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Biomedical subjects

A Cantafora

Publications and source records attributed to A Cantafora.

At least 19 recordsLinked to original sources
PubMed1992

Improved determination of individual molecular species of phosphatidylcholine in biological samples by high-performance liquid chromatography with internal standards.

Phosphatidylcholine isolated from samples of bile, liver and plasma was converted into 1,2-diradylglycerobenzoate molecular species by hydrolysis with phospholipase C and reaction with benzoic anhydride. Up to seventeen molecular species were separated and determined by reversed-phase high-performance liquid chromatography with detection at 230 nm. The major improvement introduced here was the use of distearoylphosphatidylcholine as the internal standard, which corrected the results for incomplete hydrolysis and benzoylation. Other improvements concerned the clean-up of benzoyl derivatives and the chromatographic separation. The analytical results obtained were validated by comparison with the results of either lipid phosphorus or gas chromatographic determinations.

Bile↗
PubMed1992

Changes in erythrocyte membrane lipid composition affect the transient decrease in membrane order which accompanies insulin receptor down-regulation.

We have recently demonstrated, using electron paramagnetic resonance (EPR) spectroscopy, that insulin receptor internalization in response to insulin incubation (down-regulation) in human erythrocytes is accompanied by a transient decrease in membrane order, as measured by the 2T' parallel order parameter. Since membrane lipids play such an important role in receptor internalization, we investigated the possible effects that an alteration of the normally-occurring lipid profile might have on down-regulation and the concomitant transient decrease in membrane order. Consequently, human erythrocytes enriched with cholesterol and erythrocytes from cirrhotic patients were examined, because both of these groups of cells have a higher cholesterol/phospholipid molar ratio (CH/PL) than controls. The 5-nitroxystearate spin label, which inserts into the lipid bilayer of cell membranes, was used to monitor changes in 2T' parallel for a 3-h period at 37 degrees C. We report here that both cholesterol-enriched and cirrhotic erythrocytes do not down-regulate, as demonstrated by binding assays, and that they do not show the typical transient decrease in membrane order observed in controls. The results seem to indicate that a more ordered membrane inhibits internalization of the insulin receptor in erythrocytes, and that an increase in membrane disorder is necessary for insulin receptor down-regulation.

Cholesterol↗
PubMed1992

Hepatic uptake and processing of cholesterol and cholesteryl ester from chylomicron remnants: an in vivo study in the rat.

The metabolic fate of cholesterol in chylomicron remnants was studied after intravenous infusion into biliary drained rats. The remnants were radiolabelled with [3H]cholesterol in vivo, so that radioactivity was incorporated into both the unesterified (27% of label) and esterified (73% of label) cholesterol fractions, or with 14C-labelled unesterified cholesterol after exchange in vitro. Blood and bile samples were collected at intervals for 180 min, after which the animals were killed for analysis. The total amount of radioactivity found in the liver (46%) and bile (4.5%) after infusion of [3H] remnants was higher than that found when the label was 14C (33 and 2.7%, respectively). Radioactivity from 14C-labelled unesterified cholesterol was cleared more rapidly from the blood, but the distribution of the label between the lipoprotein fractions VLDL + LDL, HDL2 and HDL3 at the end of the experiment was similar to that found when total [3H]cholesterol was used. In experiments with both types of label, approximately 94% of the total radioactivity secreted into bile was associated with the bile acid, with only about 6% in biliary unesterified cholesterol, and these proportions did not change during 180 min. When the chylomicron remnants were labelled with total [3H]cholesterol the specific radioactivity of the bile acid, taurochenodeoxycholic acid, in the bile was approximately twice that observed when the label was unesterified [14C]cholesterol. The specific radioactivity of unesterified biliary cholesterol was very low in the latter case, but higher and more comparable to that of taurochenodeoxycholic acid in the former. Thus, the metabolic fate of chylomicron remnant cholesterol differs, depending on whether it is in the esterified or unesterified form, suggesting that hepatic cholesterol originating from the two fractions may mix to a different extent with the various intracellular pools. In addition, the experiments with 14C indicate that the behaviour of chylomicron remnant unesterified cholesterol resembles that exhibited by cholesterol in HDL more than that carried in VLDL or LDL.

Animals↗
PubMed1992

Effect of HDL1 infusion on biliary secretion in perfused rat liver.

