Ascitic Plasma Research Articles

Her2-targeted pegylated stealth liposomal doxorubicin (PLD) retains its specific targeting ability to Her2-expressing tumor cells after in vivo circulation and extravasation to mouse malignant ascites

13097 Background: Receptor-directed targeting of ligand-bearing liposomes to tumor cells may enhance therapeutic efficacy by intracellular delivery of a concentrated payload of liposomal drug. The goal of this study was to assess whether Her2-targeted PLD retains its binding ability to Her2-expressing target cells through circulation in the blood and extravasation to the ascitic fluid of mice with malignant ascites. Methods: PLD was grafted with a pegylated lipophilic conjugate of an anti-Her2 scFv antibody fragment (F5) at a ratio of 15 ligands per liposome. BALB/c mice were injected with J6456 lymphoma cells into the peritoneal cavity to generate malignant ascites. When abdominal swelling developed, Her2-targeted PLD and nontargeted PLD were injected into the mice i.v. at a dose of 15 mg/kg. The ascitic fluid was collected 48 hr later, ascitic tumor cells were removed, and the doxorubicin levels in the cell-free ascitic fluid and plasma were determined. Binding of the liposome-containing ascitic fluid was tested in vitro against Her2-expressing human tumor cell lines (N87, SKBR-3) and compared to the binding of shelf formulations (not passaged in vivo) of Her2-PLD and PLD, using as parameter the amount of cell-associated doxorubicin. Results: Plasma and ascitic fluid levels of Her2-PLD were only slightly below those of PLD indicating that the Her2 ligand did not cause any significant change in the clearance rate of PLD. Her2-PLD and PLD bound to an equal extent to J6456 cells in vivo. The in vitro binding of Her2-PLD from ascites to Her2- expressing cells was increased 10 to 20-fold above that of PLD from ascites, similarly to the 20-fold difference in binding between shelf Her2-PLD and PLD. Conclusions: Her2-targeted PLD demonstrates similar circulation time to that of nontargeted PLD. After in vivo passage, the targeted liposome retains most of its original binding capacity to Her2 expressing cells indicating that the ligand is stably maintained in association with the doxorubicin liposomal carrier. Targeting of PLD using this Her2 antibody fragment should provide an important means of selective drug delivery to tumors expressing the Her2 receptor. [Table: see text]

Interleukin 6 concentration in ascitic fluid of cirrhotic patients: relationship with previous episodes of spontaneous bacterial peritonitis.

We aimed to determine whether inter-leukin (IL)-6 concentrations in plasma and ascitic fluid from cirrhotic patients with previous episodes of spontaneous bacterial peritonitis (SBP) differred from those with no previous episodes of SBP. Cirrhotic patients with ascites were divided into two groups: G1, without previous episodes of SBP (n = 6), and G2, with previous episodes of SBP (n = 6). Patients with chronic heart failure (n = 5) with ascites, without hepatic diseases, were included as controls (G3). IL-6 concentration was determined in ascitic fluid and plasma by enzyme-linked immunosorbent assay (ELISA). A group of healthy subjects (n = 8) was used as a control for the blood IL-6 assay. IL-6 concentration in ascitic fluid was significantly higher in G1 (P < 0.05) than in G2 or G3. No significant difference in plasma IL-6 concentrations was observed between the groups or between each group and the healthy subjects. A significant positive linear correlation (P < 0.002) was observed between IL-6 concentration and total protein in ascitic fluid. A low concentration of IL-6 in ascitic fluid could predispose cirrhotic patients to SBP.

