Modified Soybean Affects Cholesterol Metabolism in Rats Similarly to a Commercial Cultivar

Abstract

The consumption of soy protein lowers blood cholesterol in humans and animals. Breeding may alter the physiological effects of soybeans, such as its cholesterol-lowering property. Our hypothesis is that breeding affects the hypocholesterolemic effect of soy by modulating the expression of key hepatic enzymes related to cholesterol and bile acid biosynthesis, as well as altering fecal neutral and acidic steroid excretion. Therefore the aim of this study was to evaluate the effect of a new Brazilian soybean cultivar (UFV-116), lacking lipoxygenases 2 and 3, compared with a commercial cultivar (OCEPAR-19), on 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) and cholesterol 7α-hydroxylase (CYP7A) mRNA expression and fecal steroid output in rats. Thirty-six male rats were fed UFV-116, OCEPAR-19, or casein as the protein source, with or without addition of dietary cholesterol (0.25%). Blood and liver cholesterol, HMGR and CYP7A mRNA abundance, and fecal excretion of steroids were measured. Blood and liver cholesterol levels were lowered by both soybean cultivars, with and without cholesterol, but UFV-116 was more effective when included in the cholesterol-free diet. Both soy diets promoted lower levels of HMGR mRNA, higher levels of CYP7A mRNA, and higher excretion of fecal secondary bile acids. There was higher fecal neutral steroid output when cholesterol was added to all diets. These data show that both soybean cultivars acted similarly in lowering serum and hepatic cholesterol; therefore, breeding did not affect the hypocholesterolemic effect of the new cultivar.

Introduction

H igh levels of serum cholesterol are considered one of the most important risk factors for cardiovascular diseases.¹ There is increasing evidence that the consumption of soybean protein, in place of animal protein, lowers blood cholesterol levels. These effects have been recognized in animals for more than 80 years^2

–6 and more recently in humans.^7

–10 It has been demonstrated that the consumption of soy protein significantly lowers total cholesterol, low-density lipoprotein cholesterol, and triacylglycerols without affecting high-density lipoprotein cholesterol.¹¹ However, the effective components of soy and the mechanisms involved in its hypolipidemic actions are not fully understood. Isoflavones are biologically active compounds in soybeans that are closely associated with the proteins that have been studied. Moreover, breeding of the soybean, aiming at enhancing its nutrient values and/or to improve its sensorial properties, may lead to desirable or undesirable physiological effects because those effects cannot be attributed to any particular chemical component.¹²

Additionally, cellular and molecular biology studies have demonstrated that soy components, especially soy protein and isoflavones, modulate key transcription factors involved in the regulation of lipid metabolism. It seems they also modulate their regulated downstream gene expression in animals and in vitro cultured human cells at transcriptional or posttranslational levels,¹⁰ including 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) and cholesterol 7α-hydroxylase (CYP7A) mRNA expression.

Considering all the information about soy and its potential benefits for human health, production and consumption of soy foods within western countries have increased dramatically in the last few decades. However, in Brazil and other western countries, one of the limiting factors that affect soy consumption is its flavor, which some consider to be unappealing. To improve its sensory properties, a new cultivar of soybean (UFV-116) has been developed at the Institute of Biotechnology, Federal University of Viçosa, Viçosa, MG, Brazil, lacking lipoxygenase isozymes 2 and 3. The objective of removing these isozymes was to reduce the production of unappealing flavors that occur during processing because of oxidation of fatty acids.

Our hypothesis is that breeding may affect the hypocholesterolemic effect of soybean by modulating the expression of hepatic key enzymes related to cholesterol and bile acid biosynthesis, subsequently modifying fecal neutral and acidic steroid excretion. Considering that breeding of UFV-116 brought better sensory properties to this new cultivar, it is relevant to study its hypocholesterolemic effect because dietary intervention is one of the most manageable ways of reducing the risk of cardiovascular disease. Additionally, among soy products, the effects of full fat soy flour on cholesterol metabolism have not been sufficiently studied.

Therefore, our objective was to test this hypothesis by evaluating the effect of the UFV-116 flour, added as a protein source, on blood and liver cholesterol levels, HMGR and CYP7A mRNA abundance, and fecal steroid output in rats. In addition, we also tested a commercial cultivar (OCEPAR-19) for comparative effect in normal and hypercholesterolemic diets because it is a crop widely produced and sold in Brazil.

