Total Nondialyzable Solids (Tnds) in Human Urine. Iii. A Method for Subfractionation of Rs-1 Solids*

Abstract

The total nondialyzable solids (TNDS) of normal human urine have been found to have an approximate composition of 47 per cent protides, 16.6 per cent glucides, 9.7 per cent sialic acid, 6.2 per cent hexosamine, 3.3 per cent lipids, 12.2 per cent bound water and 8.5 per cent ash (1). The mean TNDS excretion in two series of determinations has been found to be 433 mg. and 505 mg. per 24 hours, with a value of 472 (S.D. ± 108) mg. per 24 hours for the combined series (1, 2). Methods have been described for separation of the TNDS into three reproducible fractions (2). Figure 1 is a flow sheet illustrating the technique and approximate percentage weight distribution of each fraction. Boundary electrophoretic studies, at pH 8.6, have demonstrated the presence of concentration gradients in the RS-l fraction which have mobilities closely approximating each of the gradients of normal blood plasma under similar conditions (3). The Cohn Method 10 for fractionation of plasma proteins has been modified by Lever and co-workers to permit separation of small quantities of plasma proteins (4). The present report concerns the application of this technique to the fractionation of RS-l solids from normal human urine. MATERIALS AND METHODSSubjects. Ten to 12 subjects (seven male and five female) submitted 24 hour urine specimens, which were pooled daily for rapid processing (2).Reagents. The reagents were prepared from stock solutions at room temperature and cooled to -50 C. immediately before use. The reagents A and A' of Lever gave an initial precipitation medium of pH 5. amount of acid-citrate-dextrose (ACD) plasma. The aqueous solution of RS-1 urinary fraction was found to lack the buffering action of ACD plasma; hence, in order to achieve the above conditions for urinary RS-1, the reagents A and A' of Lever and co-workers were combined to form Reagent I.Reagent I. This reagent contained 250 ml. 95 per cent ethanol, 20 ml. of M sodium acetate, 1.75 ml. of M acetic acid and water to make 1,000 ml. When necessary, the pH of this solution was adjusted to pH 5.80 ± 0.05 by addition of approximately 0.05 ml. of 10 M acetic acid.Zinc reagent. This reagent contained 54.8 Gm. of zinc acetate dihydrate dissolved in 500 ml. of water, to which 200 ml. of 95 per cent ethanol was added and then sufficient water to make 1,000 ml. The final pH was 6.5 ± 0

Total Nondialyzable Solids (Tnds) in Human Urine V. Subfractionation of the Ultrafiltrate (Uf-O) Fraction *

“…Three 24 hour urine specimens were collected, with chloroform preservative, by each of 12 normally active, healthy subjects (eight male, four female). With one exception, they were the same subjects as in preceding reports of this series (1)(2)(3). Two male subjects were Negroes.…”

“…Where necessary, pools of material from three to five individuals were used for analysis. 3 Eaton-Dikeman 42 mm. paper, No.…”

Total Nondialyzable Solids (Tnds) in Human Urine. Ix. Immunochemical Studies of the R-1 “Uromucoid” Fraction*

“…The R-1 fraction of normal human urine was recovered, as previously described (1,2), by veronal buffer (pH 8.6, ionic strength 0.1) extraction of the nonultrafiltrable solids of dialyzed urine. The fraction was suspended in distilled water and centrifuged at 40,000 G for 30 minutes, leaving a supernatant colorless opalescent gel or solution, or both, of approximately 5 mg per ml, which was used in the following experiments.…”

“…Preparation of test antigens. The fractions of total nondialyzable solids of normal urine were prepared as previously described (1,2). Samples of Tamm and Horsfall urinary mucoprotein (3), Di Ferrante-Rich mucosubstance (6), Maxfield mucoprotein (7) and AndersonMaclagan material (8) were prepared by the methods of the respective investigators.…”

Total Nondialyzable Solids (Tnds) in Human Urine. Iv. Electrophoretic Properties of Rs-1 Subfraction*

“…veronal soluble components of the total nondialyzable solids (TNDS) of normal human urine have been designated as the RS-1 fraction (1). Separation of RS-1 into three subfractions by a modification of Cohn's Method 10 for fractionation of human plasma has been reported (2). The RS-1 and subfractions RS-1A (Cohn I, II and III), RS-1B (Cohn IV and V) and RS-lC (Cohn VI) have been subjected to boundary electrophoresis at pH 8.6 and 4.5.…”