable high degree of inflammation, contained lower enzyme catalytic concentrations (median value: 2.38 mU/l, 90%-range: 0.7-5.2 mU/l), which were in the range of those in osteoarthrosis (median value: 2.50 mU/l, 90%-range: 0.8-4.8 mU/l). The higher xylosyltransferase activities in rheumatoid synovial fluids seem to be attributed to an increased cartilage destruction during the course of this disease.(ABSTRACT TRUNCATED AT 250 WORDS)

[Pathobiochemistry of joint destruction in inflammatory and degenerative joint diseases]. / Zur Pathobiochemie der Gelenkdestruktion bei entzündlichen und degenerativen Gelenkerkrankungen.

While the biochemical mechanism which leads to the destruction of joints in the course of degenerative and inflammatory arthropathies has not been cleared up completely to this day, basic differences have been noted in the way the two types of arthropathy affect the articular cartilage. The differences are described from the viewpoint of pathobiochemistry as they are fundamental to causal therapy.

Pathobiochemical significance of granulocyte elastase complexed with proteinase inhibitors: effect on glycosaminoglycan metabolism in cultured synovial cells.

Interactions between elastase inhibitor complexes and synovial cells are of special interest, since, in chronic joint diseases, granulocytes release large amounts of elastase into the synovial fluid and connective tissue, where the proteinase is bound to alpha 1-proteinase inhibitor and alpha 2-macroglobulin. To study the effect of elastase-alpha 2-macroglobulin and elastase-alpha 1-proteinase inhibitor complexes on the glycosaminoglycan metabolism of cultured synovial cells, we determined the distribution of [3H]glucosamine-labelled hyaluronate, which represents the main synthesized glycosaminoglycan, and of 35SO4(2-)-labelled chondroitin sulphate into the intracellular, pericellular and extracellular compartments of the cell culture. Exposure of the synovial cells to elastase-alpha 2-macroglobulin complexes leads to an enhanced synthesis and secretion of hyaluronate, and chondroitin sulphate, and also induces a rise of the fibronectin concentration in the medium. Analogous but less pronounced effects are observed in the presence of elastase-alpha 1-proteinase inhibitor complexes. Native uncomplexed elastase, however, causes no significant changes in hyaluronate metabolism. An increase of prostaglandin E2 in the culture medium during incubation with elastase inhibitor complexes occurs in parallel to the stimulatory effect on glycosaminoglycan metabolism. Our results demonstrate that elastase, whose enzymic activity is inactivated by the formation of complexes with alpha 1-proteinase inhibitor or alpha 2-macroglobulin, nevertheless acts as an inflammatory mediator, which in vitro induces metabolic changes closely resembling the in vivo findings in inflammatory joint diseases.

Granulocyte elastase as a new biochemical marker in the diagnosis of chronic joint diseases.

Human granulocyte elastase (EC 3.4.21.37) is released from granulocytes in large amounts in chronic inflammatory joint diseases and is therefore of special pathogenic and diagnostic importance. In order to examine the diagnostic significance of this enzyme as a clinico-chemical parameter, we determined the concentration of granulocyte elastase in complex with alpha 1-proteinase inhibitor by an enzyme immunoassay in synovial fluids and plasma of patients with chronic joint diseases. In inflammatory synovial fluids the concentration of complexed elastase correlates well with the granulocyte number and may increase to an extremely high level. In 90% of patients with manifest rheumatoid arthritis increased elastase levels are also observed in the plasma, probably due to the large gradient between the synovial fluid and plasma concentration, whereas in osteoarthrosis normal plasma concentrations were observed. Thus, these results indicate that normal plasma concentrations in patients with chronic joint diseases exclude the diagnosis of rheumatoid arthritis with high probability. The simultaneous determination of complexed elastase in plasma and synovial fluid improves the nosological differentiation of chronic joint diseases. Elastase activity on a specific chromogenic substrate, which was found in many inflammatory synovial fluids, is mainly attributed to elastase alpha 2-macroglobulin complexes. In some purulent synovial fluids, however, we were able to detect free elastase, which has been shown to play an important role in the destruction of articular cartilage.

Purification and properties of a manganese-containing superoxide dismutase from Acholeplasma laidlawii.

From the prokaryotic microorganism Acholeplasma laidlawii the major manganese-containing superoxide dismutase has been purified to homogeneity, as judged by polyacrylamide gel electrophoresis. The molecular mass of the enzyme was found to be 41 500 Da. It consists of two subunits of identical size and has an isoelectric point of 6.4. The enzyme contains 0.51 +/- 0.05 atoms of manganese per subunit. Its amino-acid composition and light absorption spectra are presented and compared with Mn- and Fe- containing superoxide dismutases from other prokaryotic organisms.

Purification and characterization of NADH oxidase from membranes of Acholeplasma laidlawii, a copper-containing iron-sulfur flavoprotein.

1. NADH oxidase was extracted from the membranes of Acholeplasma laidlawii with buffer containing 3% Triton X-100 and subsequently purified by several chromatographic steps. The final preparation was essentially homogeneous as judged by gel electrophoresis under nondenaturing conditions. 2. The enzyme appears to be a copper-containing iron-sulfur flavoprotein (FMN:CU:Fe:labile S = 1:1:6:6). The enzyme, containing a high fraction of hydrophobic amino acids, is composed of three subunits of molecular weight 65 000, 40 000 and 19 000. 3. When oxygen is used as electron acceptor the purified enzyme demonstrates a specific activity of 58.0 IU/mg of protein and catalyzes the formation of H2O2 in nearly stoichiometric amount. The apparent Km value for NADH is estimated to be 0.4 mM (pH 7.4). NADPH cannot serve as a substrate for the enzyme. In addition to the NADH oxidase activity, the enzyme is able to catalyze electron transfer from NADH to various other electron acceptors (ferricyanide, dichloroindophenol, cytochrome c). Metal-chelating agents and mercurials are shown to inhibit the activity of the enzyme. 4. From electron paramagnetic resonance and optical absorption measurements evidence was obtained that the flavin semiquinone radical in the NADH oxidase has a high air-stability, and that the flavin shuttles between the fully reduced and the semiquinone state upon electron transport from NADH to the electron acceptors. Inhibition of the NADH oxidoreductase activities by superoxide dismutase indicates that O-2 serves as an intermediate in the electron transfer from NADH to all electron acceptors used in this work. In addition to electron transfer via the superoxide radical O-2, an alternative pathway probably involving Fe-S centers is operative. From these results and literature data we present a reaction scheme for electron transport from NADH to the various electron acceptors.