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Laboratório de Metabolismo Animal, ICAM/Universidade de Évora, 7002-554 Évora, Portugal;
* Laboratoire d’Endocrinologie de la Nutrition-INRA, Université Paris Sud, 91405 Orsay Cedex, France;
Departamento de Zootecnia, Universidade de Évora, 7002-554 Évora, Portugal;
** CBA/Departamento de Biologia Animal, Faculdade de Ciências de Lisboa, 1740-016 Lisboa, Portugal; and
Departamento de Produção Agrícola e Animal, Instituto Superior de Agronomia, 1349-017 Lisboa, Portugal
2To whom correspondence should be addressed. E-mail: jmartins{at}uevora.pt.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • raw pea seeds • ileorectal anastomosis • pig • cholesterol metabolism • steroid output
The cholesterol-lowering effects of legumes in humans and animal models were found in several studies (1–4) but in others, these effects were not present (5,6). Several mechanisms were proposed to explain this cholesterol-lowering effect; these involved legume components such as proteins and their amino acid profiles (7,8), lipid fractions, fiber (3), saponins (9,10), and phytosterols (11,12). These components could act through metabolic mechanisms, usually involving plasma amino acid or hormone concentrations (13), PUFA absorption (3), or gastrointestinal mechanisms affecting either the intestinal microflora or the enterohepatic metabolism of steroids (2,14–16).
Peas are important food legumes with a world production exceeded only by soybeans, peanuts, and dry beans. For both humans and animals, dry pea seeds are a potentially rich source of protein and carbohydrates (17). A few studies were undertaken to assess the effect of peas on cholesterol metabolism, using pigs (18,19) as models for humans. This omnivorous animal is considered to be a good model for the study of diet effects on plasma cholesterol and atherosclerosis because of its physiologic and anatomical similarities to humans (20). Although a relation between cholesterolemia and intestinal digestive processes was demonstrated, the role of the hindgut and its microflora is still unclear, in contrast to the known role played by the small intestine (21,22). The microbial transformation of primary into secondary bile acids in the hindgut, for instance, could play an important regulatory role (23) because the absorption of hydrophobic secondary bile acids modulates cholesterol and bile acid synthesis in the liver (24). Thus, the by-pass of the cecum-colon section by an ileorectal anastomosis (IRA)3 allows an analysis of the role of this part of the digestive tract in cholesterol metabolism and steroid output.
The aim of the present study was to evaluate the effects of feeding whole raw pea seeds and the role of the cecum-colon section in the cholesterol metabolism and steroid output of growing pigs fed cholesterol-rich diets. To achieve this, plasma, lipoprotein, liver, bile, and fecal lipid concentrations were measured, as well as the activities of the main hepatic enzymes involved in cholesterol and bile acid synthesis.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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- and 7ß-hydroxycholesterol were synthesized according to Yamasmita et al. (25). Emulsifier-Safe was purchased from Packard Instruments. A polyclonal antibody raised against the bovine adrenal cortex LDL receptor was kindly provided by Paul Roach. An anti-rabbit IgG horseradish peroxidase-linked F(ab')2 fragment (from donkey) and the enhanced chemiluminescence reagent were acquired from Amersham Pharmacia Biotech. Animals, housing, diets, and feeding. Crossbred male pigs [n = 24; 12 wk old; Duroc boars x (Large White x Landrace sows)] from Universidade de Évora with an initial body weight (BW) of 29.8 ± 0.6 kg (mean ± SEM) were individually penned in metabolism cages (60 x 160 cm). The study was carried out in accordance with the regulations and ethical guidelines of the Portuguese Animal Nutrition and Welfare Commission (DGV, Lisboa, Portugal).
Two cholesterol-enriched diets were formulated, a semipurified casein (C) diet and a raw pea seed (RP) diet, with similar amounts of protein, essential amino acids (by supplementing with methionine and tryptophan), and gross energy. In the RP diet, 
60% of the protein supplied by casein in the C diet was replaced by protein from finely ground spring pea dry seeds (Pisum sativum cv. Solara) (Table 1). Cholesterol was solubilized in soybean oil and added to the diets (2.8 g/kg). Pigs were fed at a weekly adjusted daily rate of 50 g/kg BW in 2 equal meals (0830 and 1800 h) and had free access to water throughout the experiment.
