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Browsing by Author "NERVI, F"

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APOLIPOPROTEIN-E POLYMORPHISM IN PATIENTS WITH ACUTE-PANCREATITIS
(LIPPINCOTT-RAVEN PUBL, 1994) ROLLAN, A; LOYOLA, G; COVARRUBIAS, C; GIANCASPERO, R; ACEVEDO, K; NERVI, F
We have shown that patients with previous acute pancreatitis (AP) may have an abnormal catabolism of chylomicron remnants (CMR). Because apoprotein E (Apo E) genetic polymorphism has an important influence on CMR clearance, we compared frequency distribution of Apo E phenotypes in 52 patients with AP, 109 patients with gallstones, and 110 control subjects. Apo E phenotypes were detected by isoelectric focusing and immunoblotting. After adjusting for differences in age and gender, fasting triglyceride level was comparable between the study groups. The frequency distribution of Apo E phenotypes was not different between the three study groups and it was in Hardy-Weinberg equilibrium. The gene frequency for Apo E2 was 0.212, 0.273, and 0.243 in AP, gallstone, and control group, respectively. For Apo E3 it was 0.701, 0.627, and 0.674, and for Apo E4 0.090, 0.100, and 0.083 in the same groups, respectively. Differences were not statistically significant (chi2). In conclusion, the abnormal catabolism of CMR in patients with AP is not attributable to Apo E polymorphism. An alternative explanation may be sought in the activity of the recently identified hepatocytic Apo E receptor [LDL-related receptor protein (LRP)].
BILIARY LIPID SECRETION - IMMUNOLOCALIZATION AND IDENTIFICATION OF A PROTEIN ASSOCIATED WITH LAMELLAR CHOLESTEROL CARRIERS IN SUPERSATURATED RAT AND HUMAN BILE
(1993) RIGOTTI, A; NUNEZ, L; AMIGO, L; PUGLIELLI, L; GARRIDO, J; SANTOS, M; GONZALEZ, S; MINGRONE, G; GRECO, A; NERVI, F
Feeding a 0.5% diosgenin plus 0.02% simvastatin diet to rats increases biliary cholesterol concentration and saturation to levels generally found in human native supersaturated bile. By using preparative ultracentrifugation, gel filtration chromatography, and electron microscopy, we isolated, purified, and identified lamellar structures (unilamellar vesicles and multilamellae) as a major biliary cholesterol transport in supersaturated human and rat bile. It was estimated that more than 60% of biliary cholesterol is transported in these lamellar carriers, which were identified by transmission electron microscopy as unilamellar vesicles and multilamellar bodies within bile canaliculi of rats with cholesterol supersaturated bile. By SDS-PAGE, a characteristic and constant protein profile was found associated to the purified lamellar carriers. One of these proteins, a 130-kDa protein, was isolated from human biliary lamellae and used for preparation of a rabbit polyclonal antibody, which cross-reacted with the homologous rat protein. By Western blotting, it was established that the purified low density fraction of bile-Metrizamide gradients, containing lamellae, was enriched with the 130-kDa protein. The 130-kDa protein was characteristically detected at the canalicular membrane by Western blotting of hepatic subcellular fractions and by immunohistochemistry of rat and human liver biopsies. Amino acid sequencing of the amino terminus of the 130-kDa protein demonstrated a complete identity with aminopeptidase N, a canalicular transmembrane hydrophobic glycoprotein. These studies show that biliary lipids may acquire an ordered multilamellar structure that is present in the canaliculi of rats with supersaturated bile. These biliary lamellae are similar to lamellar bodies and surfactant-like material frequently found in other epithelia, suggesting common biogenetic, structural, and functional properties. The identification of aminopeptidase N associated with biliary lamellae is consistent with the involvement of the canalicular membrane in the secretory mechanism of biliary lipids.
