Biomedical subjects

R Masella

Publications and source records attributed to R Masella.

16 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

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

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

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

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

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↗

PubMed1989

Selective hepatic enrichment of polyunsaturated phosphatidylcholines after intravenous administration of dimethylethanolamine in the rat.

The content of polyunsaturated phosphatidylcholines (PCs) is one of the parameters which regulate membrane functions. Polyunsaturated PCs are preferentially synthesized in the liver by the microsomal enzyme phosphatidylethanolamine N-methyltransferase. The activity of this enzyme may be stimulated in vitro in isolated rat hepatocytes by supplementation with dimethylethanolamine (DME), the polar head group of the precursor of PC along this pathway. The aim of this study was to evaluate in vivo the effect of an intravenous infusion of DME in the rat on the hepatic phospholipid composition. Bile fistula rats were intravenously infused for 15 h with sodium taurocholate (1 mumol/kg per min), with or without the addition of 0.3 mg/kg per min of [14C]DME. The concentration per gram of wet liver of individual phospholipid classes, PC molecular species and of total triacylglycerols, as well as the distribution of radioactivity in liver phospholipids, in rat tissues and body fluids were analyzed. A significant (P less than 0.01) enrichment in PC was found in the liver of DME-infused rats with respect to controls. No differences in the other phospholipid classes were found. DME-infused rats showed a significant (P less than 0.01) decrease in the hepatic concentration of triacylglycerols. At HPLC analysis, the enrichment in PC in DME-infused rats was found to be selectively due to three molecular species (i.e., sn-1 stearoyl/sn-2 arachidonoyl, sn-1 stearoyl/sn-2 linoleoyl, sn-1 stearoyl/sn-2 docosahexanoyl molecular species). In agreement with quantitative data, more than 70% of hepatic radioactivity was recovered in polyunsaturated PC species, with the highest specific activity in the sn-1 stearoyl PCs. The specific activity of hepatic PC approximates that of phosphatidyldimethylethanolamine. This finding together with the effective incorporation of DME in PC suggests that this amino base is methylated after its incorporation into phosphatidyldimethylethanolamine, throughout the stimulation of hepatic N-methyltransferase activity. The selective hepatic enrichment with polyunsaturated PC species after DME infusion may offer a new experimental tool for studying hepatic membrane metabolism.

Animals↗

PubMed1989

Characterization of vesicles, containing an acylated oligopeptide, released by human colon adenocarcinoma cells. NMR and biochemical studies.

RNA-containing vesicles, recovered from the supernatant of high-density cell samples of human colon carcinoma, produce a high-resolution 1H NMR spectrum of lipids characterized by isotropic tumbling; these vesicles contain large amounts of triglycerides and cholesterol esters. Both findings have strict analogies to what is displayed by the proteolipid complexes isolated from the sera of tumor-bearing patients [(1985) Proc. Natl. Acad. Sci. USA 82, 3455-3459; (1986) FEBS Lett. 203, 164-168]. Lipid analysis and enzymatic tests indicate that these vesicles are selected micromaps of plasma membranes, analogous to those that can be recovered from culture media in which tumor cells are grown [(1985) Dev. Biol. 3, 33-57]. Peculiar lipids, an acylated oligopeptide and a modified phospholipid, are also present in the vesicles.

Acylation↗

PubMed1989

Surgical and prosthodontic residual ridge reconstruction with hydroxyapatite.

The ultimate goal of a ridge augmentation procedure is to form a bearing surface for the denture that will provide stability, retention, and support. Residual ridge reconstruction with an implanted material must be based on an understanding of the variations of ridge atrophy. A problem-oriented classification describes the different forms of residual ridges. This classification of similar types of resorbed residual ridges can be used to establish a rational plan of treatment. The proper placement of hydroxyapatite is critical to obtain the desired reconstructed residual ridge. In the mandible, a lingualized placement of the alloplast is suggested. Careful use of an anatomic matrix to support and contain the hydroxyapatite particles eliminates the need for a splint. The surgical stage includes vestibuloplasty and skin grafting to provide the patient with an increased denture-bearing surface. The ridge created in the form of a comma posteriorly enhances retention and stability. The broad residual ridge with an improved interarch relation also enhances the support and provides a better foundation for balanced occlusion.

Alveolar Process↗

PubMed1988

Determination of phospholipids in biological samples by an improved densitometric method on thin-layer chromatograms.

The quantitative determination of the different phospholipids present in samples of bile, liver or plasma has been performed by densitometric scanning of high-performance thin-layer chromatography plates. These have been developed as follows: pre-treatment with sulphuric acid-ethanol mixture, staining with ethanol-modified molybdenum blue reagent and post-treatments with water and ethanol added with a small quantity of sulphuric acid. The sequential treatment, which introduces some modifications in a previously described procedure, allowed a linear and stable colour response for phosphorous-containing lipids on uniform and colourless background. The calibration of each plate with standard mixture was required for obtaining results in good agreement with other routine methods of determination.

Bile↗

PubMed1988

On the mechanism of action of lonidamine: a study on human erythrocyte membrane.

