Isolation of hyaluronic acid from human synovial fluid by pronase digestion and gel filtration

The isolation of hyaluronic acid from human synovial fluid by passage down Sephadex G-200, following sterile digestion with pronase in Tris buffer (pH 7.9) for 8 h at 37°, affords a product without the occurrence of the enzymic or chemical degradation that may accompany other methods of isolation. Use of phosphate buffer, under similar conditions, gives turbid solutions of hyaluronic acid.     

Rheological properties of synovial fluids

Synovial fluid is the joint lubricant and shock absorber [Semin. Arthritis Rheum. 32 (2002), 10-37] as well as the source of nutrition for articular cartilage. The purpose of the present paper is to provide a comprehensive review of the rheological properties of synovial fluid as they relate to its chemical composition. Given its importance in the rheology of synovial fluid, an overview of the structure and rheology of HA (hyaluronic acid) is presented first. The rheology of synovial fluids is discussed in detail, with a focus on the possible diagnosis of joint pathology based on the observed differences in rheological parameters and trends. The deterioration of viscoelastic properties of synovial fluid in pathological states due to effects of HA concentration and molecular weight is further described. Recent findings pertaining to the composition and rheology of periprosthetic fluid, the fluid that bathes prosthetic joints in vivo are reported.     

Factors affecting the viscosity of hyaluronic acid and synovial fluid

The composition of clots formed from bovine synovial fluids was found to vary markedly with pH at 0.05 ionic strength. All hyaluronic acid was found in the clot over the range pH 2.2–4. The percentage of hyaluronic acid in the clot increased with a decrease of pH. At ionic strengths greater than about 0.22, mucin clot formation did not occur.These mucin clots belong to the class of complex coacervates of Bungenberg de Jong and appear to be combinations of a negatively charged acid (hyaluronic acid) and positively charged proteins.Synovial fluids underwent a mainly irreversible loss of viscosity upon exposure to acid conditions, especially in the pH interval 3–4. This loss in viscosity appears to have arisen from the oxidative-reductive depolymerization (ORD) reaction and a depolymerization of hyaluronic acid.The effect of pH on the ORD reaction was studied and found to be different for the four “reducing agents” studied. Three of four “reducing agents” showed a maximum effect in the range pH 4–7.     

Localization of hyaluronic acid in synovial cells by radioautography

Cultured human synovial cells secrete hyaluronic acid (HA) into the culture medium. Glucosamine-6-³H was shown to be a direct and relatively specific precursor of HA-³H by the following observations: the susceptibility of nondialyzable radioactivity in the medium to hyaluronidase, its migration with hexuronic acid on zone electrophoresis in polyvinyl chloride, its exclusion from Sephadex G-200, and the localization of radioactivity to glucosamine after hydrolysis of the labeled polysaccharide. The presence of intracellular HA-³H was established by sequential extraction of labeled cells and by radioautography of synovial cell cultures digested with hyaluronidase in situ. When cells were exposed to medium lacking glucose, glucosamine-³H-uptake was enhanced; and this made possible electron microscopic radioautographic studies. These studies demonstrate the early and continued presence of HA-³H within the Golgi apparatus.     

Isolation and properties of hyaluronic acid from bovine heart valves

1. A soluble extract of bovine heart valves was obtained after the tissue had been pulverized at liquid-nitrogen temperatures in a mill. 2. Hyaluronic acid was isolated from the crude extract by sedimentation equilibrium in a caesium chloride density gradient (Franek & Dunstone, 1966). 3. Analysis of the product indicated that it contained 15% of protein and the molar ratio of glucuronic acid to glucosamine was 1.27. 4. Its physicochemical properties, as determined by lightscattering, viscosity and sedimentation studies, suggested that its molecular size and configuration were similar to those of hyaluronic acid isolated from ox synovial fluid (Preston, Davies & Ogston, 1965).