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40 mm anterior to the ileocecal valve and the cecum-colon section was not removed. To prevent low mineral absorption, the IRA pigs were daily supplemented with sodium chloride and sodium bicarbonate (20 g each). From wk 3 onward, 6 intact (I) and 6 IRA pigs began to eat the RP diet, whereas 6 I and 6 IRA pigs continued to consume the C diet. During wk 5, total feces, ileal digesta, and urine were collected from the pigs for 5 d. Feces were collected 2 times/d and ileal digesta at 3-h intervals. Urine was collected daily in plastic containers with sulfuric acid to prevent N loss. Diet refusal, feces, ileal digesta, and urine individual samples were stored at –20°C until analyses.
At the end of wk 5 and after a food deprivation period (10 h), the pigs were killed by electronarcosis and bleeding. Blood samples were taken by cardiac puncture and plasma was obtained by centrifugation (20 min at 4°C and 1500 x g). Pigs were eviscerated immediately after slaughter and the organs were washed with physiologic saline and weighed. Samples (1 g) were created for fresh liver cellular fraction preparation to determine the HMG CoA reductase, CYP7A1, and CYP27A1 activities; samples (±20 g) were frozen (–80°C) until the determination of liver lipids and for immunoassays. Similar storage procedures were used for the gallbladder and its contents.
Diet, feces, and ileal digesta analyses. The composition of the experimental diets is given in Table 1. Diet total cholesterol and phytosterols were determined by GLC as described below for fecal and ileal digesta sterols. Freeze-dried 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 and a 2-h saponification in boiling ethanolic 2 mol/L potassium hydroxide. Neutral sterols were extracted with petroleum ether, and bile acids were deconjugated (27), extracted with diethyl ether, and 14C radioactivity measured in a Tri-carb analyzer (Packard) to account for procedural losses of bile acids. The neutral sterols and free bile acids were prepared for analysis by GLC (28) in the presence of cholestane as an external standard. A Carlo-Erba HRGC 5160 chromatograph (Thermoquest) was used, equipped with a standard fused silica WCOT capillary column (length: 25 m; film thickness: 0.2 mm) cross-linked with a OV101 (Spiral) for sterols, or with a OV1701 (Spiral) for bile acids, following the conditions described by Riottot et al. (29). The daily neutral sterol and bile acid outputs were calculated after correction for fecal and ileal flow, on the basis of a 90% theoretical recovery of dietary ß-sitosterol, a reliable marker in pigs (30).
Plasma and lipoprotein analyses. Enzymatic kits and an automatic analyzer (Hitachi 704) were used to determine plasma levels of triacylglycerols (kit 1488872, Roche Diagnostics) and phospholipids (kit MPR2 691844, Roche Diagnostics GmbH). Free cholesterol (kit 274-47109, Wako Chemicals) was measured in a UV/VIS spectrophotometer (Beckman DU-530) and total cholesterol (kit 1491458, Roche Diagnostics) in an automatic analyzer (Hitachi 917). Plasma LDL cholesterol (31) and HDL cholesterol concentrations (32) were determined in an automatic analyzer (Hitachi 917) by direct enzymatic kits (3038777 and 3045935, respectively, Roche Diagnostics).
Liver analyses
Liver lipids. Liver lipids were extracted from frozen samples (0.5 g) (28). Free and total cholesterol were measured in propanol-2 extracts, as described above. The esterified cholesterol was calculated as the difference between total and free cholesterol. Triacylglycerols and phospholipids were also analyzed as described above, in a Hitachi 917 automatic analyzer.
Liver cellular fraction and enzymatic assays. The mitochondrial and microsomal fractions were prepared from fresh liver samples (1 g) (33), using a modified buffer with 10 mmol/L of dithiothreitol to suspend the microsomal fraction for the assay of 3-hydroxy-3-methylglutaryl CoA (HMG CoA) reductase activity. The protein content of cellular fractions was determined (34) using bovine serum albumin as a standard.
Microsomal HMG CoA reductase (EC 1.1.1.34) activity was assayed by the radioisotopic technique of Philipp and Shapiro (35) with minor modifications in the preincubation time with phosphatase (60 min at 37°C) and in the incubation time after adding [14C]HMG CoA and NADPH (30 min at 37°C). Radioisotopic assays for mitochondrial sterol 27-hydroxylase (CYP27A1; EC 1.14.13.15) and microsomal cholesterol 7-hydroxylase (CYP7A1; EC 1.14.13.17) activities were described [(33,36), respectively].