Bradykinin in the ascitic fluid of patients with liver cirrhosis

Bradykinin, a nonapeptide with vasodilatory and permeabilizing activity, is generated through the cleavage of high-Mr (‘high-molecular-weight’) kininogen by kallikrein, and its generation is facilitated by plasmin. In the ascitic fluid of patients with cirrhosis, there is massive cleavage of high-Mr kininogen and activation of fibrinolysis, but bradykinin has never been measured directly. In the ascitic fluid of 24 patients with cirrhosis, we measured bradykinin-(1-9)-nonapeptide levels by RIA after liquid-phase and subsequent HPLC extraction, and those of its catabolic product bradykininin-(1-5)-pentapeptide by ELISA after liquid-phase extraction. Cleaved high-Mr kininogen, activated factor XII and plasmin-antiplasmin complexes were measured in ascitic fluid and plasma. Plasma renin activity (PRA) was also determined. As a control, we also analysed plasma from 24 healthy subjects matched for sex and age with the patients. In the ascitic fluid from patients with cirrhosis, the median bradykinin-(1-9) concentration was 3.3fmol/ml (range 0.2-29.0fmol/ml), and the median bradykinin-(1-5) concentration was 210fmol/ml (range 58-7825 fmol/ml). The levels of bradykinin-(1-5) in ascitic fluid were higher in patients with refractory ascites [median 1091fmol/ml (range 58-7825fmol/ml)] than in patients with responsive ascites [134 fmol/ml (72-1084fmol/ml)] (P = 0.010). Ascitic fluid levels of bradykinin-(1-9) were not related to the severity of ascites. PRA was higher in patients with refractory ascites [23.0ngċh-1ċml-1 (7.9-80.0ngċh-1ċml-1)] than in patients with responsive ascites [6.9ngċh-1ċml-1 (0.9-29.4ngċh-1ċml-1)] (P = 0.002). In ascitic fluid, 48% (19-68%) of high-Mr kininogen was cleaved, and plasmin-antiplasmin complexes were more concentrated than in plasma (P = 0.0001). In conclusion, in the ascitic fluid of patients with cirrhosis, both bradykinin-(1-9) and bradykinin-(1-5) are present, with cleavage of high-Mr kininogen and activation of fibrinolysis. The highest levels of the long-lived metabolite bradykinin-(1-5) were found in the ascitic fluid of patients with refractory ascites and high PRA. Activation of the kinin system may therefore be involved in decompensating cirrhosis, but a cause-effect relationship remains to be established.

Bradykinin in the ascitic fluid of patients with liver cirrhosis

Bradykinin, a nonapeptide with vasodilatory and permeabilizing activity, is generated through the cleavage of high-M(r) ('high-molecular-weight') kininogen by kallikrein, and its generation is facilitated by plasmin. In the ascitic fluid of patients with cirrhosis, there is massive cleavage of high-M(r) kininogen and activation of fibrinolysis, but bradykinin has never been measured directly. In the ascitic fluid of 24 patients with cirrhosis, we measured bradykinin-(1-9)-nonapeptide levels by RIA after liquid-phase and subsequent HPLC extraction, and those of its catabolic product bradykininin-(1-5)-pentapeptide by ELISA after liquid-phase extraction. Cleaved high-M(r) kininogen, activated factor XII and plasmin-antiplasmin complexes were measured in ascitic fluid and plasma. Plasma renin activity (PRA) was also determined. As a control, we also analysed plasma from 24 healthy subjects matched for sex and age with the patients. In the ascitic fluid from patients with cirrhosis, the median bradykinin-(1-9) concentration was 3.3 fmol/ml (range 0.2-29.0 fmol/ml), and the median bradykinin-(1-5) concentration was 210 fmol/ml (range 58-7825 fmol/ml). The levels of bradykinin-(1-5) in ascitic fluid were higher in patients with refractory ascites [median 1091 fmol/ml (range 58-7825 fmol/ml)] than in patients with responsive ascites [134 fmol/ml (72-1084 fmol/ml)] (P=0.010). Ascitic fluid levels of bradykinin-(1-9) were not related to the severity of ascites. PRA was higher in patients with refractory ascites [23.0 ng x h(-1) x ml(-1) (7.9-80.0 ng.h(-1).ml(-1))] than in patients with responsive ascites [6.9 ng x h(-1) x ml(-1) (0.9-29.4 ng x h(-1) x ml(-1))] (P=0.002). In ascitic fluid, 48% (19-68%) of high-M(r) kininogen was cleaved, and plasmin-antiplasmin complexes were more concentrated than in plasma (P=0.0001). In conclusion, in the ascitic fluid of patients with cirrhosis, both bradykinin-(1-9) and bradykinin-(1-5) are present, with cleavage of high-M(r) kininogen and activation of fibrinolysis. The highest levels of the long-lived metabolite bradykinin-(1-5) were found in the ascitic fluid of patients with refractory ascites and high PRA. Activation of the kinin system may therefore be involved in decompensating cirrhosis, but a cause-effect relationship remains to be established.

