Cholesterol-lowering effects of dietary blue lupin (Lupinus angustifolius L.) in intact and ileorectal anastomosed pigs
2005; Elsevier BV; Volume: 46; Issue: 7 Linguagem: Inglês
10.1194/jlr.m500129-jlr200
ISSN1539-7262
AutoresJ.M. Martins, Michel Riottot, Manuel Cancela d’Abreu, Ana Maria Viegas-Crespo, Maria João Lança, J. A. Afonso de Almeida, J.P.B. Freire, Ofélia Bento,
Tópico(s)Cholesterol and Lipid Metabolism
ResumoThe present study was undertaken to investigate the effect of cholesterol-enriched casein (CAS) and blue lupin seed (BL) diets on the cholesterol metabolism of intact (INT) and ileorectal anastomosed (IRA) pigs. For 3 weeks, four groups of six pigs were allocated to the treatments (CAS-INT, CAS-IRA, BL-INT, and BL-IRA). Diet-induced hypercholesterolemia was inhibited by the BL through a substantial decrease in plasma LDL-cholesterol. The BL also reduced liver esterified and total cholesterol, increased hepatic LDL receptor synthesis and HMG-CoA reductase activity, and stimulated intestinal bile acid reabsorption. The neutral sterol output was higher in BL- than in CAS-fed pigs. The bile acid output was lower in IRA than in INT pigs. Surgery also prevented steroid microbial transformation, but it did not influence plasma cholesterol levels.These results suggest that the hypocholesterolemic effect of the BL, compared with the CAS, is attributable to impaired intestinal cholesterol absorption, probably involving increased bile acid reabsorption and higher contents of dietary phytosterols, both factors that reduce the micellar solubilization of cholesterol. Furthermore, according to our data, the contribution of the large intestine to cholesterol metabolism is very weak. The present study was undertaken to investigate the effect of cholesterol-enriched casein (CAS) and blue lupin seed (BL) diets on the cholesterol metabolism of intact (INT) and ileorectal anastomosed (IRA) pigs. For 3 weeks, four groups of six pigs were allocated to the treatments (CAS-INT, CAS-IRA, BL-INT, and BL-IRA). Diet-induced hypercholesterolemia was inhibited by the BL through a substantial decrease in plasma LDL-cholesterol. The BL also reduced liver esterified and total cholesterol, increased hepatic LDL receptor synthesis and HMG-CoA reductase activity, and stimulated intestinal bile acid reabsorption. The neutral sterol output was higher in BL- than in CAS-fed pigs. The bile acid output was lower in IRA than in INT pigs. Surgery also prevented steroid microbial transformation, but it did not influence plasma cholesterol levels. These results suggest that the hypocholesterolemic effect of the BL, compared with the CAS, is attributable to impaired intestinal cholesterol absorption, probably involving increased bile acid reabsorption and higher contents of dietary phytosterols, both factors that reduce the micellar solubilization of cholesterol. Furthermore, according to our data, the contribution of the large intestine to cholesterol metabolism is very weak. Hypercholesterolemia and its implications for cardiovascular diseases is a major problem in human health, and much attention has been paid to dietary intervention as a tool for its prevention and treatment (1Kerckhoffs D.A.J.M. Brouns F. Hornstra G. Mensink R.P. Effects on the human serum lipoprotein profile of β-glucan, soy protein and isoflavones, plant sterols and stanols, garlic and tocotrienols.J. Nutr. 2002; 132: 2494-2505Google Scholar). Legumes have shown hypocholesterolemic effects in human and animal models (2Mathur K.S. Singhal S.S. Sharma R.D. Effect of Bengal gram on experimentally induced high levels of cholesterol in tissues and serum in albino rats.J. Nutr. 1964; 84: 201-204Google Scholar, 3Kingman S.M. The influence of legume seeds on human plasma lipid