John Sandson

Dialyzability, Protein Binding, and Renal Excretion of Plasma Conjugated Bilirubin*

Conjugated bilirubin is excreted in the urine, but unconjugated bilirubin is generally believed not to be excreted by the kidneys. The basis for this difference has not been resolved by past studies. Hoover and Blankenhorn reported that bilirubin was dialyzable only from the plasma of patients who had bilirubinuria, and that it was not dialyzable from the plasma of patients with acholuric jaundice (3). Most subsequent investigators, however, have maintained that neither conjugated nor unconjugated bilirubin was dialyzable or ultrafilterable from plasma (4-7). This led to the hypothesis that conjugated bilirubin was probably excreted in the urine by a process of renal tubular secretion (8). Studies that were performed in jaundiced dogs in this laboratory gave results consistent with a process of glomerular filtration of conjugated bilirubin (9). We therefore re-examined the question of the dialyzability of plasma bilirubin. A small fraction of plasma conjugated bilirubin in jaundiced humans and dogs was found to be dialyzable, and hence available for urinary excretion by glomerular filtration. The studies indicated that this dialyzable fraction was bound to low molecular weight carrier(s), which migrated in the alpha and beta globulin zones on paper electrophoresis and which sedimented on ultracentrifugation.

Cellular Origin of Hyaluronateprotein in the Human Synovial Membrane

Bright fluorescent staining, which indicates the presence of hyaluronateprotein, was observed in the lining cells of the synovial membrane following application of rabbit antiserum to hyaluronateprotein and a fluorescein-labeled antiserum to rabbit γ-globulin. Staining was shown to be specific and due to antigenic determinants on or closely associated with the protein moiety of hyaluronateprotein.

UNUSUAL PROPERTIES OF HYALURONATEPROTEIN ISOLATED FROM PATHOLOGICAL SYNOVIAL FLUIDS

In joints actively involved by rheumatoid arthritis, the nature of the synovial fluid is quite different from normal. The fluid is increased in volume (10 to 30 ml) and contains large numbers of leukocytes (> 10,000 per mm3) . The concentration of protein (4 to 5 g per 100 ml) is more than twice that of normal synovial fluid, whereas the concentration of hyaluronate (0.1 g per 100 ml) is reduced to one-third to one-half normal. Many studies have been made of the physical properties of rheumatoid synovial fluid (1), and although these reflect the physical state of the hyaluronate in the fluid, the hyaluronate has not been isolated for study. Recently, mild methods were described to isolate hyaluronate from normal synovial fluids (2). Hyaluronate containing about 2% protein was obtained, and this product was called hyaluronateprotein (HP). Similar methods were used to isolate hyaluronate from rheumatoid synovial fluids, and a number of unusual findings were observed. A compound of hyaluronate combined with more protein (about 10%o) was isolated from the majority of the fluids; some of these products formed gels during dialysis in acetate buffer at pH 4.5, and all showed immobilization at the origin during zone electrophoresis at pH 4.5. None of these properties of the hyaluronate in rheumatoid synovial fluids has been previously described. During the course of these studies, the question arose whether such findings were specific for HP from rheumatoid fluids. It appears that they are not, for in a preliminary study of fluids from a few

Synovial cell synthesis of a substance immunologically like cartilage proteinpolysaccharide.

Fluids from joints contain a substance that reacts immunologically like one of two known antigenic components of articular cartilage proteinpolysaccharide. This newly recognized substance occurs in the lining cells of synovial membranes as shown by indirect immunofluorescence. The localization of this substance in tissue culture cells derived from synovial membranes and its identification in the culture medium supports the suggestion that it is synthesized by lining cells. Rheumatoid synovial cells contain less of this substance.

Human Synovial Fluid: Detection of a New Component

A new component has been detected in synovial fluid by the agar double-diffusion technique. This component is closely related immunologically to the proteinpolysaccharides of cartilage, and is the first probable degradation product of the cartilage matrix to be consistently identified in human synovial fluid.