Correlation of high levels of hyaluronan and cytokines (IL-1beta, IL-6, and TGF-beta) in ascitic fluid of cirrhotic patients.

Our objective was to investigate the relationship between endotoxin and hyaluronan synthesis and release in serum and ascitic fluid from cirrhotic patients. We studied hyaluronan, endotoxin, albumin, and creatinine levels in ascitic fluid and plasma and cytokine levels (IL-1beta, IL-6, TGF-beta) in ascitic fluid. TGF-beta, IL-6, and IL-1beta correlation analyses indicated a strong dependence of the production of these cytokines on endotoxin levels. Correlation analyses for TGF-beta and IL-6 indicated a strong dependence of the production of hyaluronan on cytokine levels and, to a lesser extent, on IL-1beta levels. Hyaluronan analysis indicated that a certain glycosaminoglycan level is required in ascites before its appearance in plasma. Our results disclosed elevated plasma hyaluronan concentrations. The simultaneous increased hyaluronan levels in ascitic fluid do not seem to be derived from the systemic circulation. In conclusion, the high hyaluronan-ascites/hyaluronan-plasma ratio suggests an intrinsic hyaluronan production from peritoneal cells induced by endotoxins.

Hyperfibrinolysis but not hypercoagulability after a new ascites-apheresis procedure in liver cirrhosis patients with refractory ascites

Summary Given the frequent occurrence of disseminated intravascular coagulation after reinfusion of ascitic fluid in cirrhotic patients, a study involving a new ascites-apheresis apparatus was undertaken in 11 patients with decompensated liver cirrhosis and massive refractory ascites, with the aim of investigating activation of coagulation and fibrinolysis after the procedure. A large series of assays were performed in ascitic fluid and plasma before and after the apheresis sessions, and the measured values were compared with those expected and calculated on the basis of fluid and protein changes bound to the filtration and reinfusion procedures. In ascitic fluid, the measured values reflecting activation of blood clotting as prothrombin fragment 1+2 (F1+2) and thrombin-antithrombin complexes (TAT) although increased after filtration were much lower than expected. In plasma after reinfusion, F1+2 and TAT were also significantly less increased than calculated, presumably also because of their short half-life, while values reflecting activation of fibrinolysis as total, fibrin and fibrinogen degradation products were significantly increased above those calculated. Thus, the filtration-reinfusion system employed in this study appeared to efficiently remove most of the procoagulant materials from the ascitic fluid and did not significantly activate blood clotting in plasma. The observed activation of fibrinolysis was of limited extent as no relevant changes were observed in the basic clotting tests (PT and aPTT) and fibrinogen.

Decreased expression and function of Vbeta6+ and Vbeta14+ T cells is associated with decreased Th1 cytokine production in mice with plasma cell tumors.

We have found that polyclonally stimulated T cells from mice bearing ascitic plasma cell tumors demonstrate specific decreases in Th1 cytokine production. In this study we investigated whether loss of Th1 responses in the plasma cell tumor system was associated with alterations in the Vbeta T cell receptor repertoire. We examined the cell surface expression of specific Vbeta expressing splenic CD4+ or CD8+ T cells from normal and tumor bearing mice using direct three-color flowcytometry. In order to determine the Th phenotype of Vbeta expressing T cells, we enriched for Vbeta6, Vbeta14 or Vbeta8.1,8.2 cells, polyclonally stimulated them and measured the levels of the sytokines interleukin-4 (IL-4), IL-2 and interferon-gamma (IFN-gamma). We find there is a statistically significant decrease in the frequency of Vbeta6+ and Vbeta14+ CD8+ T cells in mice bearing a plasma cell tumor (B53) as compared to normal (p<0.05). Stimulated Vbeta6+ and Vbeta14+ T cells exhibit an exclusively Th1 phenotype. Stimulated Vbeta6+ and Vbeta14+ T cells from B53 mice are deficient in production of the Th1 cytokines. In contrast, stimulated Vbeta8.1,8.2+ T cells, which are not altered in B53 mice, reveal a Th2 phenotype. The significance of this study is our demonstration that decreased expression and function of Vbeta6+ and Vbeta14+ T cells may be, at least in part, responsible for the decrease in the production of IL-2 and/or IFN-gamma observed in hosts with tumors.

