Total Nondialyzable Solids (TNDS) in Human Urine. II. A Method for Reproducible Fractionation1

Abstract

The amount and nature of the total nondialyzable solids (TNDS) of normal human urine have been parametrically defined (1). This report is concerned with a technique for separation of the TNDS into three reproducible primary fractions, which are suitable for subfractionation by other methods, and their composition. MATERIALS AND METHODS MaterialsSubjects. Twelve young adult individuals (seven male, five female), who had no history or clinical evidence of renal disease or current evidence of other disease, served as subjects for this study. Each subject continued the usual daily activities and diet during the collection of three 24 hour urine specimens. Female subjects submitted specimens near the midportion of the menstrual cycle.Membranes for ultrafiltration. The solvent for collodion membranes was prepared from analytical grade reagents by mixing 96.1 ml. absolute ethanol, 40.5 ml. diethyl ether, and 13.5 ml. glacial acetic acid. One hundred ml. of collodion2 was then added and thoroughly mixed.Discs of the best grade of plate glass, 15 cm. in diameter, scratch-free, and hand-rubbed to provide a polished surface, were stored in bichromate cleaning solution. The edges of the glass plates were unbeveled. Prior to use, the plates were rinsed thoroughly with distilled water and air dried. The lint-free plate was then floated on mercury in a glass dish in a desiccator containing 75 ml. of concentrated sulfuric acid in the bottom. Exactly 12 ml. of the collodion solution was poured onto the center of the plate and allowed to dry for exactly 20 minutes in a closed desiccator. The collodion-covered disc was lifted out and slid into a vessel of distilled water. The gelled membrane floated free to the surface of the water and was transferred on a piece of filter paper for storage in distilled water saturated with thymol. The sulfuric acid was changed after preparation of each membrane.These membranes have a "T" value of 14 to 34 seconds, i.e., the period required for 100 ml. Ultrafilters. Ultrafilters of 4 L. capacity, having porous steel supporting plates 15 cm. in diameter, were used.4 Methods Fractionation and recovery. The collection and dialysis of 24 hour urine specimens was by the previously described technique (1). A 'Ao aliquot was removed for TNDS determination and the remainder of the 24 hour specimen was ultrafiltered at approximately 30 C., under a maximum pressure of 30 to 40 lbs. per sq. inch. This required about 24 hours. The solids in the ultrafiltrate were recovered by stepwise lyophilization, as previously described (1). The resultant brown powder was designated UFO (ultrafiltrate 0). The residue, designated RO (residue 0), was removed from the membrane and extracted three times with five volumes of barbital-sodium barbital buffer, ionic strength 0.1N, pH 8.6. The veronal insoluble fraction of RO, recovered by centrifugation (relative centrifugal force = 1,000 X G, 20 minutes) and designated as R1 (residue 1), was immediately suspended in distilled water and dialyzed against cold distilled water for no...

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.…”

“…Two male subjects were Negroes. The collections were made during the period September 15 to December 10. As each day's specimen was completed, the process of dialysis and subsequent fractionation of the TNDS was begun, as previously described (1,2 The supernatant, at room temperature, had an excess of ammonium sulfate added. The saturated solution was centrifuged or filtered 3 after standing about one-half hour and the precipitate was washed with saturated aqueous ammonium sulfate; the washings were added to the supernate.…”

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.…”

“…A method has been described for separation of the nondialyzable solids of normal urine into three major fractions (1). The R-1 fraction is composed of the portion which is not filtrable through collodion membranes of average pore diameter 20 mtn, and which is insoluble in veronal buffer of pH 8.6 and ionic strength 0.1.…”