CATABOLISM OF CHYLOMICRON REMNANTS IN PATIENTS WITH PREVIOUS ACUTE-PANCREATITIS
(1990) ROLLAN, A; GUZMAN, S; PIMENTEL, F; NERVI, F
A recent study reports that patients with previous acute pancreatitis commonly have an abnormal clearance of serum triglycerides after an oral fat load. This observation supports the hypothesis that patients with previous acute pancreatitis and normal fasting serum triglyceride levels may have a preexistent abnormality in the metabolism of chylomicrons. To test this hypothesis, the catabolism of chylomicrons and their remnants was studied in a series of 7 patients who had sustained an attack of pancreatitis (2, gallstone related; 2, alcohol ingestion; 1, hydatid cyst; and 3, no associated pathological condition) at least 18 mo earlier. All the patients had previously had abnormal oral-fat tolerance test results. These patients were compared with a series of 6 healthy volunteers. Chylomicrons were endogenously labeled with an oral dose of retinyl palmitate, and their plasma elimination half-life was calculated. The retinyl palmitate absorption rate constants were similar in control and pancreatitis patients. The chylomicron t1/2 were 2.3 .+-. 0.8 (SD) h and 3.9 .+-. 1.8 h in the control and pancreatitis groups, respectively (p = 0.07). The chylomicron remnant t1/2 was 2.7 .+-. 1.1 h in the control groups and 5.2 .+-. 2.4 h in the pancreatitis group (p < 0.05). This study support the hypothesis that subjects with previous acute pancreatitis may have an abnormality in the catabolism of chylomicron particles. This abnormality may represent a preexistent genetic condition expressed in either the apoprotein composition of chylomicrons or in the hepatic apolipoprotein E-receptor activity.
CHOLESTEROL CRYSTALLIZATION-PROMOTING ACTIVITY OF AMINOPEPTIDASE-N ISOLATED FROM THE VESICULAR CARRIER OF BILIARY LIPIDS
(WILEY, 1993) NUNEZ, L; AMIGO, L; RIGOTTI, A; PUGLIELLI, L; MINGRONE, G; GRECO, AV; NERVI, F
Different hydrophobic glycoproteins are associated to native biliary vesicles, which are the major carrier of biliary cholesterol. Some of these proteins promote cholesterol crystallization, a key step in cholesterol gallstone formation. This study was specifically conducted to identify the 130 kDa biliary vesicle-associated glycoprotein and to determine its in vitro effect on the cholesterol crystal formation time. The 130 kDa vesicular glycoprotein was identified as aminopeptidase-N by amino acid sequencing and specific enzymatic assay. Polyclonal antibodies raised against aminopeptidase-N allowed us to determine its concentration in human hepatic bile, which varied from 17.3 to 57.6 mug/ml. Aminopeptidase-N showed a concentration-dependent cholesterol crystallization activity when it was added to supersaturated model bile at a concentration range usually found in native bile. Because of this promoting effect on in vitro cholesterol crystal formation, we suggest that biliary aminopeptidase-N may play a critical role in the pathogenesis of cholesterol gallstone disease.
EFFECT OF BEAN INTAKE ON BILIARY LIPID SECRETION AND ON HEPATIC CHOLESTEROL-METABOLISM IN THE RAT
(1989) RIGOTTI, A; MARZOLO, MP; ULLOA, N; GONZALEZ, O; NERVI, F
We studied the effect of a bean diet on biliary lipid secretion, serum cholesterol concentration, and hepatic cholesterol metabolism in the rat. Rats fed a bean diet for 10-12 days had increased biliary cholesterol output and molar percentage by 300% and 200%, respectively, compared to rats fed an isocaloric and isoproteinic casein diet. Biliary phospholipid output increased 180%. Bile flow and biliary salt output remained in the normal range. Total serum and VLDL cholesterol concentration significantly decreased 27% and 50%, respectively, in the rats fed the bean diet. Hepatic cholesterogenesis was increased 170% in the bean-fed animals. The relative contribution of newly synthesized hepatic cholesterol to total biliary cholesterol increased 200%, and that of endogenous origin only 50%. These results suggested that newly synthetized hepatic cholesterol was preferentially channelled to the biliary cholesterol pathway in bean-fed rats. Although hepatic cholesteryl ester concentration increased 240%, the incorporation of [14C]oleate into hepatic cholesteryl esters was significantly decreased by 30% in isolated hepatocytes of bean-fed animals. These results were consistent with the possibility that the availability of hepatic free cholesterol for biliary secretion was increased in the bean-fed animals. This study demonstrates that bean intake has a profound effect on the metabolic channelling and compartmentalization of hepatic cholesterol, resulting in a significant decrease in total serum and very low density lipoprotein cholesterol concentrations and a high biliary cholesterol output.