Immunoassays.
Total membranes were prepared from frozen (–80°C) liver samples (1 g) (37). The membrane proteins were solubilized in a buffer with 2% Triton X-100 (38) and quantified (34) using bovine serum albumin as a standard. The liver membrane proteins were diluted in a dilution buffer (Tris-maleate, 125 mmol/L; CaCl2, 2 mmol/L; aprotinin, 200 kIU/L; pH 6) for the immunodetection of LDL receptors. The diluted samples (1 µg in 50 µL) were spotted onto a nitrocellulose membrane using a Dot-blot apparatus (Bio-Rad) and incubated overnight at 4°C in a quenching buffer (Tris-HCl, 60 mmol/L; NaCl, 25 mmol/L; CaCl2, 2 mmol/L; pH 8) with 5% fat-free milk. The membranes were washed 3 times with Tween-Tris buffered saline (TTBS) (NaCl, 500 mmol/L; Tris-base, 250 mmol/L; Tween 20, 0.05%; pH 7.5) and incubated for 90 min in the presence of an antibody against LDL receptors diluted 1:2000 in the incubation buffer (Tris-HCl, 60 mmol/L; NaCl, 25 mmol/L; CaCl2, 2 mmol/L; pH 8) containing 0.1% fat-free milk. The membranes were washed 3 times with TTBS, incubated for 90 min with anti-immunoglobulin antibodies conjugated with horseradish peroxidase diluted at 1:5000; after a new TTBS 3-wash cycle, they were incubated for 1 min with a chemiluminescence reagent. Sensitive films (Hyperfilm, Amersham Pharmacia Biotech) were exposed, developed, and scanned with a laser densitometer Ultroscan 2222 (LKB). The relative LDL receptor content in each spot was estimated by the scan peak height and expressed in arbitrary units/mg protein. The linearity of the response as a function of the protein quantity spotted was verified. The specific antibodies raised against the LDL receptor gave a unique band in Western blots with apparent molecular weights of 130 kDa.
Gallbladder bile analyses. Bile total lipids were extracted into propanol-2 (28), and bile total cholesterol and phospholipids were determined using enzymatic kits and a Beckman DU-530 UV/VIS spectrophotometer.
Bile samples were diluted (1:1) into propanol-2, and the total bile acid concentrations were determined (39) in an Uvicon 930 UV/VIS spectrophotometer (Kontron Instruments). The individual bile acid concentrations were assayed by GLC as described by Riottot et al. (29). The lithogenic indices were calculated according to Thomas and Hofmann (40) and Carey (41).
Calculation and data analyses. Results are presented as means ± SEM. Statistical analyses were performed by a two-way ANOVA for diet and IRA effects with the statistical software Statview 5.0 (SAS Institute). When the interaction was significant, a Tukey-Kramer multiple comparison test was used as a post-hoc test. Differences were considered significant when P < 0.05.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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-hydroxylated derivative of chenodeoxycholic acid, was the major primary bile acid and was markedly increased (P < 0.01) by the RP diet and by the surgery. The cecum-colon by-pass markedly reduced (P < 0.01) the secondary bile acids, mainly hyodeoxycholic acid, leading to an increase (P < 0.001) in the primary to secondary bile acid ratio. The hydrophobic bile acid fraction of the total bile acids (chenodeoxycholic, deoxycholic, lithocholic, 3-hydroxy-6-oxo-5ß-cholanoic, 7-oxo-lithocholic acids and other ketones), which represented 33% of these acids, was not modified by diet or surgery.
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Fecal and ileal digesta steroid output.