concentrations.Nutr. Res. Rev. 1991; 4: 97-123Google Scholar, 4Anderson J.W. Major A.W. Pulses and lipaemia, short- and long-term effect: potential in the prevention of cardiovascular disease.Br. J. Nutr. 2002; 88: 263-271Google Scholar), but these studies have mainly been done with soybean or its components. Therefore, studies involving other legumes, such as lupins, may clarify the mechanism by which plasma cholesterol is reduced and lead to the identification of new functional foods and/or components.Seeds of several species of lupins have been used as food for >3,000 years in the Mediterranean area (5Gladstones J.S. Distribution, origin, taxonomy, history and importance.in: Gladstones J.S. Atkins C.A. Hamblin J. Lupins as Crop Plants: Biology, Production, and Utilization. CAB International, Wallingford, UK1998: 1-39Google Scholar). These bitter seeds had to be soaked in water before consumption, to remove most of their alkaloid content (6Petterson D.S. Composition and food uses of lupins.in: Gladstones J.S. Atkins C.A. Hamblin J. Lupins as Crop Plants: Biology, Production, and Utilization. CAB International, Wallingford, UK1998: 353-384Google Scholar). From the second half of the 20th century onward, low-alkaloid varieties of white lupin (Lupinus albus), yellow lupin (Lupinus luteus), and blue lupin (Lupinus angustifolius) have been domesticated and selected (7Cowling W.A. Huyghe C. Swiecicki W. Lupin breeding.in: Gladstones J.S. Atkins C.A. Hamblin J. Lupins as Crop Plants: Biology, Production, and Utilization. CAB International, Wallingford, UK1998: 93-120Google Scholar). In 2004, sweet varieties of these three species were mainly cultivated in several parts of Australia, Europe, and South America (8FAO Statistics. 2004. http://faostat.fao.org/ (Accessed February 2005).Google Scholar) and used for feed and food applications. Blue lupin seeds have higher nonstarch polysaccharide (6Petterson D.S. Composition and food uses of lupins.in: Gladstones J.S. Atkins C.A. Hamblin J. Lupins as Crop Plants: Biology, Production, and Utilization. CAB International, Wallingford, UK1998: 353-384Google Scholar) and protein contents than soybean, with a similar amino acid profile (9Mellenthin O. Galensa R. Analysis of polyphenols using capillary zone electrophoresis and HPLC: detection of soy, lupin, and pea protein in meat products.J. Agric. Food Chem. 1999; 47: 594-602Google Scholar). Their use in the food industry is being developed, and lupins are beginning to replace soybean in products such as tempe, miso, fermented sauces, and cooked snack foods (6Petterson D.S. Composition and food uses of lupins.in: Gladstones J.S. Atkins C.A. Hamblin J. Lupins as Crop Plants: Biology, Production, and Utilization. CAB International, Wallingford, UK1998: 353-384Google Scholar). Lupin-based fiber supplements, cookies, bread, and spaghetti, with a high sensory quality, are also reported (10Bunger A. Soto D. Wittig E. Cariaga L. Hernández N. Development of food products containing lupin fiber and their effects in elderly people.in: Santen E. Van Wink M. Weissmann S. Romer P. Lupin, an Ancient Crop for the New Millennium: Proceedings of the 9th International Lupin Conference. International Lupin Association, Canterbury, New Zealand2000: 438-439Google Scholar).No studies have been undertaken to test the effect of blue lupin seeds on cholesterol metabolism in the pig, an animal model with a plasma lipid profile similar to that of human, which responds markedly to hyperlipidemic diets (11Miller E.R. Ullrey D.E. The pig as a model for human nutrition.Annu. Rev. Nutr. 1987; 7: 361-382Google Scholar). Moreover, although the role of the small intestine in cholesterol metabolism is well documented (12Chevallier F. Lutton C. The intestine is the major site of cholesterol synthesis in the rat.Nat. New Biol. 1973; 242: 61-62Google Scholar, 13Turley S.D. Dietschy J.M. Sterol absorption by the small intestine.Curr. Opin. Lipidol. 