FREQUENCY OF GALLBLADDER CANCER IN CHILE, A HIGH-RISK AREA
(1988) NERVI, F; DUARTE, I; GOMEZ, G; RODRIGUEZ, G; DELPINO, G; FERRERIO, O; COVARRUBIAS, C; VALDIVIESO, V; TORRES, MI; URZUA, A
We report an autopsy study of gallbladder cancer prevalence in Chile, where the risk of this disease is among the highest reported world-wide. In 14,768 autopsy protocols obtained from 3 university hospitals, 45% of women and 20% of men older than 20 years had gallstone disease (the major known risk factor for gallbladder cancer). The prevalence of gallbladder cancer in Chileans was compared to that found in a Swedish-Czechoslovakian autopsy study previously published. These countries were chosen because of their high frequency of gallstone disease. The comparison was performed by using logistic regression models adjusting for possible differences in the age-sex structure or the true incidence of gallstones in both populations. We found that the most important single risk factor for gallbladdr cancer in Chile was gallstone disease, with an estimated effect on the logistic scale meaning that the cancer risk for subjects with gallstones is seven times higher than for those without the disease. Second in importance was the risk for sex, women being 2.8 times higher than for men. The estimated difference in the sex composition and the incidence of gallstones resulted in 17.9% higher odds of cancer in Chile than in Sweden and Czechoslovakia. However, this difference was not significant. This study suggests that the major etiologic factors of gallbladder cancer in Chileans, Swedes and Czechoslovakians are primarily related to gallstone disease.
HEPATIC PRODUCTION OF VERY-LOW-DENSITY LIPOPROTEIN, CATABOLISM OF LOW-DENSITY-LIPOPROTEIN, BILIARY LIPID SECRETION, AND BILE-SALT SYNTHESIS IN RATS FED A BEAN (PHASEOLUS-VULGARIS) DIET
(1993) MARZOLO, MP; AMIGO, L; NERVI, F
Rats fed a bean diet develop a significant hypocholesterolemia. The catabolism of low density lipoprotein (LDL; d 1.019-1.063 g/ml) was studied in vivo and in vitro in the isolated perfused liver of rats fed either a casein or a bean diet. The clearance of LDL was significantly increased by 100% from 0.38 +/- 0.04 to 0.63 +/- 0.04 ml/h x 100 g body wt in vivo in the bean-fed rat. Similarly, the clearance of homologous and heterologous (human) LDL was also increased by 100% in the isolated perfused liver of bean-fed animals. Spleen, kidney, and hepatic cholesterogenesis was increased by 150% in these animals. Bile salt synthesis was increased from 1.54 +/- 0.02 to 2.84 +/- 0.09 nmol/min x g liver wt (P < 0.02) and biliary cholesterol output by 200% from 0.81 +/- 0.03 to 2.18 +/- 0.04 nmol/min x g (P < 0.02) in the isolated perfused liver of rats fed a bean diet. These results explained the depletion of hepatic cholesterol and were consistent with the LDL turnover studies, suggesting that apoB/E receptor activity was increased in these animals. ApoB and triglyceride secretion in the d < 1.060 g/ml lipoprotein fraction of liver perfusates remained normal in the bean-fed rats. In contrast, total sinusoidal cholesterol output isolated in the d < 1.060 g/ml fraction significantly decreased by 100% after 90 min of perfusion. Cholesterol output in the d > 1.060 g/ml lipoprotein fraction was unmodified by the bean diet. These data demonstrate that key metabolic pathways of hepatic cholesterol are modified in the bean-fed rat. These modifications are consistent with hypocholesterolemia induced by this legume. The marked excretion of hepatic cholesterol into the bile associated with a decreased output of sinusoidal cholesterol in apoB-containing lipoproteins suggest a functional reciprocal interrelationship between both cholesterol secretory pathways in the bean-fed animals.
