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INTRODUCTION |
A recent report from the American Heart Association (AHA)4 shows that over 30% of Americans in the 45- to 55-y-old age group have blood cholesterol levels equal to or greater than 6.20 mmol/L (240 mg/dL) (Chait et al. 1993
). The initial therapeutic approach for these individuals at increased risk for premature coronary heart disease (CHD) is use of the AHA Step I and Step II diets, which focus on reductions in dietary saturated fat and cholesterol intakes [National Cholesterol Education Program (NCEP) Expert Panel 1993] and increases in unsaturated fats (Mattson and Grundy 1985, McDonald et al. 1989, Mensink and Katan 1989). Most saturated fats (SFA) increase serum total cholesterol (TC) and LDL cholesterol (LDL-C); polyunsaturated fatty acids (PUFA) lower serum cholesterol concentrations (Grundy and Denke 1990, Mattson and Grundy 1985), whereas monounsaturated fats (MUFA) either lower (Grundy and Denke 1990, Mattson and Grundy 1985) or have no effect on plasma TC or LDL-C (Hegsted et al. 1993, Kris-Etherton et al. 1986). For many individuals, the mean reduction of 10% in plasma cholesterol, as noted in the Adult Treatment Panel II report of the NCEP Expert Panel (1993), would not be sufficient to avoid drug intervention. Equally important is that dietary interventions, which focus on reductions in dietary saturated fat and cholesterol, can also reduce HDL cholesterol (HDL-C) concentrations and therefore do not improve the lipoprotein profile, i.e., TC/HDL-C or LDL-C/HDL-C ratios (Barr et al. 1992).
Many reports have shown that consuming a diet high in vegetable vs. animal products reduces plasma cholesterol concentrations and the risk of developing CHD (Anderson et al. 1990 and 1995, Carroll 1991, Meinertz et al. 1989, Terpstra et al. 1984). Dietary plant proteins such as soy protein appear to lower plasma cholesterol concentrations in humans and animals (Anderson et al. 1995, Bakhit et al. 1994, Potter et al. 1993). However, the cholesterol-lowering effect of soy protein has not been consistently observed in all subjects, and may be more pronounced in younger subjects and in hyperlipidemic subjects (Gooderham et al. 1996, Meinertz et al. 1989).
Diets that also contain high amounts of viscous soluble fibers such as pectin or guar gum, as well as viscous nonfermentable fibers such as psyllium lower plasma cholesterol concentrations (Anderson et al. 1990). Diets containing 20-30 g of dietary fiber are not only effective at lowering plasma LDL-C concentrations, but they do so without affecting HDL-C levels (Anderson et al. 1990). Of these dietary fibers, guar gum, a gel-forming galatomannan obtained from Cyamopsis tetragonoloba, has received particular attention because of its consistent plasma cholesterol-lowering effects (Gatenby 1990, Moundras et al. 1997, Todd et al. 1990).
This study was designed to investigate the combined effects of adding soy protein and guar gum to the AHA Step I diet compared with the AAD in a non-human primate model. Soy protein isolate was added at the expense of casein, and guar gum was added at the expense of cellulose. This study was performed on non-human primates, which over several years of study in our laboratory have been shown to be rather sensitive to the effects of dietary fats on serum cholesterol and lipoprotein levels (Brousseau et al. 1993, 1994 and 1995, Nicolosi et al. 1991, Stucchi et al. 1995).
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MATERIALS AND METHODS |
Animals and diets.
Twenty adult female cynomolgus monkeys (Macaca fascicularis), ~5-7 y of age were individually housed in stainless steel cages with free access to food and water. The monkeys were fed a diet that approximated the "Average American Diet" (36% energy as fat, 15% energy as SFA, 15% energy as MUFA and 6% energy as PUFA) (AAD); a diet that approximated the standard Step I diet in which total fat and SFA are lower than in the AAD (30% energy as fat; 9% energy as SFA, 14% energy as MUFA, and 7% energy as PUFA) (standard Step I); or a modified version of the AHA Step I diet, which contains in addition to the 30% fat, a viscous soluble fiber (guar gum) and higher vegetable protein (from soy and corn) content (modified AHA Step I) for a period of 6 wk (Table 1). The monkeys were a part of a larger study in which a crossover-randomized design was used with the exception of the standard Step I diet, which was added to the study at the end as a control diet. Dietary composition of fatty acids of the various vegetable oils were analyzed as previously described (Chong et al. 1987) and are presented in Table 1. After 6 wk of dietary treatment, blood samples were taken from food-deprived monkeys, and the monkeys were given a different dietary treatment for another 6 wk. All monkeys were maintained in accordance with the guidelines of the Committee on Animals of the University of Massachusetts Lowell Research Foundation and NIH guidelines (NRC 1985). In addition, the monkeys were maintained in AAALAC (American Association for the Accreditation of Laboratory Animal Care) accredited facilities, in an environmentally controlled atmosphere (20°C) on a 12-h light:dark cycle.
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Table 1.
Composition of Average American Diet, standard Step I diet and modified AHA Step I diet fed to monkeys for 6 wk1
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Lipid measurements.