The effects of HDL1 lipoprotein infusion on biliary lipid secretion were studied in the in vitro model of rat perfused liver. A strong increase in bile flow was observed during and after lipoprotein infusion. This caused a significant rise in cholesterol, phospholipid and bile salt secretions. However, only the percentage of cholesterol increased with respect to the other bile lipids. The changes observed in the cholesterol/phospholipid molar ratio values of liver membrane subfractions (i.e., liver plasma membrane, mitochondria plus lysosomes and microsomes) isolated from the perfused rat liver after HDL1 administration were not significant.

Animals↗
PubMed1992

Molecular composition of biliary phosphatidylcholines, as related to cholesterol saturation, transport and nucleation in human gallbladder bile.

It has been suggested that qualitative changes in bile phosphatidylcholine (lecithin) play a role in the pathogenesis of cholesterol gallstones. We investigated the possible relationship between the molecular composition and hydrophobicity of biliary lecithins and bile cholesterol saturation, nucleation time and the mode of cholesterol transport in human gallbladder bile. Nineteen patients (12 with and seven without gallstones) undergoing abdominal surgery were studied. Bile cholesterol saturation ranged from 77% to 186% (median: 123%) and nucleation time from 1 to 24 days (median: 3 days). Biliary lipid carriers (vesicles and mixed micelles) were separated using Superose-6 gel chromatography and their lipid content was quantitated. Biliary lecithin composition was analyzed by HPLC. Fourteen individual molecular species were detected in the bile: none were related to cholesterol saturation or nucleation time. An arbitrary cumulative index of lecithin hydrophobicity was computed for each bile sample, based on the HPLC capacity factor of the individual species and their percent mole fraction: this index ranged from 47.0 and 58.5 (median: 49) and was unrelated to cholesterol saturation and nucleation time. The biliary concentration of sn-1 palmitoyl:sn-2 arachidonoyl lecithin was significantly correlated (p less than 0.01) with the fraction of cholesterol carried by mixed micelles. This finding suggests that arachidonoyl lecithin may play a modulatory role in the partitioning of cholesterol among biliary carriers. We conclude that major abnormalities in the composition of biliary lecithins are unlikely to play a causative role in the pathogenesis of cholesterol gallstone, although the role of arachidonoyl species requires further investigation.

Adult↗
PubMed1992

Liver metabolism of cholesterol taken up from lipoproteins in Wistar rats. An in vivo comparison between rat and human lipoproteins.

1. This study compares liver uptake, biliary secretion and blood decay of VLDL, LDL, HDL2 and HDL3 lipoprotein fractions isolated from both rat and human plasma and labelled with [14C]-cholesterol following the i.v. administration to the bile-fistulated rat model. 2. The present results demonstrate that the use of heterologous lipoproteins in bile-fistulated rat can be helpful in administering in a small volume large amounts of free and esterified cholesterol and in evaluating specific aspects of lipoprotein cholesterol metabolism by liver.

Animals↗
PubMed1992

Effect of intravenous polyunsaturated phosphatidylcholine infusion on insulin receptor processing and lipid composition of erythrocytes in patients with liver cirrhosis.

The aim of this study was to determine whether insulin receptor processing capabilities of human erythrocytes could be improved by changing the cell membrane lipid composition using an intravenous infusion of polyunsaturated phosphatidylcholine. Thirteen cirrhotics were submitted to the i.v. infusion of phosphatidylcholine (2 g day-1 for 3 days). Both erythrocyte lipid composition and insulin receptor processing ability were examined at the beginning of the study and at 0, 3 and 11 days after the end of the treatment. This treatment decreased the erythrocyte cholesterol to phospholipid molar ratio and increased the proportion of polyunsaturated fatty acids (mainly linoleic acid) immediately after the end of the treatment. The proportion of arachidonic acid increased immediately in the phosphatidylserine class and, a few days later, also in phosphatidylethanolamine. The phospholipid class distribution did not show any relevant modification in the course of the study. Surface insulin receptors, which generally were up-regulated in the untreated subject (-7.1 +/- 20.4%), showed an improvement in down regulation capabilities that appeared to be well correlated with the changes in lipid composition of cell membranes induced by i.v. infusion of polyunsaturated phosphatidylcholine. The confirmation of these findings also in target cells for insulin may open new perspectives in the treatment of diabetes mellitus.

Adult↗
PubMed1992

Altered insulin receptor processing and membrane lipid composition in erythrocytes of cirrhotic patients.