Composition of ascitic fluid in liver cirrhosis: bile acid and lipid content.

The concentrations of lipids, bile acids and proteins were evaluated in the ascitic fluid and plasma of 23 cirrhotics. Ascitic fluid density was highly correlated with its protein content, represented mostly by low molecular weight proteins. The ratio of plasma to ascitic fluid concentrations of nine examined proteins increased with molecular weight, indicating a selective ultrafiltration of the peritoneal transudate. Low density lipoproteins in ascitic fluid had modified electrophoretic mobility. Total cholesterol had a higher plasma to ascitic fluid ratio than high density lipoprotein cholesterol, whereas bile acids and proteins had similar plasma to ascitic fluid ratios. Indeed, bile acids strongly bind to circulating albumin: consequently ascitic fluid contains more cholic acid (less hydrophobic) than other bile acids. Analysis of both plasma and ascitic fluid composition in cirrhotics provides useful information on processes regulating passage of blood components into the peritoneal cavity.

Coagulation disturbances following ascites recirculation

To investigate the effects of extra-corporeal ascites recirculation on coagulation, several coagulation variables were measured in ascitic return fluid and plasma before, during and after this procedure in 16 patients with diuretic resistant ascites. Small but significant reductions in plasma fibrinogen levels and platelet counts and increases in plasma X-oligomer were observed during and after the procedure compared with before. These findings are consistent with the view that ascites recirculation induces disseminated intravascular coagulation although this would appear to be only mild and of no clinical significance in the majority of patients. Although increased levels of activated factor VII were observed in ascitic fluid indicating activation of the extrinsic pathway of coagulation, a significant increase in plasma activated factor VII during the procedure was not demonstrated. Increased fibrinolytic activity was observed in ascitic fluid due to the presence of tissue plasminogen activator. Increased X-oligomer levels were observed in ascitic fluid indicating that lysis of cross-linked fibrin is also an active process within ascites.

Insulin and C-peptide in ascitic fluid and plasma and their relative responses to glucagon in patients with cirrhosis.

Insulin (IRI) and C-peptide dynamics were studied after iv glucagon in 5 nondiabetic patients with ascites due to cirrhosis of the liver. Plasma and ascitic fluid samples for glucose, IRI and C-peptide determinations were obtained before and 6, 10, 15, 20 and 30 min after glucagon injection. Ascitic fluid volumes, estimated by dilution of ip injected PAH, were 6.2 to 20.5 L. The mean fasting plasma glucose [88 +/- 6.7 mg/dl (SE)] and C-peptide (1.40 +/- 0.42 ng/ml) levels were normal; mean plasma insulin was increased (17.4 +/- 3.0 microU/ml). After glucagon injection, there was a subnormal rise in plasma glucose (PG) compared to 5 mild diabetic patients without liver disease (8.4 +/- 3.5 vs 76 +/- 7.4 mg/dl). The plasma C-peptide rise was less than that of plasma IRI (54% vs 192%). The mean basal ascitic fluid concentration of glucose was 86 +/- 9.4 mg/dl, IRI 13.2 +/- 2.9 microU/ml and C-peptide 3.09 +/- 0.49 ng/ml. Total calculated basal ascitic fluid contents of glucose was 5.2-23.3 g, IRI 47, 120-290,000 microU and C-peptide 15,750-66,420 ng. These were 3-10 times the quantity of these substances circulating in the plasma volume. After glucagon injection, there was no significant increase in ascitic fluid glucose or IRI, but there was a 43% increase in C-peptide concentration at 10 min. In ascitic fluid, the molar concentration of IRI was lower and C-peptide higher than plasma, resulting in a C-peptide: IRI molar ratio of 11.31, markedly higher than the published normal plasma ratio of 4.63.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of dietary lipids on plasma lipoproteins and fluidity of lymphoid cell membranes in normal and leukemic mice