Diet and IRA did not modify the daily total neutral sterol and cholesterol output of the pigs (Table 6), but surgery markedly reduced (P < 0.01) the cholesterol microbial transformation into coprostanol and epicoprostanol. The same pattern was observed in the microbial transformation of dietary ß-sitosterol (data not shown). The daily total bile acid output was 1.4- and 2.5-fold higher (P < 0.01) in RP-I and RP-IRA than in C-I and C-IRA pigs, respectively. However, surgery drastically reduced (P < 0.01) the fecal excretion of bile acids, which represented 32% of that of I pigs. Primary bile acid (chenodeoxycholic and hyocholic) output was almost 5-fold higher in RP- than in C-fed pigs, primarily because of higher (P < 0.01) hyocholic acid output in the I pigs. The bulk of the bile acid output was mainly secondary bile acids, which represented 75 and 90% of the total bile acids in RP- and C-fed pigs, respectively. Surgery markedly reduced the microbial transformation of bile acids. Hyodeoxycholic acid output, the main secondary bile acid, was lower (P < 0.01) in IRA than in I pigs, representing 20 and 59%, respectively, of the total bile acids. As a result, the primary to secondary bile acid output ratio was markedly increased (P < 0.01) in IRA pigs. Finally, the total neutral and acidic steroid output was 50% higher (P < 0.05) in RP- than in C-fed pigs.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The cholesterolemia in pigs fed the cholesterol-rich C diet was 52% higher than that in similar crossbred pigs fed a diet with no added cholesterol (2.64 ± 0.08 mmol/L, unpublished data) but close to that reported in pigs fed a 3 g/kg cholesterol-rich diet (28). Compared with the C diet, the RP diet lowered the plasma total cholesterol (–22%) through a reduction in LDL cholesterol (–24%). Similar results were observed by feeding pea seeds in cholesterol-free (43) and cholesterol-rich diets to rats (4,43,44) and pigs (19), or pea fiber to normocholesterolemic humans (45). The LDL cholesterol reduction could result from a lower LDL synthesis (by conversion of VLDL into LDL or by LDL hepatic synthesis) and/or from an increased LDL catabolism (1,46). The latter process was likely involved in the observed hypocholesterolemic effect because the hepatic LDL receptor activity was 1.6-fold higher in RP- than in C-fed pigs.
The dietary cholesterol intake was very similar among the 4 groups. Cholesterol synthesis in C-fed pigs (assayed by hepatic HMG CoA reductase activity) did not differ from that observed in pigs fed a cholesterol-rich diet, although it was lower than that in pigs fed a cholesterol-free diet (28). Compared with the C-fed pigs, there was a 2-fold higher HMG CoA reductase activity in pigs fed the RP diet. The elimination of neutral sterols did not differ among dietary treatments. This elimination was also not affected by the cecum-colon by-pass, confirming that the cholesterol absorption occurs largely in the small intestine. Meanwhile, the bile acid output was 1.6-fold higher in RP- than in C-fed pigs. This increase was probably modulated by undigested components such as the pea soluble nonstarch polysaccharides (NSPs), through bile acid binding affinities or sequestration in gels (47,48). The 50% higher hemicellulose intake (calculated as the difference between NDF and acid detergent fiber intake) in RP-fed pigs supports this suggestion. Other pea bioactive components such as saponins and their high content of resistant starch [reviewed by Guillon and Champ (49)] could also have had a role in the increased bile acid output. The fecal output of bile acids may have been balanced by their hepatic synthesis from cholesterol (50). This seems to agree with the lower hepatic cholesterol storage, the upregulation of the LDL receptor, and the higher HMG CoA reductase activity in the RP-fed pigs.
Our data showed that the higher bile acid output in RP- than in C-fed pigs was not associated with increased bile acid synthesis (as estimated by the activities of CYP7A1 and CYP27A1). Nevertheless, the activity of hepatic CYP4A21, one of the key enzymes in bile acid synthesis in pigs, was not measured in this study. This enzyme is involved in the 6-hydroxylation of the chenodeoxycholic into hyocholic acid (51) and its activity in the RP-fed pigs could have been increased, as suggested by: 1) the 50% higher biliary content in hyocholic acid; and 2) the 6-fold higher elimination of this bile acid in the feces. This discrepancy between output and synthesis of bile acids is probably an artifact that resulted from not measuring the activity of CYP4A21. Moreover, the by-pass of the cecum-colon, a site of free bile acid absorption (52), should have increased the fecal output of bile acids as observed with the RP diet. Yet, IRA pigs unexpectedly excreted less bile acids than did I pigs. This reduction in the output of bile acids could have been due to their higher small intestinal absorption or to a decrease in their hepatic synthesis. This latter proposal was not supported by the activities of CYP7A1 and CYP27A1, which were similar in IRA and I pigs. Therefore, surgery probably influenced the reabsorption of bile acids through morphometric changes in the small intestinal mucosa, as previously suggested by Laplace (53). In fact, compared with I pigs, IRA pigs had an increased absorptive area in the mid-distal section of the small intestine [see (42)], a section in which bile acid reabsorption is very important (52,54). This reabsorption in IRA pigs could have been strengthened by 2 factors: 1) a higher content of trihydroxylated hyocholic acid, which is actively absorbed in the intestine (55), and 2) a lower effect of the microflora on bile acids, reducing their deconjugation and dehydroxylation and thus increasing their absorption by the intestinal bile acid transporter (56). Moreover, the RP diet and its resistant starch content could also have helped in stimulating the bile acid reabsorption as observed in rats fed amylomaize starch (57). In terms of sterol balance, the difference between the daily cholesterol input and output could represent the amount of cholesterol deposited daily in tissues (30). Taking into account the daily cholesterol intake, the estimated rate of cholesterol synthesis was higher overall in the RP- than in the C-fed pigs. These calculations, although based on theoretical assumptions, support the higher steroid output and HMG CoA reductase activity observed in the legume-fed pigs. The 50% higher total neutral and acidic steroid daily output in RP- than in C-fed pigs could thus explain the hypocholesterolemic effect of the pea diet. In fact, as previously observed by Férézou et al. (28), a 100% increase in the steroid output was required to reduce the plasma cholesterol by 50%.
