Workers in this laboratory have long been interested in the production of plasma proteins and the fluid exchange.which goes on between the circulating plasma proteins and various body tissues. Ascites comes into consideration in such relation and the experimental method of vena cava constriction was used to study the body protein exchange due to ascites. As the experiments continued it was obvious that this method presented an admirable technique for the study of ascites and some of the related medical problems. Furthermore, the ascites was not complicated by the presence of extensive liver pathology and was due to venous stasis in the portal area. Ascites was produced in the dog by placing a constricting aluminum band on the inferior vena cava between the diaphragm and the right auricle.This method of vena cava constriction was used by one of us (G. H. W.) in 19(O-10 to produce ascites. The partial obstruction was caused by sutures put in the vena cava to produce narrowing of the lumen. This condition would last many weeks and then the sutures would cut through with relief of the obstruction and complete absorption of large amounts of ascitic fluid in a matter of hours. In these experiments there was no significant liver pathology remaining after relief of the obstruction. As these earlier experiments were incidental to a study of liver abnormalities (17) they were soon discontinued.The tables below show clearly that vena cava obstruction and the resultant ascites may produce a considerable drain on protein reserve stores and a hypoproteinemia. As the ascitic fluid is removed at frequent intervals, there results a continuing internal plasraapheresis. The term plasmapkeresis as used here means the removal of whole blood with subsequent replacement of the red cells suspended in a salt solution. As the procedure is used day by day one observes an hypoproteinemia which can be maintained for months at an even level. This technique has been used extensively to study the production of new plasma proteins as influenced by diet factors

SummaryAs a corollary to our previous studies with bovine prothrombin, we have initiated a study of human prothrombin complex. This product has been isolated in crystalline form as a barium glycoprotein interaction product. Product yields were reduced compared to bovine product due to the increased solubility of the barium glycoprotein interaction product. On occasion the crystalline complex exhibited good yields. The specific activity of the crystalline complex was 1851 Iowa u/mg. Further purification of human prothrombin complex was made by removal of barium and by chromatography on Sephadex G-100 gels. The final product evidenced multiple procoagulant activities (II, VII, IX and X). The monomeric molecular weight determined by sedimentation equilibrium in a solvent of 6 M guanidine-HCl and 0.5% mercaptoethanol was 70,191 ± 3,057 and was homogeneous with respect to molecular weight. This product was characterized in regard to physical constants and chemical composition. In general, the molecular properties of human prothrombin complex are very similar to the comparable bovine product. In some preparations a reversible proteolytic enzyme inhibitor (p-aminophenylarsonic acid) was employed in the ultrafiltration step of the purification scheme to inhibit protein degradation.

Racemic lysine labeled with C14 in the epsilon carbon position was fed to dogs. The distribution of C14 in blood and tissue fractions is recorded. In normal dogs sacrificed at 24 hours, approximately one-third of the C14 was found in the urine, one-third in expired air, and one-third in the body, mostly in protein, predomantly as lysine residues. The rate of C14 excretion as CO2, hour by hour, paralleled closely the amount of non-protein C14 in the blood plasma. The liver, kidney, pancreas, and spleen all have high values for C14 in 24 hour and 17 day experiments. The gastrointestinal tract is significantly high in the 24 hour experiments. Plasma protein from animals previously fed C14 containing lysine and thus in turn labeled, was transfused into other dogs and the rate of disappearance of albumin and globulin fractions from the circulation of the recipient dog followed. The results lead to the conclusion that as a whole, plasma proteins are utilized and replaced at a rate of at least 10 per cent per 24 hours. This minimum rate is substantially faster than turnover rates commonly accepted and emphasizes the rôle played by the plasma proteins in the protein economy of the body. The exact rate determination is made uncertain by the lack of knowledge of the magnitude of the amount of protein in solution in extracellular and lymph spaces and its rate of equilibrium with circulating plasma proteins. Evidence from these transfusion studies indicates that plasma globulin is metabolized at a significantly faster rate than plasma albumin. This is confirmed by the observation that following the feeding of labeled lysine to dogs, C14 is first incorporated in globulin in high concentration but that later it also disappears more rapidly from the globulin fraction. These data suggest that the period of bone marrow maturation of the red cell during which time its related hemoglobin is synthesized does not exceed 3 to 5 days.

It has already been found that a considerable number of enzymes occur in nuclei of rat liver cells which have been isolated at pH 6.0 by a procedure involving the use of the Waring blendor with very dilute citric acid for breaking the cells, followed by differential centrifugation (1-3). As a complementary type of work, studies have been started to determine the relative concentrations of some important substrates in the cell nucleus as compared to the corresponding concentrations in the whole cell or in the cytoplasm. Since many of the substrates of the cell are water-soluble it was necessary to avoid aqueous solutions in the preparation of the nuclei. For this purpose a modification of the method of Behrens (4), which employs only non-polar organic solvents, was developed. The free amino acid pattern of these nuclei was determined by paper partition chromatography, and their nucleic acid content estimated. The method of Behrens has already been used in a study of the distribution of vitamins between nucleus and cytoplasm (5). EXPERIMENTALA. Source of Material.--Wistar strain rats fed ad libitum on a "fox chow" diet were killed by decapitation. The livers were removed as quickly as possible, easily visible fibrous tissue was removed, and the livers were then immediately frozen by being placed in beakers cooled in an acetone-dry ice bath. The frozen livers were allowed to stand at -15 ° in a cold room for about an hour, and then were broken into as small pieces as possible. The resulting material was then immediately lyophilized continuously during an interval of 36 to 48 hours. When nearly all the water had been removed, the material was powdered with a mortar and pestle and lyophilization was continued for 5 to 6 hours. The final powder was immediately used in the cell fractionation procedure described below. B. Preparation of Cell Nuclei, Whole Cells, and the Cytoplasmic Fractions.--Fivepreparations of nuclei were isolated from liver powder lyophilized as above, three from rat liver and two from rabbit liver. Fifty gin. of the lyophilized powder were ground in a ball mill of 1 liter capacity at -15 ° for 24 hours in the presence of 200 ml. of 50-4)0 ° petroleum ether with about 160 irregular pebbles of 15 to 20 ram. average diameter. The petroleum ether facilitated grinding so that relatively small proportions of unbroken cells remained even though the grinding time was not prolonged. The 629on May 12, 2018 jgp.rupress.org Downloaded from

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