INFLUENCE OF LEGUME INTAKE ON BILIARY LIPIDS AND CHOLESTEROL SATURATION IN YOUNG CHILEAN MEN - IDENTIFICATION OF A DIETARY RISK FACTOR FOR CHOLESTEROL GALLSTONE FORMATION IN A HIGHLY PREVALENT AREA
(1989) NERVI, F; COVARRUBIAS, C; BRAVO, P; VELASCO, N; ULLOA, N; CRUZ, F; FAVA, M; SEVERIN, C; DELPOZO, R; ANTEZANA, C; VALDIVIESO, V; ARTEAGA, A
Chileans and North American Indians have one of the highest prevalence rates of cholesterol gallstones in the world. The most common theory to explain this has been the operation of some as yet undefined genetic risk factor in these populations. Searching for some common environmental factor for gallstones in Chileans and North American Indians, we found that beans and other legumes are common foods consumed by both populations. In this study we tested the hypothesis that legume intake may favor the production of biliary cholesterol supersaturation. We studied 20 young men subjected to a diet containing 120 g/day of legumes and a control diet without legumes for a period of 1 mo each. Both diets supplied identical quantities of energy, carbohydrates, protein, total fat, fiber, and cholesterol. Low-density lipoprotein cholesterol concentration decreased by 16% (p < 0.001) after the legume diet. Biliary cholesterol saturation increased in 19 of the 20 subjects; the mean of the group markedly increased from 110% to 169% (p < 0.001) after the legume diet. These results are consistent with the hypothesis that legume intake is a potential risk factor for cholesterol gallstone disease.
ISOLATION AND PURIFICATION OF HUMAN BILIARY VESICLES WITH POTENT CHOLESTEROL-NUCLEATION-PROMOTING ACTIVITY
(1992) MIQUEL, JF; RIGOTTI, A; ROJAS, E; BRANDAN, E; NERVI, F
1. Cholesterol nucleation is a critical step in the formation of cholesterol gallstones. This nucleation takes place after aggregation and fusion of cholesterol-rich biliary vesicles, a process probably modulated by biliary proteins. The present study was conducted to identify specific proteins associated with native cholesterol-rich biliary vesicles and to explore their effect on the cholesterol-nucleation time of supersaturated artificial bile.
MECHANISM AND KINETIC CHARACTERISTICS OF THE UNCOUPLING BY PLANT STEROIDS OF BILIARY CHOLESTEROL FROM BILE-SALT OUTPUT
(1985) ULLOA, N; NERVI, F
In male Wistar rats fed diets containing different plant steroids, including sitosterols, diosgenin, digitonin and saponin from gypsophila, biliary cholesterol secretion significantly increased 50% to 300%, whereas biliary bile salt and phospholipid showed minor changes. Both cholesterol and phospholipid outputs were coupled to biliary bile salt output in a curvi-linear relationship which could be fitted by rectangular hyperbolae, in the animals fed with different plant steroids. The theoretical maximal biliary cholesterol output significantly increased by 200% in sitosterol-fed rats and 500% in diosgenin-fed animals. No changes were found in the kinetic characteristics of biliary phospholipid outputs. Adding 2% cholesterol to the diosgenin diet abolished the increment of biliary cholesterol output induced by the plant steroid. The intraperitoneal injection of 45 .mu.mol/kg body wt per day(3 days) diosgenin, a C27-sapogenin, and 65 .mu.mol/kg body wt. per day (3 days) tomatidin, a C27-alkaloid, incorporated in phosphatidylcholine-taurocholate liposomes significantly increased biliary cholesterol output by 70%. These experiments indicated that the plant steroid-induced biliary cholesterol output was independent of the inputs of cholesterol from the diet and from hepatic cholesterogenesis modified by the plant steroid. It was apparent that the profound changes of biliary cholesterol secretion were the consequence of direct effects of the steroids on the intrahepatocytic regulatory mechanisms of biliary cholesterol secretion. This novel effect appears to be a universal characteristic of plant steroids, since it can be elicited by sitosterols, C27-sapogenins, C27-alkaloids, and saponins of the cholanic and .beta.-amirinic group.
MUTAGENIC SUBSTANCES IN RED AND WHITE WINE IN CHILE, A HIGH-RISK AREA FOR GASTRIC-CANCER
(1987) BULL, P; YANEZ, L; NERVI, F
Chilean home-made and commercial wines were analyzed for the presence of mutagenic substances using the Salmonella mutagenicity test with preincubation. Strains TA98 and TA100 were used in the absence and in the presence of S9 mix. 90% of red wines from a total of 30 samples and 54% of white wines from a total of 22 were found to be mutagenic. In all cases, S9 mix did not affect the mutagenicity of the samples. At least in one case, more than one mutagen was present, since the mutagenicity with TA98 could be selectively inactivated without affecting that with TA100. This study supports the hypothesis that wine consumption may be an important risk factor for upper gastrointestinal cancer, particularly for adenocarcinoma of the stomach, which is highly prevalent in Chile.
