Howard L. Holley

An electrophoretic study of normal and post-mortem human and bovine synovial fluids

Abstract Electrophoretic studies of normal and post-mortem human synovial fluids showed no appreciable difference in their electrophoretic characteristics, but both differ in some characteristics from traumatic and arthritic fluids. A new rapidly moving peak was found frequently in the patterns for normal and post-mortem fluids and occasionally in bovine synovial fluids. This has been called the π peak. Bovine synovial fluid differed from the human fluids in respect to the number of protein components present. The “protein” content of the normal fluids agreed with the few values previously reported (2.8 g./100 ml.). The “protein” content of bovine synovial fluid was approximately one-third of the value obtained for the normal human fluids. The post-mortem human fluids have a total nitrogen value similar to the normal fluid value, but the NPN value for the post-mortem fluids was approximately three times that of the normal fluids.

Interactions of hyaluronic acid with serum albumin

Abstract The Pi peak observed in the electrophoretic patterns of many synovial fluids has been demonstrated to be principally a complex between hyaluronic acid and plasma albumin. It could be best shown at pH 8.6 in veronal buffers. The viscosity of hyalunoric interactions occurred at pH 7.1 to 7.6 in phosphate buffers. The viscosity of hyaluronic acid decreased in phosphate solutions, and the Pi component was not formed with depolymerized hyaluronic acid. Hyalunoric acid also produced abnormal patterns of the globulins when added to blood serum. In their ultracentrifuge patterns, such solutions exhibited small rapidly sedimenting peaks. These results are interpreted as indicating that hyaluronic acid solutions exist in a loose gel structure. In the presence of some proteins, a slowly dissociable bond is formed between the protein and the gel structure. This interaction may be important in the permeability of the inter-cellular ground substance of connective tissue.

Factors affecting the viscosity of hyaluronic acid and synovial fluid.

Abstract 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.

Some factors affecting the interaction of hyaluronic acid with bovine-plasma albumin

Abstract In veronal buffer at pH 8.6, hyaluronic acid and bovine plasma albumin may form a complex which is evident in electrophoretic patterns as a peak intermediate between those for the components. This peak is called the Pi complex. The present study was undertaken to determine the effects of relative viscosity of the solution, intrinsic viscosity of the hyaluronic acid and the relative concentrations of the components on Pi complex formation. With solutions in which the ratio of albumin to hyaluronic acid was low, all the albumin was bound to hyaluronic acid to form a Pi complex. In solutions that contained higher ratios of albumin to hyaluronic acid, the binding capacity of hyaluronic acid was apparently exceeded and free albumin was demonstrated in the solution. In contrast, some hyaluronic acid remained free even in the presence of an excess of albumin. Complex formation seemed to be directly related to the intrinsic viscosity of the hyaluronic acid.

Preparation and stability of hyaluronic acid

The Roseman electrodeposition method provided a convenient method for the preparation of hyaluronic acid. Five repetitions of the process were required, and yields of about 20% were obtained. The products obtained from bovine synovial fluid appeared to be free of protein and to be only slightly degraded relative to that in the original synovial fluid. The best materials had an intrinsic viscosity in 0.2 M phosphate buffer of about 50. The preparation was somewhat improved if the solutions contained 5–10% ethanol or were saturated with toluene. Merthiolate produced degradation of purified hyaluronic acid. Degradation of hyaluronic acid on lyophilization or storage could be made very slight by lyophilization of salt-free (dialyzed) gels and storage of gels or dry products at −10°. Solutions containing 20% ethanol could be kept for long times at room temperature without change of viscosity.

Synovial fluid. I. Comparison of sodium and potassium concentrations in normal and diseased joint fluid.

Conclusions 1. Data have been presented on the sodium and potassium concentrations of normal human synovial fluid. From the values obtained it is apparent that the normal values for sodium previously reported on material obtained from cattle joints compares favorably with those found in the living human subject and in postmortem fluids. The potassium values were quite variable, using the present technics. In the synovial fluid obtained from rheumatoid arthritics, there was an increase in sodium concentration with a corresponding increase in the synovial fluid/serum ratio for sodium. 2. Technics are described for the collection and analysis of synovial fluid.

An ultracentrifugal study of synovial fluid

Abstract Fifteen human post-morten synovial fluids having a wide range of “viscosity” were analyzed in the ultracentrifuge. All the fluids contained three major components which were albumin, globulin, and hyaluronic acid. In six of the fluids, minor components with sedimentation constants ranging from 7.7 to 13.6 S were detected. Six synovial fluids were analyzed prior to and after treatment with testicular hyaluronidase. No significant difference in the sedimentation constants of the albumin and hyaluronic acid components could be noted although the viscosity of the samples was markedly decreased by the enzymic treatment. A slight increase in the sedimentation of the globulin component was noted. The addition of hyaluronic acid to serum and to arthritic and postmortem synovial fluids also did not appear to affect the sedimentation behavior of the albumin significantly, whereas the presence of protein caused a decrease in the rate of sedimentation of hyaluronic acid. The hyaluronic acid appeared to cause a decrease in the sedimentation constant of the globulin component or a disturbance in the component. These results suggest that albumin sediments freely through a gel structure of hyaluronic acid, but globulin (or a component) may associate to some degree. The presence of protein of both types appears to slow down the collapse of the gel lattice.