2003; 14: 233-240Google Scholar), the role of the large intestine and its microflora is still unclear. The absorption of free bile acids by the colon was demonstrated (14Schiff E.R. Small N.C. Dietschy J.M. Characterization of the kinetics of the passive and active transport mechanisms for bile acid absorption in the small intestine and colon of the rat.J. Clin. Invest. 1972; 51: 1351-1362Scopus (214) Google Scholar), but its contribution to the enterohepatic circulation is poorly understood (15Hoffman N.E. Hofmann A.F. Metabolism of steroid and amino acid moieties of conjugated bile acids in man. V. Equations for the perturbed enterohepatic circulation and their application.Gastroenterology. 1977; 72: 141-148Google Scholar). Furthermore, the microbial transformation of primary into secondary bile acids in the hindgut (16Kellogg T.F. Microbiological aspect of enterohepatic sterol and bile acid metabolism.Fed. Proc. 1971; 30: 1808-1814Google Scholar) could affect cholesterol metabolism, because the absorption of hydrophobic secondary bile acids modulates hepatic cholesterol and bile acid synthesis (17Pandak W.M. Vlahcevic Z.R. Heuman D.M. Redford K.S. Chiang J.Y. Hylemon P.B. Effects of different bile salts on steady-state mRNA levels and transcriptional activity of cholesterol 7α-hydroxylase.Hepatology. 1994; 19: 941-947Google Scholar). Therefore, bypass of the cecum-colon section was used as a tool to obtain more information concerning the effect of the hindgut in cholesterol metabolism and steroid output.In this study, we examined the effects of feeding whole blue lupin seeds and the role of cecum-colon bypass on the cholesterol metabolism and neutral and acidic steroid output of growing pigs fed cholesterol-rich diets. Analyses of sterols in the plasma, liver, bile, and feces were carried out.MATERIALS AND METHODSChemicals and isotopesChemicals and solvents of the highest purity available were purchased from Merck (Darmstadt, Germany), Prolabo (Paris, France), and Sigma-Aldrich (St. Louis, MO). Enzymatic assay kits were purchased from Roche Diagnostics GmbH (Mannheim, Germany) and Wako Chemicals GmbH (Neuss, Germany). Hydroxypropyl-β-cyclodextrin was a kind gift of Societé Roquette Frères (Lestrem, France). Anion-exchange AG1-X8 resin was purchased from Bio-Rad (Ivry-Seine, France), and l-3-[glutaryl-3-14C]hydroxymethylglutaryl-CoA, [5-3H]mevalonolactone, [4-14C]cholesterol, 25-[26,27-3H2]hydroxycholesterol, and [14C]taurocholate sodium salt were purchased from DuPont-NEN Products (Les Ulis, France). 25-Hydroxycholesterol was provided by Roussel-Uclaf (Romainville, France), and 7α- and 7β-hydroxycholesterol were synthesized as described previously (18Yamasmita H. Kuroki S. Nakayama F. An assay of cholesterol 7α-hydroxylase utilizing a silica cartridge column and 5α-cholesten-3β,7β-diol as an internal standard.J. Chromatogr. 1989; 496: 255-268Google Scholar). Emulsifier-Safe was acquired from Packard Instruments Co. (Meriden, CA). A polyclonal antibody raised against the bovine adrenal cortex LDL receptor was kindly provided by Paul Roach (University of Adelaide, Adelaide, Australia). The anti-rabbit IgG horseradish peroxidase-linked F(ab′)2 fragment (from donkey) and the enhanced chemiluminescence reagent were purchased from Amersham Pharmacia Biotech (Les Ulis, France).Animals and dietsTwenty-four 12 week old crossbred male pigs [Duroc boars × (Large White × Landrace sows)] from Universidade de Évora (Évora, Portugal) with an initial body weight of 30.3 ± 0.5 kg (mean ± SEM) were individually penned in metabolism cages (60 × 160 