Materials and Methods

Soy flours

The modified cultivar was obtained by classical breeding using backcrossing procedures in which the mutant and recessive genes that determine the loss of lipoxygenase 2 and 3 were transferred from natural mutants to the new cultivar. The natural and not adapted mutants are recessive and transferred as unique and unlinked genes. Seeds from the two soy cultivars were heat-treated, had their hulls removed, and were ground in a hammer mill with a 20-mesh sieve (0.84 mm), and the flours were stored under refrigeration.

Rat study

Thirty-six male Sprague–Dawley rats (Harlan Sprague Dawley, Indianapolis, IN, USA) weighing 90 g were housed individually in a light (12-hour light)- and temperature (24°C)-controlled room and given diet and deionized water ad libitum. After arrival, all animals were fed ground chow for a 1-week stabilization period to adapt to diet and room conditions. Next, animals were randomized into groups of six and fed with experimental diets based on the AIN93M formulation.¹³ The diets contained 14% protein provided by one of the two soy flours, UFV-116 and OCEPAR-19, or casein (Table 1).

Table 1.

Composition of the Experimental Diets

	Content (g/kg of diet)
Ingredient	Casein	UFV-116	OCEPAR-19
Casein (>85% protein)	140.0	0.0	0.0
Soy flour	0.0	350.0	370.0
Cornstarch	420.0	291.1	282.1
Dextrose	155.0	155.0	155.0
Sucrose	100.0	100.0	100.0
Cellulose (fiber)	50.0	42.6	42.6
Soybean oil	85.0	12.0	1.0
Mineral mix	35.0	35.0	35.0
Vitamin mix	10.0	10.0	10.0
l-Cystine	1.8	1.8	1.8
Choline bitartrate	2.5	2.5	2.5

Experimental diets are based on the AIN93-M diet.¹³

All soybean diets had their fiber, lipid, and carbohydrate contents adjusted according to the soy flour composition. Lipids were adjusted for 85 g/kg in each diet to account for the lipid content of soy flours compared with the usual 50 g/kg for the AIN93M diet (Table 1). Each of these diets was fed with and without added 0.25% dietary cholesterol for 28 days: C, casein diet; C⁽⁺⁾, casein diet plus 0.25% cholesterol; U, UFV-116 diet; U⁽⁺⁾, UFV-116 diet plus 0.25% cholesterol; O, OCEPAR-19 diet; and O⁽⁺⁾, OCEPAR-19 diet plus 0.25% cholesterol. Weight gain and feed intake were monitored throughout the experimental period. Feces were collected for a 72-hour period during the final week of the study, lyophilized, and stored at −20°C until analysis of bile acid and neutral steroid concentrations. At the end of the experiment, the rats were euthanized by decapitation while under light anesthesia with CO₂. Blood and liver samples were collected on ice and stored at −20°C. The animal study complied with the Guide for the Care and Use of Laboratory Animals ¹⁴ and was approved by the Purdue University Animal Care and Use Committee.

Cholesterol and steroid analyses

Blood and liver cholesterol concentrations were determined using the o-phthalaldeyde method.¹⁵ Fecal bile acids and neutral steroids were quantified by the method of Chezem and Story.¹⁶ Trimethylsilyl ethers of neutral steroids were quantified using gas–liquid chromatography (Agilent Technologies, Palo Alto, CA, USA) with a 30-m DB-1701 capillary column (JW Scientific, Folsom, CA, USA), whereas quantification of trimethylsilyl ethers of bile acids utilized a 30-m DB-5 capillary column (J&W Scientific).

Northern blot analysis

Total RNA was isolated from 1 g of liver using the guanidium thiocyanate–phenol–chloroform method.¹⁷ Northern blot analysis was performed with standard procedures¹⁸ as modified by Tsang et al.¹⁹ Total mRNA was expressed as arbitrary units of CYP7A or HMGR mRNA per arbitrary unit of 18S rRNA to normalize for differences in loading and transfer efficiency. The plasmids pSK-7a²⁰ (containing a 1.64-kb EcoRI fragment that contained the entire coding region of the rat CYP7A gene), pGEM-HMGR (containing a 1.2-kb EcoRI fragment that contains the entire coding region of the rat HMGR gene), and pDF8²¹ (containing a 1.06-kb BamHI–EcoRI fragment corresponding to the central region of the rat 18S rRNA gene) were used.