Mice of the GR/A strain were fed four different isocaloric semipurified diets, enriched in either (1) saturated fatty acids (palm oil), or (2) polyunsaturated fatty acids (corn oil), or (3) palm oil plus cholesterol, or (4) a fat-poor diet containing only a minimal amount of essential fatty acids. We have studied the effects of these dietary lipids on the density profile and composition of the plasma lipoproteins and on the lipid composition and fluidity of (purified) lymphoid cell membranes in healthy mice and in mice bearing a transplanted lymphoid leukemia (GRSL). Tumor development in these mice occurred in the spleen and in ascites. While the fatty acid composition of the VLDL-triacylglycerols still strongly resembled the dietary lipids, the effects of the diets decreased in the order VLDL-triacylglycerols > HDL-phospholipids > plasma membrane phospholipids. Diet-induced differences in the latter fraction were virtually confined to the content of oleic acid and linoleic acid, and they were too small to affect the membrane fluidity, as measured by fluorescence polarization using the probe 1,6-diphenyl-1,3,5-hexatrine. Healthy mice were almost irresponsive to dietary cholesterol, but in the tumor bearers, where lipoprotein metabolism has been shown to be disturbed, the cholesterol diet caused a substantial increase in the low- and very-low density regions of both blood and ascites plasma lipoproteins. The cholesterol-rich diet also increased the cholesterol/phospholipid molar ratio and lipid structural order (decreased fluidity) in GRSL ascites cell membranes, but not in the splenic GRSL cell membranes. We conclude that the composition of plasma lipoproteins and cell membrane lipids in GR/A mice is subject to exquisite homeostatic control. However, in these low-responders to dietary lipids the development of an ascites tumor may lead to increased responsiveness to dietary cholesterol. The elevated level of membrane cholesterol thus obtained in GRSL ascites cells did not affect the expression of various cell surface antigens or tumor cell growth.

Inhibition of the cathepsin B like proteinase by a low molecular weight cysteine-proteinase inhibitor from ascitic fluid and plasma α2 macroglobulin

The cathepsin B like proteinase present in ascitic fluid of a patient with neoplasia has been purified and characterized after pepsin activation. From this fluid we have prepared the low molecular weight (LMW) cysteine-proteinase inhibitors. Three major inhibitor forms were found with isoelectric points of 7.4, 5.4, and 5.1, respectively. The interaction of the enzyme with the former inhibitor was studied because this inhibitor was the most abundant. The Ki value was found to be 4.3 X 10(-8) M. Two molecules of this proteinase were bound by one molecule of plasma alpha 2 macroglobulin (alpha 2M). The LMW inhibitor was able to bind to the enzyme entrapped in alpha 2M and reduced its endopeptidase activity on benzyloxycarbonyl-L-phenylalanyl-L-arginine-4-methyl-7-coumarylamide. These results may have a physiological significance in the regulation of the enzyme which, among other extracellular hydrolases, probably plays an important role in tumor invasion.

エールリッヒ腹水がん移植マウス腹水中のパラグアニジノフェニルアラニンに特異的な新しいセリンプロテアーゼ

A new enzyme which hydrolyzes anilide substrates of p-guanidino-L-phenylalanine in preference to those of arginine was found in the ascitic plasma from Ehrlich ascites tumor-bearing mice. The activity of this enzyme on Nα-benzyloxycarbonyl-p-guanidino-L-phenylalanine p-nitroanilide was strongly inhibited by diisopropyl fluorophosphate and phenylmethanesulfonyl fluoride but not by sulfhydryl-reactive reagents and metal chelating agents. Peptide substrates containing p-guanidino-L-phenylalanine were hydrolyzed by this enzyme much faster than those containing arginine. These results suggest that this enzyme is a different type of serine protease from trypsin and thrombin. This enzyme was also found in the human gastric and colon cancer cells and their surrounding ascitic plasmas.