PROTECTIVE ROLE OF BILIARY CHOLESTEROL AND PHOSPHOLIPID LAMELLAE AGAINST BILE ACID-INDUCED CELL-DAMAGE
(W B SAUNDERS CO-ELSEVIER INC, 1994) PUGLIELLI, L; AMIGO, L; ARRESE, M; NUNEZ, L; RIGOTTI, A; GARRIDO, J; GONZALEZ, S; MINGRONE, G; GRECO, AV; ACCATINO, L; NERVI, F
RAPID ISOLATION OF VESICULAR AND MICELLAR CARRIERS OF BILIARY LIPIDS BY ULTRACENTRIFUGATION
(LIPID RESEARCH INC, 1990) AMIGO, L; COVARRUBIAS, C; NERVI, F
REGULATION OF BILIARY CHOLESTEROL SECRETION - FUNCTIONAL-RELATIONSHIP BETWEEN THE CANALICULAR AND SINUSOIDAL CHOLESTEROL SECRETORY PATHWAYS IN THE RAT
(1988) NERVI, F; MARINOVIC, I; RIGOTTI, A; ULLOA, N
The functional interrelationship between biliary cholesterol secretion, sinusoidal lipoprotein cholesterol secretion and bile salt synthesis was studied in the rat. Diosgenin, fructose, and colestipol in the diet were used to, respectively, influence biliary cholesterol output, VLDL production and bile salt synthesis. In the acute bile fistula rat, biliary cholesterol output was 700% increased by diosgenin and 50% decreased by fructose. In the rats fed both diosgenin and fructose, biliary cholesterol secretion was increased only by .apprx. 200%, whereas biliary bile salts and phospholipid outputs were unchanged. In the isolated perfused liver, VLDL-cholesterol output was 50% reduced by diosgenin alone, but was unchanged following feeding of diosgenin plus fructose. However, the livers of rats fed diosgenin plus fructose exhibited a 700% increase in VLDL-triglyceride production and a 200% increase in VLDL-cholesterol output. A significant reciprocal relationship between VLDL-cholesterol secretion and the coupling ratio of cholesterol to bile salts in bile was observed. Colestipol added to the diet maintained both sinusoidal and biliary cholesterol outputs within the normal range. In the chronic bile fistula rat, colestipol increased bile salt synthesis by 100% while diosgenin and fructose diets had no effect. Similarly, the addition of fructose to the colestipol diet did not decrease bile salt synthesis. These data suggest a reciprocal relationship between biliary cholesterol secretion and hepatic secretion of cholesterol as VLDL particles. The free cholesterol pool used for bile salt synthesis seems functionally unrelated to the pool from which VLDL-cholesterol and biliary cholesterol originate. These findings support the idea that metabolic compartmentalization of hepatic cholesterol is a major determinant of the quantity of cholesterol available for recruitment by the bile salt-dependent biliary cholesterol secretory mechanism.