Statistical analysis

The experiment was carried out in a completely randomized design with a 3×2 factorial arrangement: three different proteins (from casein diet [C], from UFV-116 diet [U], and from OCEPAR-19 diet [O]) and two levels of dietary cholesterol (0% and 0.25%). The data were analyzed using two-way analysis of variance and contrast analysis. UFV-116 was compared with OCEPAR-19 with and without added cholesterol ([U⁽⁺⁾×O⁽⁺⁾] and [U×O]). The effect of cholesterol addition on both soybean diets was also compared ([U + O]×[U⁽⁺⁾ + O⁽⁺⁾]). Finally, the effect of casein compared with soybean proteins was examined with and without additional cholesterol to verify the effects of both soybean proteins related to an animal protein [C×(U + O)] and [C⁽⁺⁾×(U⁽⁺⁾ + O⁽⁺⁾)]. All analyses were carried out using Statistica software,²² and results were significant when P<.05.

Results

Animals fed soybean protein showed lower body weight and food intake compared with those fed casein. There was no difference among soybean diets with and without additional cholesterol for these parameters. The feed efficiency ratio (g of weight gain/g of food intake) was higher for casein compared with soybean diets when cholesterol was added. UFV-116 showed a higher feed efficiency ratio than OCEPAR-19 (Table 2).

Table 2.

Means, Standard Deviations, and Statistical Contrasts for Food Intake, Weight Gain, and Feed Efficiency Ratio from Rats Fed Soybean Proteins or Casein Diets Supplemented or Not with Cholesterol

Diets	FI (g/day)	WG (g)	FER (%)
C	23.89±1.59	216.67±31.75	29.98±12.08
C⁽⁺⁾	23.94±1.76	225.83±28.43	37.51±4.21
U	22.67±2.03	128.33±24.61	29.22±4.44
U⁽⁺⁾	22.05±1.43	131.00±14.30	32.05±9.67
O	19.27±1.66	91.67±6.83	20.20±4.46
O⁽⁺⁾	19.42±1.58	93.67±13.23	23.11±4.48
Contrasts
U×O	^*	NS	^*
U⁽⁺⁾×O⁽⁺⁾	^*	NS	^*
[U + O]×[U⁽⁺⁾ + O⁽⁺⁾]	NS	NS	NS
C×[U + O]	^*	^*	NS
C⁽⁺⁾×[U⁽⁺⁾+ O⁽⁺⁾]	^*	^*	^*

Significant by contrast analysis (P<.05).

C, casein diet; C⁽⁺⁾, casein diet + 0.25% cholesterol; FER, feed efficiency ratio; FI, food intake; NS, not significant; O, OCEPAR-19 diet; O⁽⁺⁾, OCEPAR-19 diet + 0.25% cholesterol; U, UFV-116 diet; U⁽⁺⁾, UFV-116 diet + 0.25% cholesterol; WG, weight gain.

The UFV-116 diet was more effective in lowering blood cholesterol when cholesterol was not added to the diet. Animals fed a casein diet showed blood and liver cholesterol levels higher than animals fed either soy diet. The C⁽⁺⁾ group had 9.27% more liver cholesterol compared with the C group. The relative and absolute liver weights of rats fed soybean diets were lower, with or without cholesterol added, compared with rats fed a casein diet (Table 3).

Table 3.

Means, Standard Deviations, and Contrasts for Blood Cholesterol, Liver Cholesterol, and Liver Relative Weight from Rats Fed Soybean Proteins or Casein Diets Supplemented or Not with Cholesterol

Diets	BC (mg/dL)	LC (mg/g)	LRW
C	97.02±24.57	6.02±0.28	3.83±0.04
C⁽⁺⁾	106.37±10.63	8.50±2.21	4.10±0.06
U	63.53±3.73	3.95±0.75	3.11±0.11
U⁽⁺⁾	76.89±9.99	4.10±0.38	3.21±0.09
O	67.28±9.17	3.44±0.31	3.14±0.12
O⁽⁺⁾	87.05±9.68	4.07±0.55	3.31±0.11
Contrasts
U×O	^*	NS	NS
U⁽⁺⁾×O⁽⁺⁾	NS	NS	NS
[U + O]×[U⁽⁺⁾ + O⁽⁺⁾]	NS	NS	NS
C×[U + O]	^*	^*	^*
C⁽⁺⁾×[U⁽⁺⁾ + O⁽⁺⁾]	^*	^*	^*

Significant by contrast analysis (P<.05).