Alterations in biosynthesis and homeostatis of cholesterol and in lipoprotein patterns in mice bearing a transplanted lymphoid tumor

The membrane fluidity of murine lymphoid GRSL tumor cells has been shown to depend on their site of growth in the host. Tumor cells located in ascites, in contrast to those in the enlarged spleen, show a very high plasma membrane fluidity, mainly due to a decreased level of cellular (membrane) cholesterol. Yet, the rate of cholesterol biosynthesis in the tumor cells as estimated by the activity of HMG-CoA reductase and the incorporation of [ 14C]acetate into cholesterol was extremely high when compared to various lymphoid cells in normal control mice. Also the rate of biosynthesis and the cholesterol content in liver and spleen of tumor-bearing mice were substantially higher than in the organs of control mice. Blood plasma cholesterol, however, was decreased in tumor-bearing mice as a result of altered lipoprotein patterns. Outgrowth of the tumor was accompanied by a strong reduction in plasma high-density lipoproteins. Low-density lipoproteins became transiently increased, but eventually all lipoproteins, and consequently the plasma cholesterol content decreased to very low levels, especially so in the ascites plasma. The low transfer of [ 14C]cholesteryl ester-labeled lipoproteins between blood and ascites plasma after either intravenous or intraperitoneal injection suggested a hampered flow between the two compartments. Also apparent differences in cholesteryl ester fatty acid composition between lipoproteins of the blood and ascites plasma indicated the lack of a rapid equilibration between the two compartments. The results suggest that the limited availability of lipoproteins as an additional source of cholesterol to the rapidly growing ascites cells promotes their high membrane fluidity.

A new serine protease which preferentially recognizes p-guanidino-L-phenylalanyl residue in ascitic plasma from Ehrlich ascites tumor-bearing mice

A new enzyme which hydrolyzes anilide substrates of p-guanidino-L-phenyl-alanine in preference to those of arginine was found in the ascitic plasma from Ehrlich ascites tumor-bearing mice. The activity of this enzyme on N α-benzyloxycarbonyl-p-guanidino-L-phenylalanine p-nitroanilide was strongly inhibited by diisopropyl fluorophosphate and phenylmethanesulfonyl fluoride but not by sulfhydryl-reactive reagents and metal chelating agents. Peptide substrates containing p-guanidino-L-phenylalanine were hydrolyzed by this enzyme much faster than those containing arginine. These results suggest that this enzyme is a different type of serine protease from trypsin and thrombin. This enzyme was also found in the human gastric and colon cancer cells and their surrounding ascitic plasmas.

Evidence for an Enzyme which Cleaves the Guanidinobenzoate Moiety from Active‐Site Titrants Specifically Designed to Inhibit and Quantify Trypsin

The serum and ascitic plasma surrounding Ehrlich ascites tumour cells grown in mice contains an enzyme which cleaves 4‐nitropheny‐p‐guanidinobenzoate and 4‐methylumbelliferyl‐p‐guanidinobenzoate. We refer to this enzyme as guanidinobenzoatase. Either of the substrates acts as a competitive substrate for the enzyme when it is assayed with the other substrate. Arginyl‐containing peptide substrates of typsin and thrombin also act as competitive inhibitors of the enzyme but are not cleaved by it. The properties of this enzyme are remarkable in the continuous production of cleavage products from the two substrates, which are both active‐site titrants for trypsin‐like enzymes, rather than stoichiometric cleavage and inhibition of one molecule of enzyme by one molecule of active‐site titrant. The enzyme contains serine in its active centre, being inhibited by both phenylmethylsulphonyl fluoride and diisopropyl‐fluorophosphate.

The role of respiration in tumor cell transition from the noncycling to the cycling state.

