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

Abstract

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. A mean excretion rate of 90 mg per 24 hours (range, 51 to 168 mg per 24 hours) has been established for both male and female adults. Attempts to separate the R-1 into subfractions by electrophoresis, various solvents, and ultracentrifugation have failed to provide subfractions which were distinctly different in composition from the original material. The present paper reports a further characterization of the R-1 mucosubstance by immunological methods. MATERIALS AND METHODSThe 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. The analyses of the centrifugate and supernate are given in Table I; the brown pellet is composed at least partly of cellular detritus and some inorganic crystalline material. The supernatant colloid migrated electrophoretically as a single hypersharp peak of ascending mobility 9.6 X 10' cm2 sect-volt' in veronal buffer of pH 8.18 and ionic strength 0.01 at a potential gradient of 5.8 v per cm. A frozen but unlyophilized sample of R-1 in water (5 mg per ml) was ultracentrifuged by M. C. Davies, Lederle Laboratories. A single hypersharp

Immunofluorescent Study on the Origin of Tamm-Horsfall Glycoprotein

“…The fluorescent materials were observed also in tubular lumina and the cell membranes were remained completely unstained. DISCUSSION Boyce et al (1) have demonstrated that the uromucoid antigen was contained in the kidney with a titre decreasing in the order of the cortex, medulla and pelvis. They found it also in the skin, subcutaneous fat and striated muscle, but not in the erythrocyte membrane, saliva, bone matrix and seminal plasma.…”

The Composition and Mode of Formation of Renal Calculi

“…These substances were unginally known as protective colloids but the term is probably not justified as our knowledge of them is incomplete. There are a number of methods of isolation that produce different fractions, some of which have been shown immunologically to be a mixture of mucoproteins (Boyce, King and fielden, 1961). There appear to be at least three types of urinary mucoprotein.…”

Total Nondialyzable Solids (Tnds) in Human Urine. Xiii. Immunological Detection of a Component Peculiar to Renal Calculous Matrix and to Urine of Calculous Patients*

“…The rabbit matrix immune serum was prepared by foot-pad injections of suspensions of lyophilized matrix or other antigen in complete Freund's adjuvant (10). Sera from adult rabbits, which had been injected with the same antigen, were pooled.…”

“…Previously described methods (10), utilizing the Ouchterlony agar gel plates, were followed. The plates with 6 peripheral circular wells of 0.3 ml capacity and 10 mm distance from the center well were suitable for this study, in which comparison of precipitin reactions of a large number of unknowns was desirable.…”

“…Uromucoid (R-1) fraction from normal urine. The R-1 fraction of urinary mucosubstances is 1183 highly antigenic and regularly produces rabbit immune serum of high titer (10). In the ten preparations of matrix immune sera, involving 36 rabbits, and in tests of samples of R-1 from 21 normal persons, purified by reprecipitation from distilled water with 0.58 M NaCl, only two positive precipitin reactions between R-1 and rabbit antimatrix serum were observed.…”