REGULATION OF BILIARY CHOLESTEROL SECRETION IN THE RAT - ROLE OF HEPATIC CHOLESTEROL ESTERIFICATION
(1984) NERVI, F; BRONFMAN, M; ALLALON, W; DEPIEREUX, E; DELPOZO, R
Although the significance of the enterohepatic circulation of bile salts in the solubilization and biliary excretion of cholesterol is well established, little is known about the intrahepatic determinants of biliary cholesterol output. Studies were undertaken to elucidate some of these determinants in the rat. Feeding 1% diosgenin for 1 wk increased biliary cholesterol output and saturation by 400%. Bile flow, biliary bile salt, phospholipid and protein outputs remained in the normal range. When ethynyl estradiol (EE) was injected into these animals, biliary cholesterol output decreased to almost normal levels under circumstances of minor changes in the rates of biliary bile salt and phospholipid outputs. Similarly, when chylomicron cholesterol was i.v. injected into diosgenin-fed animals, biliary cholesterol output significantly decreased as a function of the dose of chylomicron cholesterol administered. Relative rates of hepatic cholesterol synthesis and esterification were measured in isolated hepatocytes. Although, hepatic cholesterogenesis increased 300% in diosgenin-fed animals, the contribution of newly synthesized cholesterol to total biliary cholesterol output was only 19 .+-. 9%, compared with 12 .+-. 6% in control and 15 .+-. 5% in diosgenin-fed and EE-injected rats. The rate of oleate incorporation into hepatocytic cholesterol esters was 30% inhibited in diosgenin-fed rats. When EE was injected into these animals, the rate of cholesterol esterification increased to almost 300%. To investigate further the interrelationship between hepatic cholesterol esterification and biliary cholesterol output, 21 diosgenin-fed rats were studied. Six of them received in addition EE and 10 received chylomicron cholesterol. The relationships between biliary cholesterol output as a function of both microsomal acyl-CoA:cholesterol acyltransferase (ACAT) activity and hepatic cholesterol ester concentration were significantly correlated in a reciprocal manner. The size of the biliary cholesterol precursor pool can be rapidly modified through changes in the activity of the hepatic ACAT. [Implications with respect to the role of high cholesterol secretion to gallstone disease were presented.].
REVERSAL OF PROGESTERONE-INDUCED BILIARY CHOLESTEROL OUTPUT BY DIETARY-CHOLESTEROL AND ETHYNYLESTRADIOL
(1983) DELPOZO, R; NERVI, F; COVARRUBIAS, C; RONCO, B
SUB-CELLULAR DISTRIBUTION OF CHOLESTEROL ESTER HYDROLASE IN HUMAN-LIVER
(1981) DELPOZO, R; BRONFMAN, M; BULL, P; NERVI, F
ULTRACENTRIFUGAL ISOLATION OF VESICULAR CARRIERS OF BILIARY CHOLESTEROL IN NATIVE HUMAN AND RAT BILE
(1987) ULLOA, N; GARRIDO, J; NERVI, F
We have utilized ultracentrifugation of native bile-Metrizamide density gradients to isolate a vesicular transport system of biliary lipids in both man and rat. We identified vesicular structures by electron microscopy. Fresh bile specimens were obtained from bile fistula rats (unsaturated bile) and from patients 1 week after bile duct surgery (supersaturated bile). Metrizamide was dissolved in bile (33% w/v), and continuous density gradients were performed with undiluted bile (density limits = 1.020 to 1.300 gm per ml). The relative distribution of biliary cholesterol, phospholipid and bile salt was studied as a function of the density of the fractions. Approximately 50% of total rat biliary cholesterol and between 61 and 90% of human biliary cholesterol was concentrated in the lightest fractions of the gradients (density < 1.060 gm per ml). In contrast, less than 20% of bile salts was present in fractions with densities lwoer than 1.060 gm per ml. The highest amounts of bile salts and phospholipids of the bile-Metrizamide density gradients were found in the density range of 1.075 to 1.100 gm per ml in both human and rat bile. More than 80% of biliary proteins was found in fractions with densities > 1.075 gm per ml, and only 2% was found in cholesterol-rich fraction with density < 1.060 gm per ml in both species. When bile salt concentration was raised in rat bile from 38 to 97 mM by adding taurocholate, and low density cholesterol-rich fraction almost disappeared. Electron microscopy of negatively stained preparations of the fractions with density < 1.060 gm per ml showed 40 to 120 nm vesicles, which were not apparent in the other fractions. Similar vesicles were demonstrated also in fresh rat bile and within the canaliculi after acute depletion of the bile salt pool (biliary bile salt concentration of 3.45 mM; total biliary lipid concentration of 0.25 gm%). The structure of these vesicles was shown in thin sections of liver specimens. They appeared as internal cavities surrounded by a single, continuous 6-nm-thick bilayer. These studies demonstrate that a high proportion of biliary cholesterol is transported in vesicles in human supersaturated native bile and that vesicular carriers are also responsible for the transport of a significant amount of biliary cholesterol in unsaturated rat bile. The presence of vesicles in unsaturated hepatic bile strongly supports the thesis that biliary lipids may be secreted as vesicles from the hepatocyte into the canaliculi.

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