Insulin binding and insulin receptor down-regulation characteristics were evaluated, as well as cholesterol and phospholipid levels in erythrocytes from 22 patients with liver cirrhosis. These parameters were correlated with clinical characteristics and with scores related to severity of the liver injury. Nine healthy subjects were studied as a control group. It was observed that insulin binding was generally greater in patients than in controls because of an increase in surface insulin receptor numbers, rather than from a change in receptor affinities. In cirrhotic patients, the erythrocyte insulin receptors did not undergo the normal down-regulation process but, in most cases, increased in response to insulin incubation. The alterations in insulin processing characteristics were more frequent in patients with alcoholic cirrhosis and more severe liver impairment and correlated with the changes in the lipid composition of erythrocyte membranes. In particular, an increase in the cholesterol to phospholipid molar ratio and a decrease in polyunsaturated fatty acid content of phospholipids in erythrocyte membranes of cirrhotic patients was associated with impairments in insulin receptor processing. Similar changes in insulin receptor processing were observed when the molar ratio of cholesterol to phospholipid in normal erythrocytes was modified in vitro by incubation with cholesterol-rich liposomes.

Adult↗
PubMed1991

The cesium-induced delay in myoblast membrane fusion is accompanied by changes in cellular subfraction lipid composition.

We have recently demonstrated that the delay in myoblast membrane fusion induced by cesium is accompanied by changes in isolated membrane lipids (Santini, M.T., Indovina, P.L., Cantafora, A. and Blotta, I. (1990) Biochim. Biophys. Acta 1023, 298-304). In the present study, we have investigated changes in the lipid profile of total cell homogenates and microsomal membrane fractions during myoblast membrane fusion as well as the effects that addition of cesium may have on these lipid variations in order to try to understand the production and translocation of lipids during this myogenic process. The data presented here indicate that the lipid composition of cell homogenates and microsomes varies in a different manner from isolated plasma membranes during myogenic fusion. In addition, cesium affects, in a different manner, the normally-occurring lipid production and distribution which takes place in each subcellular fraction.

Animals↗
PubMed1991

Gallstones in cystic fibrosis: a critical reappraisal.

Radiolucent gallstones are common in young adults with cystic fibrosis. In the mid-1970s, it was suggested that gallstones are made of cholesterol, but this hypothesis has never been tested. Several recent studies have shown that the detection of cholesterol monohydrate crystals in bile has high sensitivity and specificity for the diagnosis of cholesterol gallstones. We therefore used this approach to study 17 young adults with cystic fibrosis, 10 of whom had radiolucent gallstones. The two groups of patients were comparable in age and gender (all patients but one were male). Duodenal bile was obtained after gallbladder contraction with intravenous cerulein; it was used for lipid and protein chemistry studies and for polarizing microscopy. The latter was performed both in whole bile and in the postultracentrifugation (100,000 g) sediment. Bile cholesterol saturation did not significantly differ between patients with (1.21 +/- 0.28) or without gallstones (0.99 +/- 0.54). Slight cholesterol supersaturation was found in 7 of 10 gallstone and three of seven nongallstone patients. At no time were cholesterol crystals detected in either the group, even after bile ultracentrifugation. Two more cystic fibrosis patients with gallstones died of severe bronchopneumopathy, and small pigment gallstones were obtained at autopsy. At stone analysis, cholesterol content was 44% and 28% of dry weight, respectively. Infrared spectroscopy of stone powder was compatible with the presence of calcium bilirubinate and proteins as major components. We conclude that radiolucent gallstones of cystic fibrosis are not of the conventional cholesterol type.

Adolescent↗
PubMed1991

Impaired hepatic handling and processing of lysophosphatidylcholine in rats with liver cirrhosis.

Lysophosphatidylcholine is a major metabolic product in the plasma and cellular turnover of phospholipids, with well-known membrane-toxic and proinflammatory properties. Because the liver plays a key role in plasma lysophosphatidylcholine removal and biotransformation and because virtually nothing is known of these processes in a diseased organ, the hepatobiliary metabolism of lysophosphatidylcholine was investigated in rats with carbon tetrachloride-induced liver cirrhosis. Twelve adult male Wistar rats with histologically confirmed cirrhosis and 8 control animals were fitted with jugular and biliary catheters and allowed to recover. The animals were kept under constant IV infusion of taurocholate (1 mumol/min). Two microcuries of sn-1[14C]palmitoyl-lysophosphatidylcholine was administered as a single bolus. The fate of the injected radioactivity, including removal from plasma, uptake, and subcellular location in the liver and molecular and aggregative forms, was studied by combined chromatographic and radiochemical methods. Major findings were (a) that lysophosphatidylcholine has a prolonged permanence in plasma of cirrhotic rats, due both to decreased hepatic clearance and to depressed conversion into phosphatidylcholine; (b) that the rate of lysophosphatidylcholine acylation is much slower in the cirrhotic than in the normal liver, both at the microsomal and at the cytosolic level; (c) that cytosolic lysophosphatidylcholine in the cirrhotic liver, but not in the normal liver, is predominantly non-protein bound; (d) that the strict molecular selectivity of lysophosphatidylcholine acylation observed in controls is partially lost in cirrhosis; and (e) that a consistent fraction of lysophosphatidylcholine is converted into triacylglycerols in cirrhotics but not in controls. These findings show a profound derangment of lysophosphatidylcholine handling and processing in the cirrhotic liver, which is of potential pathogenetic significance.