The incubation of human erythrocytes with increasing levels of the antineoplastic drug Lonidamine clearly indicated a dose-dependent effect on the lipid composition of the plasma membranes. A selective transfer of phosphatidylcholine and cholesterol from membrane to incubation medium and the consequent enrichment in phosphatidylethanolamine of the membrane itself was observed. Moreover, the membranes were found to contain increasing levels of the drug which was incorporated at a constant membrane/medium partition ratio. The changes in composition appeared to be consistent with morphological alterations observed by scanning and freeze-fracture electron microscopy demonstrating changes in cell shape, the presence of numerous intracellular vesicles, and a membrane protein rearrangement. The analysis of intact red cells by nuclear magnetic resonance ruled out the possibility that the alterations described above could be due to an ATP depletion. This further confirmed that cell membranes were the primary target of the Lonidamine action, the previously described energy metabolism impairment being a consequence of a selective damage of cellular membranes, probably originating from the incorporation of the drug into the lipid bilayer.

Cholesterol↗

PubMed1987

Transport, utilization and biliary secretion of lysophosphatidylcholine in the rat liver.

The hepatic uptake, transport and utilization of plasma lysophosphatidylcholine (lysoPC) and its contribution to biliary lipid secretion have been investigated in bile-fistula rats. The animals were given a single intravenous dose of sn-1-[1-14C]palmitoyl-lysoPC, under constant intravenous sodium taurocholate infusion (1 mumol/min), and the fate of the label was followed in blood, bile and liver for up to 3 h. The livers were excised at given time points, extracted and/or homogenized to determine the lipid distribution and subcellular location of radioactivity. LysoPC was rapidly cleared from plasma, though a consistent fraction of the label persisted in plasma over the experimental time-period in the form of either lysoPC or PC. Recovery of radioactivity in the liver varied from 15.6% after 5 min to 19.5% after 3 h. Hepatic lysoPC underwent rapid microsomal acylation to form specific PC molecular species (mainly 16:0-20:4 and, to a lesser extent, 16:0-18:2 and 16:0-16:1). Ultrafiltration, dialysis and gel-chromatographic analyses of cytosolic fractions (post 105,000 X g supernatants) indicated that lysoPC is transported to the site of acylation mostly as a macromolecular aggregate with an approx. Mr of 14,400. Small amounts of radioactivity were secreted into bile over 3 h (20% in the form of lysoPC and the remainder as 16:0-18:2 and 16:0-20:4 PC species). Plasma lysoPC, taken up by the liver, is mostly transported by a cytosolic carrier with a molecular weight close to fatty-acid-binding proteins; it then enters a distinct acylation pathway, selective for some polyunsaturated-PC species and does not contribute significantly to biliary secretion, either directly, or through its products.

Animals↗

PubMed1987

Studies of structural modifications induced by gamma-irradiation in distearoylphosphatidylcholine liposomes.

An investigation of the structural and thermodynamical modifications induced by gamma-irradiation in model membranes is reported. Differential scanning calorimetry and X-ray diffraction were used to study the different phases and associated transitions of distearoylphosphatidylcholine multilamellar liposomes after 60Co gamma-irradiation. Changes were observed in the shape of the calorimetric peaks and in the corresponding phase transition temperatures. In particular a shoulder was observed at about 20 kGy. The three phases characteristic of lecithins with identical acyl chains were detected also for the highest radiation dose. The formation of lysolecithin and stearic acid upon phospholipid degradation was observed. The lysolecithin concentration increases as a function of irradiation dose, until a saturation value is reached at 40 kGy. These results correlate quite well with those obtained for interlayer and interchain distances and for the width of the main phase transition calorimetric peak. At the highest dose (approximately equal to 80 kGy) cross-linked adjacent radicals and other molecular species are also formed. Appreciable differences, and some similarities, in the behaviour of DSPC and DPPC liposomes under gamma-irradiation were observed.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1987

Composition of free fatty acids and adipose tissue triglycerides in portacaval shunted rats.

This study was designed to determine the individual fatty-acid pattern in plasma and in two adipose tissue sites of rats submitted to portacaval shunt (PCS) using the button technique. The animals were studied both in a fed state and after a 48-hour fast. Glucose, ketone bodies (KB), lactate and pyruvate levels were also measured. The results of the shunted animals were compared with pair-fed and freely fed control groups. In PCS rats, plasma free fatty-acid (FFA) levels were not significantly higher than in freely fed controls. beta-Hydroxybutyrate was significantly lower in PCS rats compared with both control groups. A 48-hour fast caused an elevation of plasma FFA and KB in all groups, but in PCS rats this increase was less pronounced. Lactate levels did not decrease during fasting in PCS rats, as they did in the control groups. PCS did not modify plasma FFA or the adipose tissue composition, neither in fed nor in fasted rats. We conclude that the PCS rats operated upon by using the button technique show mild modifications in lipid metabolism, affecting the fatty-acid composition neither in plasma FFA nor in adipose tissue.

Adipose Tissue↗