Resting Yoshida AH130 hepatoma cells, harvested at the plateau of tumor development in vivo, were recruited into the cycling state following transfer to an in vitro system whereby these cells were incubated in the autologous ascites plasma diluted with buffered saline and enriched with glucose. In this system, cell recruitment into the phase of DNA synthesis (S phase) strictly depends on the activity of the respiratory chain and is abolished by anaerobiosis as well as by antimycin A, although the intracellular levels of ATP and the rate of protein synthesis are practically unaffected by these treatments. Furthermore, 2,4-dinitrophenol, at concentrations which uncouple the respiratory phosphorylation and hence enhance both glycolysis and oxygen consumption, does not hinder cell promotion into S phase. Thus, the absolute respiration dependence of cycling resumption by resting ascites cells does not seem to rely on respiratory ATP supply, but rather is linked to the electron flow through the respiratory chain.

Low Density Lipoproteins from Human Ascites Plasma&lt;subtitle&gt;Characterization and Degradation by Sodium Deoxycholate&lt;/subtitle&gt;

The low density lipoproteins (LDL) in human ascites plasma were separated into two sublcasses by ultracentrifugation. The chemical compositions of the two LDLs were determined. On a weight percentage basis, LDL1 contained cholesterol ester, 20.8%; cholesterol, 19.3%; triacylglycerol, 19.7%; phospholipid, 22.1%; and protein, 10.8%. LDL2 contained cholesterol ester, 21.6%; cholesterol, 14.5%; triacylglycerol, 20.1%; phospholipid, 20.6%; and protein, 18.0%. The relative content of triacylglycerol and the ratio of unesterified cholesterol to esterified cholesterol in both LDLs were very high compared to corresponding human serum LDLs. Following the treatment of these LDLs with 575 mg of sodium deoxycholate per 10 mg of lipoprotein protein, a triacylglycerol-protein rich fraction was obtained by gel filtration in the presence of a micellar concentration of the detergent.

Vanadate inhibition of sarcoplasmic reticulum Ca 2+-ATPase and other ATPases

Vanadate is a potent inhibitor of the Ca 2+-ATPase activity of sarcoplasmic reticulum in the presence of A-23187. The purified enzyme is sensitive to vanadate even in the absence of the ionophore. Ca 2+ and norepinephrine protect the enzyme against inhibition of vanadate. The nonspecificity of vanadate is emphasized by the finding of inhibition of several other ATPases including the Ca 2+Mg 2+-ATPases of the ascites and human red cell plasma membranes, Mg 2+-ATPase of the ascites plasma membrane, and the K +-ATPases of E. coli and hog gastric mucosal cell membranes. The ascites plasma membrane Ca 2+-ATPase (an ecto ATPase) and mitochondrial ATPase are not inhibited by vanadate.

Lipid composition of yoshida ascites hepatoma and of livers and blood plasma from host and normal rats

The lipid composition of Yoshida ascites hepatoma cells was analyzed together with that of ascitic plasma and of livers and blood plasma from host and normal rats. In comparison to normal livers, host livers showed no significant differences in the content of the various lipid classes, but contained a higher percentage of palmitic acid and a lower proportion of arachidonic acid in the major phospholipid classes. In addition, tumor growth induced a marked hypertriglyceridemia in host animals; changes in the concentration of other plasma lipid classes were not statistically significant. The ascitic plasma contained small amounts of lipids mainly constituted by cholesteryl esters and phospholipids. Yoshida hepatoma cells contained less phospholipids in comparison to both host and normal liver, while the increased level of triglycerides and the decrease of free fatty acids were not statistically significant. Hepatoma cells showed appreciable amounts of ether-linked lipids associated in part to neutral lipids (as glyceryl ether diesters) and, in part, to ethanolamine and choline phosphoglycerides. The alkyl groups in GEDE as well as in ethanolamine and choline phosphoglycerides were mainly constituted by C16:0 and C18:0 followed by C18:1. The alk-1-enyl groups in ethanolamine and choline phosphoglycerides were C16:0 and C18:0 with only a minor proportion of C18:1. In comparison to both host and normal liver, Yoshida hepatoma cells showed significant changes in the fatty acid composition of neutral lipids and phospholipids. Some of the major changes consisted of an increase of monoenoic acids associated with a decrease of arachidonic and docosahexaenoic acids in phosphatidylethanolamine, phosphatidylcholine, and phosphatidylinositol.