Grace P. Kerby

Enzymatic Activity in Human Synovial Fluid from Rheumatoid and Non-Rheumatoid Patients.

Summary Enzyme assays have been done on sonicated cells after separation from synovial fluid (I), supernatant synovial fluid (II), and sonicated whole synovial fluid including cells (III). 2 lysosomal enzymes (acid phosphatase and lysozyme), 2 glycolytic enzymes (lactic dehydrogenase and pyruvate kinase), and adenylate kinase levels were measured in each of the 3 samples from each synovial fluid. Rheumatoid and non-rheumatoid fluids were studied. Results suggested that lysozyme derived at least in part from connective tissue. Lysozyme released from suspended cells by sonication was partially inhibited by supernatant synovial fluid (both rheumatoid and non-rheumatoid), as was commercial egg-white lysozyme added to synovial fluid. Lactic dehydrogenase was inhibited somewhat by rheumatoid synovial fluid and by 2 gouty synovial fluids studied. Commercial lactic dehydrogenase added to rheumatoid and non-rheumatoid synovial fluid was partially inhibited by the rheumatoid and not by the non-rheumatoid fluid.

Release of enzyme from human leukocytes on damage by bacterial derivatives.

Summary By a method which detects cell injury by measurement of a lysozyme-like enzyme released from the cell, the adverse effect of various bacterial derivatives on human leukocytes is demonstrated. Variation in degree of effect from individual to individual is evident.

Inhibition of Lysozyme by Heparin.

Summary The inhibition of lysozyme by heparin is shown by the usual criteria of the Michaelis-Menton formulation to be competitive in type.

Effect of Adrenal Hormone Overdosage on Bacterial Removal by the Splanchnic Viscera.

Summary Chronic administration of adrenocorticotropic hormone and cortisone had no effect on the removal of M. aureus var pyogenes by the splanchnic vascular system. It is suggested that the increased incidence of bacteremia seen after ACTH and cortisone as well as that seen in the leucopenic states is the result of poor localization of the organisms in the tissues. In the former, the reduction in the inflammatory reaction and the change in the metabolism of leucocytes are important. In the latter, white blood cells are not present to migrate into the lesion. There was a questionable increase in removal rate after acute administration (4 hours).

A method for detection of leukocyte injury based on release of a lysozyme-like enzyme.

Summary A method is described for the detection of cell injury by the measurement of intracellular substances released upon injury. The cells studied have been human leukocytes, and the intracellular substance selected for measurement has been an enzyme with the properties of lysozyme. Application of the method is illustrated by data concerning the adverse effect of hypotonicity and of an antiserum to human leukocytes on the cells. In further studies beyond the scope of the present report, the method has been found to be useful in screening a variety of physical, chemical and biologic agents for adverse effect on the metabolism of human leukocytes, as well as for detecting reversal of such injurious effects by protective agents.

Chromatographic Separation of Heparin and Ghondroitin Sulfate.

In a recent communication, Rienitis(1) has contributed interesting and useful data concerning the electroporesis of acid mucopolysaccharides on filter paper. Certain mixtures of acid mucopolysaccharides have been resolved by the method, but the separation of heparin and chondroitin sulfate was not accomplished, due to the slightly different mobilities of the substances. It is of interest to note that such separation can be achieved chromatographically. The writers current investigations are concerned with acid mucopolysaccharides as found in biological materials of human and animal source. In the course of these studies, chromatographic technics have been used, the filter paper strips (Whatman No. 1, 44 cm) being stained by a method developed in this laboratory and similar to that used by Rienitis. The accompanying photograph (Fig. 1)shows the distinct separation of a mixture of chondroitin sulfate and heparin. The ascending chromatogram was developed with 25% propanol in M/15 phosphate buffer at pH 6.4 for 33 hours at 4°C. Heparin Sodium (Liquaemin; Organon, Inc.) and Chondroitin Sulfate (General Biochemicals, Inc.) were used in the present study. Results of the application of this technic in the investigation of biological materials will be incorporated in later reports.

Urinary Excretion of Acid Mucopolysaccharides by Rabbits Injected with Chelating Agents and with Chondroitin Sulfate.

Summary (1) Introduction of chondroitin sulfate into the blood stream of rabbits was followed by elevated excretion of acid mucopolysaccharides in urine totalling 5-10% of injected amount over 2-4 days thereafter. Repeated injections were followed by excretions which were still 5-fold greater than baseline levels one week after third and last injection. (2) Repeated subcutaneous injection of disodium EDTA and disodium calcium EDTA into rabbits was followed by a brief and frequently mild rise in urinary excretion of acid mucopolysaccharides; onset, degree and duration of rise varied widely from animal to animal. The authors are indebted to Harry T. Tully and W. J. K. Rockwell for management of daily urine collections and injection of rabbits and to Sadie Taylor for skillful technical assistance; also to Philip Pickett for preparation and special staining of histologic sections.

A Method for Measuring Removal of Bacteria from the Blood by the Various Organs of the Intact Animal.

Summary 1. A method of constant intravenous infusion of bacteria combined with the determination of bacterial counts in the circulation at various sites by venous catheterization is described, which provides a means of determining the site and of quantitating the rate of removal of bacteria from the blood stream of the intact animal. 2. The total canine splanchnic removal of hemolytic Micrococcus aureus averaged 74 ± 16% (S.D.); splenic removal, 78 ± 17% (S.D.). No organisms were lost in circulation through the lungs. 3. The total rabbit splanchnic removal of encapsulated Klebsiella pneumoniae type B averaged 20 ± 10% (S.D.).

In vitro Absorption of Serotonin by Thrombocytes of Rheumatoid Arthritic and Non-Arthritic Individuals.

Summary Under controlled in vitro conditions (involving equal numbers of human thrombocytes in autologous plasma to which was added an amount of serotonin creatinine sulfate that could be absorbed completely by a few normal specimens in the limited time period allowed), a possibly significant decrease in absorption of serotonin was noted with thrombocytes from patients with non-rheumatoid inflammatory states (p <.02) and with rheumatoid arthritis (p <.05), as compared to individuals with no demonstrable inflammation.

The Influence of Buffers on Human Platelet Aggregation and Energy Metabolism

Summary Washed human platelets suspended in Krebs-Ringer-bicarbonate and Krebs-Ringer-phosphate buffers, respectively, showed significant differences in lactate production, in 14CO2 production from pyruvate-2-14C, and in degree of aggregation by ADP. Degree of aggregation of platelets by ADP or palmitate was greater in bicarbonate than in phosphate buffer, as was production of lactate by platelets from added glucose or pyruvate. The reverse was true of 14CO2 production. The effect of exogenous ATP previously reported (2) was shown to be in large part but not completely due to addition of phosphate moiety. The effect of added palmitate previously reported (3) was shown to require inorganic phosphate for significance and to be due chiefly to competition with pyruvate in furnishing acetyl CoA to the tricarboxylic acid cycle.