The mechanisms by which the pea seeds influenced the steroid metabolism were not confined to the increased bile acid output. In fact, the RP-fed pigs had a biliary bile acid concentration 40% higher than the C-fed pigs. These pigs, food-deprived for 10 h before slaughter, probably had the majority of their bile acid pool in the gallbladder, as observed previously in hamsters (58). Therefore, when estimating the gallbladder bile acid pool at slaughter, this pool was 1.5-fold higher in the RP group than in the C group (14.4 vs. 5.8 mmol, respectively). This effect, also observed in humans consuming legume-based diets (59), could have been due to a higher intestinal bile acid absorption rate in the RP-fed pigs, probably due to the same mechanisms (higher absorptive area and bile content in hyocholic acid) previously suggested for the increased intestinal bile acid reabsorption in IRA pigs. The negligible effect of this enlarged bile acid pool on hepatic acidic steroid synthesis, which is modulated by hydrophobic bile acids (24), could be due to the predominantly hydrophilic bile acid profile of pigs.
Both diets induced similar microbial transformations of cholesterol and ß-sitosterol, as determined in feces. In the RP-I and C-I pigs, the microbial transformation of cholesterol (11 and 16% of the total neutral sterols, respectively) and ß-sitosterol (24 and 32% of the total ß-sitosterol) was low. This fact may have been because the microflora were encompassed by an excess of cholesterol in the intestinal lumen, as previously observed in pigs fed cholesterol-rich diets (28,30). Surgery almost completely prevented the microbial transformation of cholesterol and ß-sitosterol. The reduction in microbial transformation of bile acids was not as high as that for cholesterol, but it was nevertheless very important. In fact, the secondary bile acids of the RP-IRA and C-IRA pigs reached 45 and 62% of the total bile acid output and represented 7 and 10% of the biliary bile acids, respectively. These results agree with those reported in ileoanal anastomosed patients (60) and suggest that the distal part of the small intestine of our pigs had a microbial population that was able to deconjugate and dehydroxylate bile acids.
Cecum and colon by-pass did not modify cholesterol metabolism even though it reduced the microbial transformation of steroids and probably exerted a trophic effect on the small intestinal epithelium. Thus, under the conditions here, the role of the hindgut in cholesterol metabolism was negligible. In contrast, feeding whole pea seeds to pigs for 3 wk reduced plasma cholesterol, probably through 2 mechanisms: 1) a higher bile acid output, most likely modulated by pea undigested components (soluble NSP, resistant starch, and saponins); and 2) an increased hydrophilic bile acid pool. These 2 different effects are the result of the highly complex pea composition; further studies of the fractioned legume’s bioactive components will be pursued to understand their mode of action more fully.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations used: BW, body weight; C, casein; CYP7A1, cholesterol 7-hydroxylase; CYP27A1, sterol 27-hydroxylase; HMG CoA, 3-hydroxy-3-methylglutaryl CoA; I, intact; IRA, ileorectal anastomosis; NDF, neutral detergent fiber; NSP, nonstarch polysaccharide; RP, raw pea seed; TTBS, Tween-Tris buffered saline.
Manuscript received 21 May 2004. Initial review completed 9 August 2004. Revision accepted 10 September 2004.
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LITERATURE CITED |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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