BC, blood cholesterol; LC, liver cholesterol; LRW, liver relative weight (liver weight×100/final body weight).

Both soy cultivars lowered HMGR mRNA abundance, with or without cholesterol added to the diet, compared with the casein diet. There was an increase in CYP7A mRNA abundance in rats fed soybean cultivars compared with rats fed a casein diet; however, there were no differences between the two soybean cultivar diet groups (Table 4).

Table 4.

Means, Standard Deviations, and Contrasts for HGMR and CYP7A mRNA in Rats Fed Soybean Proteins or Casein Diets Supplemented or Not with Cholesterol

	Arbitrary units
Diets	HMGR	CYP7A
C	3.27±0.61	0.49±0.36
C⁽⁺⁾	2.48±0.74	0.82±0.19
U	1.92±0.61	1.37±0.27
U⁽⁺⁾	1.59±0.53	1.43±0.61
O	1.97±0.84	1.25±0.61
O⁽⁺⁾	1.71±0.71	1.45±0.49
Contrasts
U×O	NS	NS
U⁽⁺⁾×O⁽⁺⁾	NS	NS
[U + O]×[U⁽⁺⁾ + O⁽⁺⁾]	NS	NS
C×[U + O]	^*	^*
C⁽⁺⁾×[U⁽⁺⁾ + O⁽⁺⁾]	^*	^*

Arbitrary units are expressed in units of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) or cholesterol 7α-hydroxylase (CYP7A) mRNA per unit of 18S rRNA.

Significant by contrast analysis (P<.05).

Fecal neutral and acidic steroid excretion was higher in the rats fed soybeans than in those fed casein and was greater in both groups that were fed the cholesterol-enriched diets compared with the groups without additional cholesterol (P<.05). There was an increase of secondary bile acids in all diets that had added cholesterol, but the difference was significant only when the soy diets were compared with each other. Soybean diets that had added cholesterol led to a higher output of total neutral steroids, and C⁽⁺⁾ promoted the higher excretion of total neutral steroids (Table 5).

Table 5.

Means, Standard Deviations, and Contrasts for Fecal Steroid Excretion of Rats Fed Soybean Proteins or Casein Diets Supplemented or not with Cholesterol

	Level (mg/g of dried feces)
Diets	PBA	SBA	TBA	TNS
C	0.19±0.02	1.73±0.46	1.92±0.11	20.99±3.74
C⁽⁺⁾	1.01±0.09	7.53±1.32	8.54±1.91	72.55±9.06
U	1.08±0.12	3.72±0.74	4.80±1.96	9.70±3.45
U⁽⁺⁾	1.08±0.17	9.43±2.56	10.51±2.05	17.25±4.33
O	1.42±0.54	6.41±1.89	7.83±2.53	14.09±4.21
O⁽⁺⁾	1.19±0.38	10.70±2.97	11.89±3.04	36.31±5.97
Contrasts
U×O	NS	NS	NS	NS
U⁽⁺⁾×O⁽⁺⁾	NS	NS	NS	^*
[U + O]×[U⁽⁺⁾ + O⁽⁺⁾]	NS	^*	^*	^*
C×[U + O]	^*	NS	NS	NS
C⁽⁺⁾×[U⁽⁺⁾ + O⁽⁺⁾]	NS	NS	NS	^*

Significant by contrast analysis (P<.05).

PBA, primary bile acids; SBA, secondary bile acids; TBA, total bile acids; TNS, total neutral steroids.

