Effect of Red Cell Age on Its Susceptibility to Lysis by Rabbit Hemolytic Factor

Abstract

A hemolytic factor capable of lysing human and other mammalian red cells in vitro has been previously isolated from rabbit erythrocytes(l-3). This factor requires exogenous ATP, Mg++, and Na+ or K+ for its lytic activity, but is not dependent on glycolysis, choline esterase, or active ion transport. In addition, the action of this factor on intact erythrocytes is completely inhibited by preincubation with purified human stroma in the presence of the above co-factors. It appears, therefore, that its substrate is some component of the red cell membrane and that lysis of the cell is the result of membrane degradation.

Since it has been demonstrated that some relationship exists between the age of the red cell and its susceptibility to osmotic(4), im-munologic(5), and primaquine-induced he-molysis(6) it was of interest to investigate the effect of red cell age on its lysis by rabbit hemolytic factor.

Materials and methods. ⁵⁹Fe citrate: Abbott Laboratories; 100 microcuries per ml.

Glycine-2-¹⁴C: Prepared by the Isotopes Specialties Co.; specific activity of 4.2 mC per mM.

Buffered saline:99 parts isotonic saline + 1 part isotonic Tris (hydroxymethyl) amino-methane-Histidine HC1 buffer, pH 7.0.

Incubation medium. Sucrose (0.25 M) containing adenosine triphosphate (0.001 M), MgCl₂ (0.002 M), glutathione (0.0016 M), Ethylenediamine tetraacetic acid (0.0002 M), and potassium phosphate buffer, pH 7.0 (0.01 M).

Hemolytic jactor from rabbit erythrocytes. Partially purified rabbit hemolytic factor was prepared by ammonium sulfate fractionation of hemolyzed rabbit red cells as previously described and the hemolytic titer of each preparation was determined before use as described under “Hemolysin Test”(2), substituting washed rat for human red cells.

Glutamic oxaloacetic transaminase activity (GOT). Assayed by the method of Steinberg and Astrow(7).

Specific radioactivity of hemoglobin (Hgb). Hgb concentration was determined by measuring the optical density of stroma-free he-molysates at 540 mμ, in a Beckman model DU Spectrophotometer. The Hgb was precipitated (carrier Hgb added where necessary) 3 times from CCVsaturated water with acetone at 0°. ⁵⁹Fe was measured in a Tracer Lab well-type scintillation counter and ¹⁴C in an automatic Nuclear D-47 gas-flow counter. Radioactivity was expressed as CPM per mg of Hgb.

Experimental and results. Radioactively tagged red blood cells were obtained from male albino rats 4 to 40 days post intravenous injection of 11 to 20 μc of either ⁵¹Fecitrate or glycine-2-¹⁴C. The blood was mixed with an equal volume of ACD solution, plasma and buffy coat were removed and the cells were washed with buffered saline at 4°C until no radioactivity remained in the supernatant wash fluid.

In the beginning of this study the cells were then separated into a relatively “young” and a relatively “old” fraction after centrifugation in 2 volumes of buffered saline at 2,000 g for 30 minutes, the young erythrocytes being less dense than older cells(8). This was done in an attempt to emphasize any differences in behavior of the hemolytic factor toward cells of different ages. However, this was found to be unnecessary, and therefore in later experiments the total cell population was used without prior fractionation.