Biochemical events in joint disease

FORMERLY untouchable human joints are now receiving the attention of workers in widely separated fields. This is not surprising for there has been a growing realization that in the synovial joint, biochemistry, anatomy and mechanics converge and influence each other in many direct and intimate ways. A movable joint is composed of the synovial membrane and capsule, articular cartilage and subchondral bone, ligaments, tendons and fibrocartilages, and synovial fluid. The most urgent need has been a means to obtain specimens of these joint components from living subjects. This need was perhaps first met by the introduction of safe and effective needles [24] that permitted biopsies of synovial membrane from closed joints of ambulatory patients. Specimens of synovial membrane, cartilage, or bone are now obtained with increasing frequency by orthopedic surgeons during arthrotomy [5, 61, or from amputated limbs [7]. A great deal of information is accumulating from studies on joint components using techniques of chemistry, immunology, histology, cytochemistry, electron microscopy, and immunohistology. Integration and synthesis of this information into a coherent view of joint function is making progress, especially in the normal joint [8, 91. New studies and ideas relate the structure and metabolism of the synovial membrane to its secretion of synovial fluid, and explore the function of this viscous fluid as a nutrient source and lubricant for the moving articular cartilages. This paper will concentrate on some aspects of the altered structure and function of synovial joints involved by rheumatoid arthritis.

Paper Electrophoresis of Human Synovial Fluid.

Summary After treatment of human synovial fluid with hyaluronidase, the hyaluronate and proteins can be separated by paper electrophoresis. The electrophoretic distribution of the protein fractions of normal synovial fluid differs from that of the protein fractions of both normal serum and osteoarthritic synovial fluid.

EFFECT OF ASCORBIC ACID ON RHEUMATOID SYNOVIAL FLUID.

Robertson, Ropes, and Bauer (1939) were the first to show that L-ascorbic acid could cause a marked decrease in the viscosity of solutions of hyaluronate. Ascorbic acid is present in the blood of normal persons in concentration high enough to cause a decrease in the viscosity of solutions of purified hyaluronate. It is not known whether ascorbic acid plays a role in the pathogenesis of diseases in which depolymerization of hyaluronate is thought to occur. The concentration of ascorbic acid in the synovial fluid of patients with rheumatoid synovitis was determined and correlated with (1) the concentration of ascorbic acid in the serum, and (2) the reduced viscosity of the synovial fluid.

Binding of an Alpha Globulin to Hyaluronateprotein in Pathological Synovial Fluids

Rabbits were immunized with hyaluronateprotein isolated from normal or pathological synovial fluids. Immunoelectrophoresis of hyaluroni-dase-digested pathological hyaluronateprotein and its antiserum showed a single precipitin arc extending through both alpha globulin zones. Digested normal hyaluronateprotein formed no lines with either its antiserum or with that to pathological hyaluronateprotein. The antigenic component present in pathological hyaluronateprotein formed a reaction of complete fusion with a serum protein which was identified by immunoelectrophoresis as an alpha globulin.

NONDIALYZABLE HEXOSE OF HUMAN SYNOVIAL FLUID

Studies that show differences in the components of synovial fluids in osteoarthritis and rheumatoid arthritis may be helpful diagnostically. Synovial fluids from joints involved by rheumatoid arthritis contain a high concentration of protein, and electrophoretic studies reveal increased a2-globulins (1, 2). Since this protein fraction contains the highest concentration of hexose among the, electrophoretically separated proteins of serum (3), determination of nondialyzable hexose concentration in synovial fluid was undertaken. In synovial fluid from osteoarthritic joints the nondialyzable hexose concentration, determined with the anthrone reagent, was similar to or lower than the normal level, while in synovial fluid from joints involved by rheumatoid arthritis it was elevated. The first step in this study was to identify the carbohydrate components of dialyzed synovial fluid that contributed to the anthrone reaction. Synovial fluid was dialyzed and then lyophilized. The lyophilized solids were hydrolyzed in sulfuric acid, and the carbohydrates in the hydrolysate were separated by a combination of zone electrophoresis in borate buffer and paper chromatography. Galactose, mannose, small amounts of fucose and glucose, and hyalobiuronic acid were found.