1-Acylglycerophosphocholine O-Acyltransferase↗
PubMed1991

Dietary taurine content changes liver lipids in cats.

Adult female cats were fed a completely defined purified diet (taurine-free) alone or containing 0.05% taurine (the normal dietary requirement) or 1% taurine (20-fold the normal dietary requirement) for greater than 2 y. The relative composition of conjugated biliary bile acids was not different among the three groups and virtually all bile acids were conjugated with taurine. The taurine concentration in liver varied dramatically with the amount of taurine in the diet. Total liver lipid content decreased with increasing dietary taurine. Individual lipid components also varied, especially free fatty acids (which decreased with increasing dietary taurine) and triglycerides (which increased with increasing dietary taurine), indicating that taurine has a metabolic effect on lipid metabolism. Taurine deficiency also caused significant changes in the fatty acid distribution of sphingomyelin. In particular, a decrease of lignoceric acid and an increase of nervonic acid were observed. The present data suggest that hepatocellular levels of taurine can modulate the mobilization of liver lipid stores and the utilization by the liver of circulating free fatty acids. These effects are probably mediated by factors affecting membrane fluidity, such as the ratio of cholesterol to phospholipids, the degree of unsaturation of phospholipids and the changes in sphingomyelin fatty acid composition.

Administration, Oral↗
PubMed1991

Improvement of estradiol 17 beta-D-glucuronide cholestasis by intravenous administration of dimethylethanolamine in the rat.

The intravenous administration of dimethylethanolamine in the rat promotes a selective enrichment of liver membranes with polyunsaturated phosphatidylcholines. The effect of dimethylethanolamine pretreatment on cholestasis induced by estradiol 17 beta-D-glucuronide, a potent cholestatic agent, was assessed in this study. Dimethylethanolamine, dissolved in sodium-taurocholate was infused intravenously (0.3 mg/kg/min) for 15 hr. One group of control rats (estradiol 17 beta-D-glucuronide controls) received the bile salt only. An estradiol 17 beta-D-glucuronide bolus was then injected intravenously (10.4 mg/kg) into dimethylethanolamine-pretreated and estradiol 17 beta-D-control rats, and its effect on bile flow and biliary lipid secretion was compared for 3 hr. The estradiol 17 beta-D-glucuronide inhibitory effect on bile flow and biliary lipid secretion was significantly antagonized by dimethylethanolamine pretreatment. The maximum inhibition of bile flow was found 30 min after estradiol 17 beta-D-glucuronide administration, when it decreased from 3.5 +/- 0.4 microliters/min/100 gm (basal) to 0.9 +/- 0.3 microliters/min/100 gm in estradiol 17 beta-D-glucuronide controls, whereas in dimethylethanolamine-pretreated rats this decreased only from 3.2 +/- 0.4 (basal) to 2.3 +/- 0.4 microliters/min/100 gm. Bile flow and the biliary secretion of cholesterol, phosphatidylcholine and bile salts were significantly higher in the dimethylethanolamine-pretreated rats than in estradiol 17 beta-D-glucuronide controls (p less than 0.02) during the cholestatic phase. The inhibitory effect of estradiol 17 beta-D-glucuronide on bile flow was associated with a marked decrease of membrane fluidity (p less than 0.001) assessed by 1,6-diphenyl-1,3,5-hexatriene fluorescence anisotropy and with a cholesterol enrichment of microsomes, sinusoidal and canalicular liver plasma membranes and inhibition of sinusoidal Na+,K(+)-ATPase activity (p less than 0.05). These membrane alterations persisted 180 min after estradiol 17 beta-D-glucuronide administration despite complete normalization of bile flow. Dimethylethanolamine pretreatment significantly counteracted the reduction of membrane fluidity (p less than 0.001), the cholesterol enrichment and the inhibition of Na+,K(+)-ATPase (p less than 0.05) promoted by estradiol 17 beta-D-glucuronide administration in all membrane subfractions 30 and 180 min after administration. In addition, dimethylethanolamine-pretreated rats had more polyunsaturated fatty acids in membrane phosphatidylcholine with respect to the control groups. Dilatation of canaliculi and loss of microvilli were evident in estradiol 17 beta-D-glucuronide controls 180 min after estradiol 17 beta-D-glucuronide administration.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1990

