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...

Two major mechanisms for the biosynthesis of lecilthins (phosphatidyl choline) have been shown to occur: the utilization of intact choline through the intermediate stages of phosphoryl choline and CDP-choline ( 1 ) , and the "activation" of L-methionine ( Me)2 with formation of S-adenosylmethionine ( AdMe) , followed by the stepwise transfer of methyls to the N of an acceptor phospholipid (2-6).3In a previous investigation (9) the activities of enzymes involved in the former pathway have been determined on the livers, lungs, and brains of developing rats. We have now carried out a similar study of the enzymes responsible for the other pathway, that is: (a) Me-activating enzyme (ATP: L-methionine S-adenosyltransferase, EC 2.5.1.6), catalyzing the reaction:(1) Me + ATP + AdMe + PP + PI, and (b) phospholipid methyltransferase( s), catalyzing the reactions: 1 With the technical collaboration of Thelma T. 3 Of the other pathways which have been described, the reacylation of lysolecithins (7) should be regarded as part of a mechanism for the conversion of a lecithin species into another rather than a process for the de nozw formation of lecithins. The physiological significance of the Ca-activated incorporation of choline into lecithin by liver preparations (8) remains to be ascertained.(2) phosphatidyl EA + AdMe + phosphatidyl MEA + AdH phosphatidyl MEA + AdMe + phosphatidyl DME + AdH phosphatidyl DME + AdMe -+ lecithin + AdH (3 )

A number of high molecular weight compounds have been separated as constituents of normal urine. Regardless of the investigators' intention, in practically all of these studies only one or a few components were isolated. Interpretation and correlation of these numerous data, obtained by various procedures, would be greatly facilitated by the quantitative determination of the amount and composition of the total biocolloid content of normal 24 hour urine specimens. Hamerman, Hatch, Reife, and Bartz (1) have reported the recovery of the total nondialyzable solids (TNDS) from three 24 hour urine specimens by alternate dialysis and vacuum distillation with final lyophilization. The following experiments were designed to expand these data and to study individual variations in TNDS, in order to establish the limits of such variation in normal human urine. MATERIALS AND METHODSSubjects for the study were laboratory workers who were in excellent health, had no history or clinical evidence of renal or urinary tract disease, and who were within the average weight and height range for their age group. Each followed his daily routine without special diet, variation in fluid intake, or alteration in physical activity.Twenty-four hour urine specimens were collected into chemically clean containers, with 10 ml. of phenylmercuric nitrate 1: 1,000 as an inhibitor of enzymatic and bacterial activity. As soon as the collection was completed, each specimen was measured and the total specimen (or a 1,200 ml. aliquot) divided into 150 ml. portions. Each portion was placed in a 35 cm. Visking cellulose tubing 2 of 2.8 cm. inflated diameter. The sacs were knotted to include a 35 ml. volume of air in each bag. This arrangement kept the sac upright, absorbed mechanical shock, and permitted agitation of the urine during dialysis. The sacs were completely submerged in a closed carboy con-

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