C3 Degradation Research Articles

Influence of time, temperature and coagulation on the measurement of C3, C3 split products and C4

Quantitative and qualitative immunoelectrophoretic analyses of circulating C3, C3 split products and C4 were performed in matched EDTA plasma and serum obtained from 5 normal subjects and stored for up to 48 h at room temperature (18°C–22°C) and 4°C. Fluctuations in apparent levels of C3 were greater in serum than plasma stored at room temperature, a fall in levels seen by 24 h being followed by a significant increase. By contrast, levels of C3 did not alter if stored at 4°C. C4 levels in both EDTA plasma and serum remained unchanged for 24 h, a slight decrease being seen at 48 h. Levels of C4 remained constant if samples were stored at 4°C. Crossed immunoelectrophoresis revealed a significant progressive decrease in C3 levels and a simultaneous increase in C3c occuring after 4 h in serum and 8 h in EDTA plasma, stored at room temperature. In studies conducted at 4°C, similar but delayed fluctuations were seen. A progressive and significant increase in C3d levels was seen in both plasma and serum samples stored at room temperature, levels rising to 276% (plasma) and 308% (serum) of levels seen at zero time. At 4°C marginal increases in C3d levels only were observed. These results suggests that in vitro degradation of C3 and C4 are readily facilitated by temperature, time and coagulation, and that conditions of collection and storage of samples must be optimized for the accurate definition of activation of the complement cascade.

Metabolism of leukotriene C3 in the guinea pig. Identification of metabolites formed by lung, liver, and kidney.

[5,6,8,9,11,12-3H6]Leukotriene C3 was converted to polar metabolites which ere excreted in feces and urine for 4-5 days after subcutaneous administration to guinea pigs. Lung homogenates converted leukotriene C3 to leukotriene D3. Liver and kidney homogenates did not catabolize leukotriene C3 appreciably due to inhibition of gamma-glutamyl transpeptidase in these tissues by endogenous glutathione. Liver and kidney homogenates metabolized [3H6]leukotriene D3 to the cysteine analog, leukotriene E3 (Bernström, K., and Hammarström, S. (1981) J. Biol. Chem. 256, 9579-9582). In addition leukotriene C3 and products of greater polarity were formed. The conversion of leukotriene D3 to leukotriene C3 was catalyzed by gamma-glutamyl transpeptidase which performed a reverse reaction due to the high tissue concentrations of glutathione. The results suggest that the degradation of leukotriene C3 to leukotriene D3 is important for the further metabolism of this leukotriene in liver and kidney.

Acute hemorrhagic pancreatic necrosis in mice: alterations of serum complement.

Induction of acute hemorrhagic pancreatic necrosis by dietary means in mice produces alterations in the serum complement system. Total hemolytic complement, i.e., CH50, and native C3 levels fall during the development of pancreatitis while, at the same time, what could be immunoreactive C3 degradation products are demonstrable both in the circulation and in the urine. No evidence of renal deposition of C3 was obtained by immunofluorescence analysis, although marked alterations in proteinuria were observed, suggesting that renal dysfunction(s) is a feature of acute hemorrhagic pancreatic necrosis. Lack of renal complement deposition, together with our earlier negative findings with respect to pancreatic localization, suggests that serum complement alterations are side effects of the pancreatitis, attributable to intravascular, pancreatic enzyme-mediated degradation of serum complement components.

Inactivation of complement by mouse saliva: Enzymatic degradation of C2, C3, C4, C5, and C7

While searching for lymphocyte surface antigens in mouse saliva we have observed that this secretion prevented the complement-mediated lysis of lymphocyte in a microlymphocytotoxicity test. We show in this report that mouse saliva has a specific action on C2, C4, C3, C5, and C7 components which are inactivated very rapidly while C1, C6, C8, and C9 are not modified after 15 min of incubation. The action of mouse saliva is inhibited by PMSF, TLCK, and the Kunitz bovine trypsin inhibitor, which indicates the role of a tryptic-like enzyme. After fractionation on G-75, the main activity is recovered in a low molecular weight peak which is composed of a group of three to four proteins of 10–25,000 molecular weight. When mouse saliva is injected iv into rats, their complement hemolytic activity is completely abolished in the first 24 hr and then recovers to its normal level on the third day after injection. These preliminary results suggest that mouse saliva may become a useful tool for in vivo decomplementation experiments. This inhibition of the complement reaction is obtained with rat saliva but was never observed with any of the human saliva tested.

679 KINETIC ANALYSIS OF SOLID PHASE C3 FRAGMENTS DEPOSITED ON CROSS-LINKED DEXTRAN GEL BEADS

Upon activation of the third component of complement (C3) by the classical or alternative pathway convertases, an opsonically active form of C3 gets firmly bound to a wide variety of surfaces. To analyze the forms of solid phase C3, dextran gel beads (675,superfine) were incubated with normal human serum in veronal-buffered saline 5 mM Mg++, 10 mM ethylene glycol tetraacetic acid (EGTA) for timed intervals, and samples washed with phosphate-buffered saline (200 × volume) at room temperature. Gel beads were solubilized with Dextranase, and solutions analyzed on sodium dodecyl sulfate gels in unreduced and reduced (2-Mercaptoethanol) form. C3 fragments accounted for more than 90% of the total proteins present. A 175,000 M.W. fragment was the predominant species at 5′, 10′, 15′, 20′ and 30 minute intervals, and was maximum at 15′. Other fragments were detected as early as 5′ (155,000 M.W.), 10′ (138,000 and 23,000), 15′ (110,000), and at 60′ only these and not the 175,000 M.W. fragment were detected. Solubilization of dextran gel beads with Dextranase appears to be an ideal vehicle for the analysis of solid phase activation and degradation of C3 and the relationship of the deposited fragments to these various biological activities including opsonization and immune adherence. Supported by U.S.P.H.S. Research Grant AM 16392.

Urinary excretion of C3 antigen in glomerulonephritis.

C3 and fibrin degradation products (F.D.P.) have been measured in early morning urine samples from 38 normal people and 123 patients with glomerulonephritis. Normal urine contained less than 0.3 mug of either antigen per ml. C3 and F.D.P. were both detected in the urine of many patients with glomerulonephritis. Levels above 1 mug/ml were exceptional in patients with "minimal change," and the highest excretion of both antigens occurred in mesangiocapillary glomerulonephritis, membranous nephropathy, and focal glomerulosclerosis.Both C3 and F.D.P. excretion showed considerable variation with time, with parellel fluctuations in the two antigens. These fluctuations did not depend on the total protein leakage and suggest that the complement and clotting sequence are closely related in these glomerular disorders.