cm). All procedures were approved by the Portuguese Animal Nutrition and Welfare Commission (Lisboa, Portugal).Two experimental cholesterol-enriched diets, a semipurified casein diet (CAS) and a raw blue lupin seed diet (BL), were formulated to have similar amounts of crude protein, essential amino acids (lysine, methionine, and tryptophan), and gross energy. In the BL, ∼60% of the protein supplied by casein in the CAS was replaced by protein from finely ground whole blue lupin dry seeds (Table 1). Cholesterol was included in both diets at a rate of 2.8 g/kg, after solubilization in soybean oil. Pigs were fed at a weekly adjusted daily rate of 50 g/kg body weight in two equal meals (8:30 AM and 6:00 PM) and had free access to water throughout the experimental period.TABLE 1Ingredients and average composition of the experimental dietsIngredients and CompositionCASBLg/kgIngredientsCornstarch464.0342.3Sucrose100.0100.0Wheat straw100.0100.0Soybean oil100.085.0Acid hydrolyzed casein180.072.0Blue lupin whole seeds—244.0l-Lysine—1.5dl-Methionine1.02.0l-Tryptophan—0.5Calcium carbonate16.218.4Dicalcium phosphate30.826.3Sodium chloride5.05.0Vitamin and mineral mixtureaSupplied (mg/kg diet): retinol, 30; cholecalciferol, 5.4; dl-α-tocopherol, 22.5; thiamine, 0.75; riboflavin, 4.5; cyanocobalamin, 0.023; biotin, 0.15; menadione, 1.5; folic acid, 0.75; nicotinic acid, 30; pantothenic acid, 15; potassium iodide, 1.62; manganese oxide, 99; zinc oxide, 198; sodium selenite, 0.39; cobalt sulfate·7H2O, 0.72; copper sulfate·5H2O, 63; iron carbonate, 342; butylated hydroxytoluene, 0.375.3.03.0Cholesterol2.82.8Dry matter g/kgNutrient contentDry matter (g/kg)911.9928.6Ashes74.979.0Crude protein (N × 6.25)163.7155.1Crude fat96.593.4Gross energy (MJ/kg DM)17.817.6Total dietary fiber84.0150.0Soluble dietary fiber1.05.0Insoluble dietary fiber83.0145.0Cholesterol (g/kg)2.82.7Phytosterols (g/kg)0.3bComposed of 0.2 g of β-sitosterol, 0.05 g of campesterol, and 0.05 g/kg stigmasterol.0.9cComposed of 0.3 g of β-sitosterol, 0.2 g of campesterol, 0.1 g of stigmasterol, and 0.3 g/kg other phytosterols.BL, blue lupin seed diet; CAS, casein diet.a Supplied (mg/kg diet): retinol, 30; cholecalciferol, 5.4; dl-α-tocopherol, 22.5; thiamine, 0.75; riboflavin, 4.5; cyanocobalamin, 0.023; biotin, 0.15; menadione, 1.5; folic acid, 0.75; nicotinic acid, 30; pantothenic acid, 15; potassium iodide, 1.62; manganese oxide, 99; zinc oxide, 198; sodium selenite, 0.39; cobalt sulfate·7H2O, 0.72; copper sulfate·5H2O, 63; iron carbonate, 342; butylated hydroxytoluene, 0.375.b Composed of 0.2 g of β-sitosterol, 0.05 g of campesterol, and 0.05 g/kg stigmasterol.c Composed of 0.3 g of β-sitosterol, 0.2 g of campesterol, 0.1 g of stigmasterol, and 0.3 g/kg other phytosterols. Open table in a new tab Experimental design and sampling proceduresAfter a postweaning period consuming a commercial diet (S801; Rações Veríssimo, Leiria, Portugal), the pigs (n = 24) were made hypercholesterolemic by feeding the CAS for 2 weeks. At the beginning of week 3, 12 pigs underwent an end-to-side ileorectal anastomosis procedure (19Green S.A. A note on amino acid digestibility measured in pigs with pre- or post-valve ileo-rectal anastomoses, fed soyabean, pea and meat meals.Anim. Prod. 1988; 47: 317-320Google Scholar) ∼40 mm before the ileocecal valve and without removing the hindgut. The ileorectal anastomosed (IRA) pigs were supplemented daily with sodium chloride (20 g) and sodium bicarbonate (20 g) to prevent low mineral absorption. From week 3 onward, six intact (INT) and six IRA pigs were fed the BL, whereas six INT and six IRA pigs continued to consume the CAS.During week 5, all of the pigs were subjected to 5 d total feces, ileal digesta, and urine collection. Feces were collected twice