Blood from food-deprived monkeys was collected via the saphenous vein at wk 4, 5 and 6 of dietary treatment into heparin-containing tubes; plasma was harvested after low speed centrifugation at 2500 × g for 15 min at 4°C. Plasma cholesterol (Allain et al. 1974) and triacylglycerols (TAG) (Bucolo and David 1973) were measured enzymatically; after the apolipoprotein (apo) B-containing lipoproteins VLDL and LDL were precipitated with phosphotungstate reagent (Weingand and Daggy 1990), the supernatant was assayed for HDL-C (Sigma Chemical, St. Louis, MO). The LDL-C fraction [very low density lipoprotein cholesterol (VLDL-C) + LDL-C] in monkeys was determined by subtracting the HDL-C from the TC. Plasma lipid measurements in our laboratory are standardized by participation in the Center for Disease Control-National Heart, Lung and Blood Institute Lipid Standardization Program.
Statistical analyses.
SigmaStat software (Jandel Scientific, San Rafael, CA) was used for all statistical evaluations. A repeated measures one-way ANOVA (RM ANOVA) was used to analyze all data. When significance was found by RM ANOVA, the Student-Newman-Keuls separation of means was used to determine group differences. All values are expressed as means ± SEM and significance was set at P < 0.05 (Snedecor and Cochran 1980).
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RESULTS |
Animals and diets.
The monkeys adapted well to all diets and all monkeys survived all three dietary treatments. The mean daily food intake over the 6 wk of each dietary period did not differ (data not shown). The mean body weights for the 20 monkeys during each dietary treatment period were AAD, 6.25 ± 0.27 kg; standard Step I, 6.53 ± 0.30 kg; and modified AHA Step I, 6.30 ± 0.36 kg.
Plasma lipid concentrations.
Plasma TC, LDL-C, HDL-C and TAG concentrations were not significantly different in wk 4, 5 and 6 within any of the diet periods; thus the three measurements were averaged and are presented in Table 2. After consuming the standard Step I diet for 6 wk, the monkeys had lower plasma TC (
19%, P < 0.05) and LDL-C (24%, P < 0.09) relative to when they consumed the AAD (Table 2). There were no differences for plasma HDL-C and TAG concentrations, nor were there differences in the TC/HDL-C and LDL-C/HDL-C ratios when the monkeys were fed the AAD and the standard Step I dietary treatments (Table 2). When the monkeys were fed the modified AHA Step I diet, plasma TC and LDL-C were 38% (P < 0.0001) and 55% (P < 0.0001) lower and the TC/HDL-C and LDL-C/HDL-C ratios were 43% (P < 0.003) and 57% (P < 0.003) lower than when they consumed the AAD (Table 2). The key finding is that the modified AHA Step I diet reduced plasma cholesterol beyond the effect of the standard Step I diet, i.e., compared with the standard Step I diet, the modified AHA Step I diet resulted in lower plasma TC and LDL-C concentrations (24%, P < 0.004 and 40%, P < 0.005) and TC/HDL-C and LDL-C/HDL-C ratios (37%, P < 0.02 and 52%, P < 0.02) (Table 2). There were no differences in plasma HDL-C and TAG between the modified AHA Step I diet and the AAD and the standard Step I diet periods (Table 2).
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Table 2.
Plasma lipid concentrations in monkeys after consuming the Average American Diet, standard Step I diet
and modified AHA Step I diet for 6 wk1,2,3
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DISCUSSION |
The primary purpose of this study was to investigate whether plasma lipoprotein cholesterol would respond to modifications of dietary protein and fiber at the dietary fat level of 30%, recommended by guidelines of the AHA (NCEP Expert Panel). Adult female cynomolgus monkeys were chosen as the animal model for this study because when fed hypercholesterolemia-producing diets, they have lipoprotein distribution profiles similar to those observed in humans (Marzetta and Rudel 1986). In addition, cynomolgus monkeys are sensitive to dietary lipid modifications (Hennessy et al. 1992, Marzetta and Rudel 1986), making them a good model for studying the effects of dietary manipulations on plasma lipoprotein cholesterol.
The AHA Step I diet is defined as a diet that contains <30% of energy from fat, <10% of energy from SFA, and <300 mg cholesterol/d (NCEP Expert Panel 1993). The AHA Step I diet is recommended as the first step to lower plasma cholesterol concentrations. In practice, it is variable in its effect on plasma cholesterol, with decreases typically ranging from ~5 to 10% compared with the AAD (NCEP Expert Panel 1993). It also may lower HDL-C concentrations, thereby not improving the lipoprotein profile (Barr et al. 1992). In previous carefully controlled studies (Mattson and Grundy 1985, McDonald et al. 1989, Mensink and Katan 1989), changes in dietary fat composition have resulted in very similar changes in plasma cholesterol concentrations in human subjects and cynomolgus monkeys as was observed in this study with the AAD and standard Step I diet. Numerous studies have also demonstrated that oils containing SFA raise serum TC and LDL-C (Barr et al. 1992, Brousseau et al. 1994, Hegsted et al. 1993). In this monkey study, the AAD contained 15% of fat from SFA, and the standard Step I and modified AHA Step I diets contained 9% SFA. The standard Step I diet reduced plasma TC by 19% and LDL-C by 24%, relative to the AAD. This confirms previous studies (Kuo et al. 1989, Nicolosi et al. 1990, Spady and Dietschy 1985 and 1988) in which unsaturated fatty acids have been shown to prevent the down-regulation of LDL receptor activity normally resulting from saturated fat and cholesterol intake, thus resulting in a lowering of plasma cholesterol concentrations. In contrast, the modified AHA Step I diet reduced plasma TC and LDL-C by 38 and 55%, respectively, and the TC/HDL-C and LDL-C/HDL-C ratios by 43 and 57%, respectively, relative to the AAD (Table 2).
