Azorubin-Binding Capacity and Protein Composition of Serum of Rats Subjected to Tourniquet Shock and to Treatment With Carbon Tetrachloride

Abstract

The cause of a decreased azorubin-binding capacity (ABC) of serum albumin, previously observed in humans under certain pathological conditions (1), is not known. The present paper is part of an experimental study into the mechanism of a lowering of the ABC (2). It describes the production of decreased ABC values of serum albumin of rats by subjecting them to tourniquet shock or to treatment with carbon tetrachloride.In addition, the serum protein composition of these experimental rats, was analyzed, since changes in the protein metabolism have been observed in traumatic shock (3, 3a). EXPERIMENTALChemical procedures. For the determination of the protein and non-protein nitrogen, the micro-Kjeldahl method of Hiller, Plazin, and van Slyke (4) was used, with some modifications which are described elsewhere (5). The non-protein nitrogen was separated from the serum proteins by precipitation with uranyl acetate according to the procedure of Neubauer (6). One ml. of serum was diluted with 3 ml. of water, and 1 ml. of 1. practically the same patterns and the same albumin percentages were obtained. The albumin concentrations were defined according to Wiedemann' (13) and determined from both the descending and ascending'boundaries. The globulin area was divided, by the method of Tiselius and Kabat (14), into three parts corresponding approximately to the alpha-, beta-, and gamma-globulin of human sera. More details on the electrophoretic analysis are given elsewhere (5).For the electrophoretic analysis of normal rat serum in the presence of azorubin, a solution of 5 mg. purified azorubin (for purification see [15]) in 8 ml. buffer was added to 4 ml. of serum and dialysed. The buffers used were Michaelis buffer pH 8.6 and phosphate buffers of pH 8.6, 7.7, 7.2, and 6.1, I = 0.1. The patterns were recorded on Kodak Ektachrome Color Film, using the optical system of Philpot-Svensson and a diagonal bar in the standard size electrophoresis apparatus.Determination of ABC. The chromatographic method for estimating the ABC (16) was modified so that only 1 ml. of serum was needed for the single chromatographic runs. A total of 4 or 3 ml. of serum was required, therefore, for each analysis, which consisted of a blank run and triplicate, or at least duplicate, runs of the serum-azorubin mixtures (see below). The chromatographic tubes employed were about 20 cm. long, with an inner diameter of 5 mm., and terminated in a 0.5 mm. capillary of 2 to 3 cm. length. A funnel was sealed to the upper end. The columns were formed using a slurry of 500 mg. of anionotropic aluminum oxide in freshly prepared, twice-distilled water. Methods for preparation and deactivation of anionotropic aluminum oxide have been described previously (15,16). The length of the column obtained was about 25 mm. The average total flow time for the serum-azorubin mixture described below was 9.5 minutes.For each run, 1 ml. of serum and 0.25 ml. of an azorubin solution (mostly 0.5 per cent) in 0.6 per cent sodium chloride (pH 7.8) were combined at least 30 minutes prior ...

Increased Electrophoretic Mobility of Albumin in the Serum of Rats Subjected to Carbon-Tetrachloride Treatment or to Tourniquet Shock

“…The blood was obtained on the tenth day. Other details have been given previously (3). The average weight of the rats declined from 336 gm.…”

“…under the injections of CCI4. Tourniquet shock was produced by applying rubber bands to both hind legs (3). The average weight of the animals at the time of sacrificing was 269 gm.…”

“…Total protein and non-protein nitrogen were determined by a micro-Kjeldahl procedure as outlined previously (3).…”

“…It was assumed then that the low ABC of serum albumin observed in clinical cases (2) or in experimental animals (3,4) was produced by a firm binding, in vivo, of increased amounts of certain anions, possibly higher fatty acids.…”

“…The electrophoretic mobilities of albumin were therefore determined in sera of rats subjected to carbon-tetrachloride poisoning and to tourniquet shock since these conditions are known to lower the ABC of serum albumin (3). Studies on the influence of dialysis on the ABC of sera and other protein solutions were included as supporting data.…”