per day, and ileal digesta was collected at 3 h intervals. Urine was collected daily in plastic containers with sulfuric acid to prevent nitrogen loss. Individual samples of diet refusals, feces, ileal digesta, and urine were stored (−20°C) until analyses.At the end of week 5 and after a ∼10 h food deprivation period, the pigs were killed by electronarcosis and bleeding. Blood samples were taken by cardiac puncture. Plasma was obtained by immediate centrifugation (20 min at 4°C and 1,500 g) (J-6B; Beckman, Buckinghamshire, UK) and frozen (−80°C) (UF460; Heto, Brondby, Denmark) until analysis. Pigs were eviscerated immediately after slaughter, and their organs were washed with physiologic saline and weighed. The liver was apportioned for the preparation of fresh cellular fractions (1 g) or for storage (±20 g at −80°C) under vacuum until analysis. Gallbladders and their contents were stored using similar procedures.Diet, feces, and ileal digesta analysesThe compositions of the experimental diets are shown in Table 1. Total, soluble, and insoluble dietary fibers were analyzed according to Association of Official Analytical Chemists Official Method 985.29 (20Association of Official Analytical ChemistsAOAC official method 985.29. Total dietary fiber in foods—enzymatic-gravimetric method.in: Official Methods of Analysis. 16th edition. AOAC International, Gaithersburg, MD1995: 18-19Google Scholar). Total alkaloids from the blue lupin seeds were determined according to European Commission directive 71/250/EEC (21EEC.Commission directive 71/250/EEC establishing Community methods of analysis for the official control of feedingstuffs.Official Journal of the European Communities. 1971; L155: 13-37Google Scholar). Diet total cholesterol and phytosterols were determined by gas-liquid chromatography (GLC) as described below for fecal and ileal digesta sterols.Lyophilized feces and ileal digesta samples (2 g) homogenized in distilled water (20 ml) were extracted with ethanol for 48 h in a Soxhlet apparatus before the addition of known amounts (20 μl) of [14C]taurocholate sodium salt. Lipid fractions were saponified for 2 h in boiling ethanolic potassium hydroxide (2 mol/l). Neutral sterols were extracted with petroleum ether. Bile acids in the aqueous phase were deconjugated (22Grundy S.M. Ahrens E.H. Miettinen T.A. Quantitative isolation and gas liquid chromatography analysis of total fecal bile acids.J. Lipid Res. 1965; 6: 397-410Google Scholar) and extracted with diethyl ether, and 14C radioactivity was measured by liquid scintillation in a Tri-carb analyzer (Packard, Rungis, France) to account for procedure losses. The neutral sterols and free bile acids were prepared for analysis by GLC (23Férézou J. Riottot M. Sérougne C. Cohen-Solal C. Catala I. Alquier C. Parquet M. Juste C. Lafont H. Mathé D. et al.Hypocholesterolemic action of β-cyclodextrin and its effects on cholesterol metabolism in pigs fed a cholesterol-enriched diet.J. Lipid Res. 1997; 38: 86-100Google Scholar), using cholestane as an external standard. The assays were done in a Carlo-Erba HRGC 5160 chromatograph (Thermoquest, Les Ulis, France), equipped with a standard fused silica WCOT capillary column (length, 25 m; film thickness, 0.2 μm) cross-linked with OV101 (Spiral, Dijon, France) for sterols and with OV1701 silicone (Spiral) for bile acids, according to the conditions described by Riottot et al. (24Riottot M. Olivier P. Huet A. Caboche J.J. Parquet M. Khallou J. Lutton C. Hypolipidemic effects of β-cyclodextrin in the hamster and in the genetically hypercholesterolemic Rico rat.Lipids. 