Thus, the standard Step I diet was only ~50-60% as effective as the modified AHA Step I diet at lowering plasma TC and LDL-C concentrations. The modified AHA Step I diet significantly reduced plasma lipids beyond the level of the standard Step I diet, TC and LDL-C by 24 and 40%, respectively, and the TC/HDL-C and LDL-C/HDL-C ratios by 37 and 52%, respectively (Table 2). This additional reduction in plasma cholesterol concentrations with the modified AHA Step I diet is due either to the increase in dietary soy protein and/or guar gum that are contained in the modified AHA Step I diet vs. the AAD and the standard Step I diet.
The lowering of plasma TC and LDL-C by the modified AHA Step I diet in this study is in agreement with previous observations that replacing casein with soy protein in the diet leads to significant reductions in plasma cholesterol concentrations (Anderson et al. 1990 and 1995, Carroll 1991, Meinertz et al. 1989, Terpstra et al. 1984). The cholesterol-lowering mechanism of soy protein is still far from clear. This effect on serum cholesterol concentrations is thought to be due largely to the amino acid composition of soy protein. However, studies in which amino acids patterned after soy protein or casein were fed to experimental animals have suggested that other nonprotein components present in soy may be partially responsible for the hypocholesterolemic effect (Huff et al. 1977, Nagata et al. 1982, Tasker and Potter 1993). There are a number of non-amino acid components or effects of soy products that have been associated with lowering blood lipids (Potter 1995). These include saponins (Topping et al. 1980), protein digestibility (Woodward and Carroll 1985), protein phosphorylation (Samman and Roberts 1984) and phytoestrogens (Anthony et al. 1996 and 1997). In these studies, Anthony et al. (1996 and 1997) showed that alcohol-extracted soy protein, which did not contain phytoestrogens, was less effective at lowering plasma cholesterol levels and preventing the development of atherosclerosis in non-human primates than nonalcohol-washed soy protein diets. In this study, intact soy protein isolate was fed in the AAD and modified AHA Step I diet.
The mechanism(s) by which soy produces its cholesterol-lowering effect is thought to be due to the reduced absorption of steroids, including dietary cholesterol and bile acids, in the gastrointestinal tract, presumably by increasing binding to bile acids and excretion (Benyen 1990, Sugano et al. 1990, Yamashita et al. 1990). The physiologic effect of increased bile acid excretion is that cholesterol is removed from the body. This in turn causes an increase in metabolism of cholesterol for enhanced bile acid synthesis. Endogenous synthesis of cholesterol is increased, as is hepatic LDL receptor activity, the net result of which is increased removal of cholesterol from the blood (Potter 1996). However, in this study, fecal steroid excretion was not measured.
Another cholesterol-lowering effect of the modified AHA Step I diet is the substitution of guar gum for cellulose. In this study, the AAD contained ~8% soy protein, compared with 9% in the modified AHA Step I. This suggests that the reduction in plasma TC and LDL-C by the modified AHA Step I diet was due to the addition of guar gum rather than to the soy protein. Previous studies have shown that diets containing high amounts of viscous soluble fibers such as pectin or guar gum, as well as viscous nonfermentable fibers such as psyllium and modified celluloses lower plasma cholesterol concentrations (Anderson et al. 1990, Fernandez et al. 1995, Gatenby 1990, Moundras et al. 1997, Todd et al. 1990). However, to our knowledge, there is no previous information on the cholesterol-lowering effect of guar gum in non-human primates. Another advantage of soluble fibers is that diets containing 20-30 g of dietary fiber are not only effective at lowering plasma LDL-C concentrations, but they do so without affecting HDL-C levels (Anderson et al. 1990). The mechanisms underlying these effects of guar gum are not fully understood, but one common hypothesis is that guar gum interferes with the intestinal absorption of sterols because of its viscosity or its binding properties (Moundras et al. 1997), leading to uptake of cholesterol from the plasma.
This study suggests that AHA Step I diets supplemented with guar gum and soy protein could lower plasma cholesterol concentrations twice as effectively as the AHA Step I diets that are currently recommended. Whether the cholesterol-lowering effect is the result of soy protein or guar gum or a synergistic effect of both remains to be determined.
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Abstract
Introduction
Abstract
review of the literature. In: Dietary Fibre Perspectives No. 2 (Leeds, T., ed.), pp. 100-115. John Libbey, London, UK.