Hepatic uptake and processing of free cholesterol from different lipoproteins with and without sodium taurocholate administration. An in vivo study in the rat.

The metabolic fate of [14C]cholesterol carried in the VLDL, LDL, HDL2 and HDL3 lipoprotein fractions has been investigated in bile-fistula rats with jugular vein cannulation. After the depletion of bile salt pool, the labelled lipoprotein fraction was administered either with or without the continuous infusion of sodium taurocholate. The proportion of labelled steroids secreted in the bile within 3 h after the lipoprotein administration was generally higher when the exogenous bile salt was infused. Specifically, the HDL2 fraction induced the secretion of relatively high proportions of labelled steroids, mainly bile salts, either with or without the taurocholate infusion. The other lipoprotein fractions increased the proportion of free cholesterol in steroid bile secretion under taurocholate administration. The label associated to the LDL fraction showed a higher biliary secretion and a more steady clearance from plasma if the exogenous bile salt was not administered. In this same condition, the administration of HDL3 fraction gave the highest values of radioactivity recovered in the liver (mainly as free cholesterol) and a significant increase of cholesterol in the biliary secretion. The present results suggest that each lipoprotein fraction tested may contribute in a peculiar manner to bile salt and cholesterol biliary secretion. Both the expression of apo-E and apo B,E receptors and the levels of circulating bile salts appear to have a role in this process.

Animals↗
PubMed1990

Separation and determination of molecular species of phosphatidylcholine in biological samples by high-performance liquid chromatography.

A method for determining the molecular species composition of phosphatidylcholine in biological samples by reversed-phase high-performance liquid chromatography with dual-wavelength ultraviolet detection is described. The optimum compromise between analysis time and chromatographic resolution under isocratic and isothermal conditions (0.8 ml/min and 32 degrees C, respectively) was obtained with the mobile phase methanol-ethanol (6:4, v/v) containing 20 mM choline chloride-water-acetonitrile (90:7:3, v/v/v). The problems of quantification at 205 nm, due to large differences in the detector response with the degree of unsaturation, were resolved by using the appropriate calibration factors chosen with the ratio of absorbances at 205 and 215 nm. The proposed procedure gave results in good agreement with fatty acid composition in samples of rat bile, liver, liver mitochondria and microsomes determined by gas-liquid chromatography.

Animals↗
PubMed1990

The cesium-induced delay in myoblast membrane fusion is accompanied by changes in isolated membrane lipids.

We have recently demonstrated that cesium ions delay the sharp decrease in both membrane conductivity and membrane permittivity of chick embryo myoblasts seen at fusion (Santini, M.T., Bonincontro, A., Cametti, C. and Indovina, P.L. (1988) Biochim. Biophys. Acta 945, 56-64). Analysis of the conductivity dispersion data (obtained in the radiowave frequency range) indicated that cesium delays fusion by about 30 h. We suggested that cesium is affecting both active ionic transport by blocking potassium channels as well as interfering with membrane lipid and/or protein charges. In the present study, we have investigated both the possible role of membrane lipids in myoblast fusion and the possible effects of cesium on these lipids. Our data indicate that lipid changes do occur in the isolated myoblast plasma membrane of controls during myogenic differentiation especially prior to fusion and that in cesium cultures these variations do not occur. These variations are in accordance with current membrane fusion theory. Specifically, there is a decrease in bilayer-stabilizing lipids (phosphatidylcholine) and an increase in bilayer-destabilizing ones (phosphatidylethanolamine and phosphatidic acid) and cholesterol during the fusion process. In addition, although slight, during fusion there appears to be a decrease in phosphatidylinositol which is believed to be involved in the inositol phosphate second messenger system. In cesium cultures, in which fusion is greatly delayed, the same lipid changes do not take place and those that are observed seem to reflect the fusion delay.

Animals↗