1993; 28: 181-188Google Scholar). Daily neutral sterol and bile acid outputs were calculated after correction for fecal and ileal flow, on the basis of a theoretical 90% recovery of dietary β-sitosterol, a reliable marker in pigs (25Marsh A. Kim D.N. Lee K.T. Reiner J.M. Thomas W.A. Cholesterol turnover, synthesis, and retention in hypercholesterolemic growing swine.J. Lipid Res. 1972; 13: 600-615Google Scholar).Plasma and lipoprotein analysesThe determination of plasma levels of triacylglycerols and phospholipids was made with enzymatic kits and an automatic analyzer (704; Hitachi, Tokyo, Japan). Free and total cholesterol were measured with enzymatic kits in an ultraviolet/visible spectrophotometer (DU-530; Beckman, Fullerton, CA) and a Hitachi 917 automatic analyzer, respectively. Plasma LDL-cholesterol (26Nakamura M. Tanigichi Y. Yamamoto M. Hino K. Manabe M. Homogenous assay of serum LDL-cholesterol on an automatic analyzer.Clin. Chem. 1997; 43: 260-261Google Scholar) and HDL-cholesterol (27Sugiuchi H. Uji Y. Okabe H. Irie T. Uekama K. Kayahara N. Miyauchi K. Direct measurement of high-density lipoprotein cholesterol in serum with polyethylene glycol-modified enzymes and sulphated α-cyclodextrin.Clin. Chem. 1995; 41: 717-723Google Scholar) concentrations were measured with direct enzymatic kits using a Hitachi 917 analyzer.Liver analysesLiver lipidsTotal liver lipids were extracted from frozen samples (0.5 g) (23Férézou J. Riottot M. Sérougne C. Cohen-Solal C. Catala I. Alquier C. Parquet M. Juste C. Lafont H. Mathé D. et al.Hypocholesterolemic action of β-cyclodextrin and its effects on cholesterol metabolism in pigs fed a cholesterol-enriched diet.J. Lipid Res. 1997; 38: 86-100Google Scholar), and free and total cholesterol were measured in propanol-2 extracts, as described above. Esterified cholesterol was calculated as the difference between total and free cholesterol. Triacylglycerols and phospholipids were also determined in a Hitachi 917 analyzer, as described above.Liver cellular fraction and enzymatic assaysMitochondrial and microsomal fractions were prepared from fresh liver samples (1 g) (28Souidi M. Parquet M. Férézou J. Lutton C. Modulation of cholesterol 7α-hydroxylase and sterol 27-hydroxylase activities by steroids and physiological conditions in the hamster liver.Life Sci. 1999; 64: 1585-1593Google Scholar). The microsomal fraction for the assay of HMG-CoA reductase activity was suspended in a modified buffer with 10 mmol/l DTT. The protein content from cellular fractions was determined (29Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. Protein measurement with the Folin phenol reagent.J. Biol. Chem. 1951; 192: 265-275Google Scholar) using BSA as a standard.Microsomal HMG-CoA reductase (EC 1.1.1.34) activity was assayed by the radioisotopic technique of Philipp and Shapiro (30Philipp B.W. Shapiro D.J. Improved method for the assay and activation of hydroxy-3-methylglutaryl CoA reductase.J. Lipid Res. 1979; 20: 588-593Google Scholar) with minor adjustments in the preincubation time with phosphatase (60 min at 37°C) and in the incubation time after the addition of [14C]HMG-CoA and NADPH (30 min at 37°C). The microsomal cholesterol 7α-hydroxylase (CYP7A1; EC 1.14.13.17) and mitochondrial sterol 27-hydroxylase (CYP27A1; EC 1.14.13.15) activities were assayed according to Souidi, Parquet, and Lutton (31Souidi M. Parquet M. Lutton C. Improved assay of hepatic microsomal cholesterol 7α-hydroxylase activity by the use of hydroxypropyl β-cyclodextrin and an NADPH-regenerating system.Clin. Chim. Acta. 1998; 269: 201-217Google Scholar) and Souidi et al. (28Souidi M. Parquet M. Férézou J. Lutton C. Modulation of cholesterol 7α-hydroxylase and sterol 27-hydroxylase activities by steroids and physiological conditions in the hamster liver.Life Sci. 1999; 64: 1585-1593Google Scholar), respectively.ImmunoassaysTotal membranes were prepared from frozen (−80°C) liver samples (1 g) according to Kovanen, Brown, and Goldstein (32Kovanen P.T. Brown M.S. Goldstein J.L. Increased binding of low density lipoprotein to liver membranes from rats treated with 17α-ethinyl estradiol.J. Biol. Chem. 1979; 254: 11367-11373Google Scholar). Membrane proteins, solubilized in a buffer containing Triton X-100 (2%) (33Schneider W.J. Beisiegel U. Goldstein J.L. Brown M.S. Purification of the low density lipoprotein receptor, an acidic glycoprotein of 164,000 molecular weight.J. Biol. Chem. 1982; 257: 2664-2673Google Scholar), were assayed (29Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. Protein measurement with the Folin phenol reagent.J. Biol. Chem. 1951; 192: 265-275Google Scholar) using BSA as a standard. The immunodetection of LDL receptors was done as described previously (34Martins J.M. Riottot M. de Abreu M.C. Lança M.J. Viegas-Crespo A.M. Almeida J.A. Freire J.B. Bento O.P. Dietary raw peas (Pisum sativum L.) reduce plasma total and LDL cholesterol and hepatic esterified cholesterol in intact and ileorectal anastomosed pigs fed cholesterol-rich diets.J. Nutr. 2004; 134: 3305-3312Google Scholar). Relative LDL receptor contents were expressed in arbitrary units per milligram of protein and arbitrary units per organ. The linearity of the response as a function of the protein quantity spotted was verified. Specific antibodies raised against the LDL receptor gave a unique band on Western blots with apparent molecular masses of ∼130 kDa.Gallbladder bile analysesBile total lipids were extracted into propanol-2 according to Férézou et al. (23Férézou J. Riottot M. Sérougne C. Cohen-Solal C. Catala I. Alquier C. Parquet M. Juste C. Lafont H. Mathé D. et al.Hypocholesterolemic action of β-cyclodextrin and its effects on cholesterol metabolism in pigs fed a cholesterol-enriched diet.J. Lipid Res. 1997; 38: 86-100Google Scholar), and bile total cholesterol and phospholipids were measured using enzymatic kits and a Beckman DU-530 spectrophotometer.Bile samples were diluted (1:1) into propanol-2, and the total bile acid concentrations were determined by the method of Turley and Dietschy (35Turley S.D. Dietschy J.M. Reevaluation of the 3α-hydroxysteroid dehydrogenase assay for total bile acids in bile.J. Lipid Res. 1978; 19: 924-928Google Scholar) in a Uvicon 930 ultraviolet/visible spectrophotometer (Kontron Instruments, Ltd., Watford, Hertfordshire, UK). The individual bile acid concentrations were assayed by GLC as described previously (24Riottot M. Olivier P. Huet A. Caboche J.J. Parquet M. Khallou J. Lutton C. Hypolipidemic effects of β-cyclodextrin in the hamster and in the genetically hypercholesterolemic Rico rat.Lipids. 1993; 28: 181-188Google Scholar). The lithogenic indices were calculated according to Carey (36Carey M.C. Critical tables for calculating the cholesterol saturation of native bile.J. Lipid Res. 1978; 19: 945-956Google Scholar).Calculations and data analysesResults are presented as means ± SEM. Statistical analysis was performed by two-way ANOVA for diet and IRA effects using the software package Statview 5.0 (SAS Institute, Inc., Cary, NC).RESULTSPhysiological and organ weight dataPigs remained in good health throughout the experimental period. Daily food intake was higher (P < 0.05) in BL- than in CAS-fed pigs, but it did not influence the daily weight gain and the final body weight of pigs (Table 2). Total dietary fiber intake was also higher (P < 0.001) in BL- than in CAS-fed pigs, but cholesterol intake was not different between treatments. Surgery had no significant effect on these parameters. Finally, relative liver weights were lighter (P < 0.05) in BL- than in CAS-fed pigs.TABLE 2Physiological data and relative organ weights in INT and IRT pigs fed cholesterol-rich CAS or BL for 3 weeksCASBLANOVAWeight and IntakeINTIRAINTIRADietIRAFinal weight (kg)41.8 ± 3.438.0 ± 3.043.7 ± 3.042.3 ± 1.1NSNSDaily weight gain (g/day)aValues reported to the 5 day collection period.537.3 ± 127.7390.7 ± 76.8606.5 ± 111.5458.8 ± 98.7NSNSDaily food intake (g DM/day/kg BW)aValues reported to the 5 day collection period.37.0 ± 3.137.9 ± 1.843.6 ± 2.642.2 ± 1.6*NSTotal dietary fiber intake (g/day/kg BW)aValues reported to the 5 day collection period.3.39 ± 0.193.31 ± 0.216.55 ± 0.406.65 ± 0.31***NSCholesterol intake (g/day/kg BW)aValues reported to the 5 day collection period.0.11 ± 0.010.10 ± 0.010.11 ± 0.010.11 ± 0.01NSNSLiver (g/kg BW)21.7 ± 0.621.5 ± 1.019.6 ± 1.318.5 ± 0.9*NSGallbladder (g/kg BW)0.8 ± 0.20.8 ± 0.11.0 ± 0.21.0 ± 0.2NSNSBW, body weight; INT, intact; IRA, ileorectal anastomosed. Values shown are means ± SEM (n = 6, except for CAS-IRA, where n = 5). Significance: * P < 0.05; *** P < 0.001. No significant interactions between diet and IRA effects were recorded.a Values reported to the 5 day collection period. Open table in a new tab Fasting plasma lipids and lipoproteinsPlasma total cholesterol concentration was decreased (P < 0.01) in BL- compared with CAS-fed pigs because of a lower (P < 0.01) LDL-cholesterol concentration (Table 3). The LDL- to HDL-cholesterol ratio was also lower (P < 0.05) in BL-fed pigs. A tendency to lower LDL-cholesterol (P = 0.07) in IRA compared with INT pigs was also observed. All other lipid parameters were unaffected by the treatments (Table 3).TABLE 3Plasma and lipoprotein lipid concentrations in INT and IRA pigs fed cholesterol-rich CAS or BL for 3 weeksCASBLANOVALipidINTIRAINTIRADietIRAmmol/lTriacylglycerols0.65 ± 0.040.62 ± 0.050.63 ± 0.120.77 ± 0.11NSNSPhospholipids1.86 ± 0.191.80 ± 0.191.80 ± 0.181.77 ± 0.16NSNSFree cholesterol0.73 ± 0.060.64 ± 0.050.70 ± 0.100.72 ± 0.09NSNSTotal cholesterol4.52 ± 0.223.74 ± 0.273.20 ± 0.383.10 ± 0.36**NSLDL-cholesterol2.69 ± 0.161.95 ± 0.281.67 ± 0.201.62 ± 0.14**0.07HDL-cholesterol1.36 ± 0.091.33 ± 0.141.18 ± 0.161.22 ± 0.20NSNSratioLDL/HDLaLDL- to HDL-cholesterol ratio.2.01 ± 0.141.63 ± 0.291.45 ± 0.081.42 ± 0.14*NSValues shown are means ± SEM (n = 6, except for CAS-IRA, where n = 5). Significance: * P < 0.05; ** P < 0.01. No significant interactions between diet and IRA effects were recorded.a LDL- to HDL-cholesterol ratio. Open table in a new tab Liver lipids, enzymatic activities, and LDL receptor abundanceThe BL tended to decrease (P = 0.07) hepatic free cholesterol and decreased (P < 0.001) esterified and total cholesterol levels (−56% and −20%, respectively) compared with the CAS, but surgery did not affect the liver lipids (Table 4). HMG-CoA reductase specific and total activities were 16- and 20-fold higher, respectively (P < 0.001), in BL- than in CAS-fed pigs. CYP7A1 activities were not affected by diet and surgery, but CYP27A1 total activity was lower (P = 0.05) in IRA than in INT pigs. Finally, LDL receptor level per liver was ∼2-fold higher (P < 0.001) in BL- than in CAS-fed pigs.TABLE 4Liver lipid concentrations, enzymatic activities, and LDL receptor abundance in INT and IRA pigs fed cholesterol-rich CAS or BL for 3 weeksCASBLANOVAParameterINTIRAINTIRADietIRAmg/gFree cholesterol 4.13 ± 0.22 4.06 ± 0.10 3.69 ± 0.20 3.75 ± 0.240.07NSEsterified cholesterol 1.16 ± 0.07 1.07 ± 0.09 0.47 ± 0.08 0.51 ± 0.02***NSTotal cholesterol 5.29 ± 0.25 5.13 ± 0.14 4.16 ± 0.20 4.26 ± 0.24***NSTriacylglycerols 10.75 ± 1.58 9.96 ± 0.60 11.35 ± 1.08 10.79 ± 1.92NSNSPhospholipids 20.99 ± 0.70 20.78 ± 0.24 20.28 ± 0.37 20.83 ± 0.69NSNSpmol/min/mg proteinHMG-CoA reductase 2.05 ± 0.37 1.80 ± 0.39 40.74 ± 10.30 20.55 ± 4.93***NS10,736 ± 1,336aThese values are in pmol/min/organ
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