Discussion

Soy protein has been known for many decades to reduce blood cholesterol;^23
–25 however, the specific components and the mechanisms involved on its hypolipidemic actions are not fully understood. Studies have pointed out different effects on different soy products,²⁶ feeding with the intact protein or amino acid mixtures,²⁷ lysine/arginine ratio and lysine content compared with casein,^28,29 and presence of specific soy peptides,³⁰ among others. Our data showed that UFV-116 was more effective in reducing serum cholesterol. However, previous studies with these cultivars showed that UFV-116 and OCEPAR-19 flours have similar amounts of proteins. Additionally, it seems that breeding did not promote significant changes in the amino acid profile for UFV-116 compared with OCEPAR-19,³¹ so this could not probably be a reason for differences on the cholesterol-lowering effect between the two cultivars.

Indeed, many researchers have pointed out that the alcohol-extractable components of soy proteins, especially isoflavones, are one of the major factors responsible for its cholesterol-lowering effect.³⁰ In a recent meta-analysis, Taku et al.³² showed that soy isoflavones significantly lowered serum total and low-density lipoprotein cholesterol but did not change high-density lipoprotein cholesterol and triacylglycerol in humans. They concluded that, when provided concurrently with soy protein, soy isoflavones would have synergistic or additive effects on the cholesterol-lowering effect. According to Esteves et al.,³¹ the UFV-116 cultivar had higher genistein and daidzein contents than OCEPAR-19. This could suggest that the isoflavone content from that cultivar might have influenced its cholesterol-lowering properties.

HMGR, which catalyzes the reduction of 3-hydroxy-3-methylglutaryl-coenzyme A to mevalonate at an early stage of cholesterol biosynthesis, represents a major target for regulation of the overall pathway. Regulation of cholesterol synthesis is exerted near the beginning of the pathway,³³ and cholesterol supply influences its levels as well as its activity. Madani et al.³⁴ reported that soy protein increases HMGR activity but that adding cholesterol to the diet promoted its reduction. In our study, both cultivars lowered mRNA for HMGR when compared with casein, regardless of the addition of cholesterol to the diets. However, many studies relating soy protein to cholesterol biosynthesis or metabolism have used experimental diets with a soy protein isolate and low lipid levels or no added cholesterol.^35,36 It is important to consider that full fat soy flours, which are high in lipids, were used in the experimental diets, so bioactive compounds can have an influence.

Moreover, isoflavones have been pointed out as inhibitors of HMGR.³⁷ Genistein has been shown to decrease cholesterol synthesis in cultured human hepatoma cells³⁸ and inhibit HMGR activity in MCF-7 human breast cancer cells.³⁹ Consumption of soy isoflavones significantly decreased HMGR mRNA abundance and cholesterol synthesis in the liver of nephrotic rats.⁴⁰ Sung et al.³³ suggested that the hypocholesterolemic effects of soy foods, previously reported in several epidemiological studies, are in part due to the inhibition of HMGR activity by isoflavones. So, the isoflavones could be responsible for reducing HMGR mRNA abundance by both cultivars.

The conversion of cholesterol to bile acids produces sufficient amounts of detergent necessary for the digestion and absorption of lipid nutrients (e.g., triacylglycerols and fat-soluble vitamins) and provides a metabolic pathway through which excess cholesterol can be removed from the body.⁴¹ CYP7A is the only mammalian enzyme capable of initiating the multi-organelle pathway through which cholesterol is converted into bile acids. This enzyme is subjected to feedback regulation by bile acids fluxing through the liver via enterohepatic circulation. Disruption of the enterohepatic circulation of bile acids has been shown to suppress the reabsorption of bile acids, resulting in an up-regulation of CYP7A and leading to an increase in bile acid synthesis from cholesterol.⁴²

In our study, both soybean cultivars increased CYP7A mRNA, regardless of the cholesterol addition. Although several studies have pointed out that cholesterol addition increases both CYP7A activity and mRNA levels^15,36,43 in soy protein diets, we can infer that the presence of several components aside from soy protein may have played an important role in this result.

Studies in rabbits, rats, and monkeys have yielded evidence that soy protein increases fecal excretion of bile acids relative to casein.^44
–46 In our study, the soy diets with added cholesterol promoted increased total bile acid output in a similar way. However, in absolute values, adding cholesterol to the experimental diets led to an extensive increase of these compounds: 442.79% for C⁽⁺⁾, 218.96% for U⁽⁺⁾, and 151.85% for O⁽⁺⁾. Similar results were observed for secondary bile acids: 435.26% for C⁽⁺⁾, 253.49% for U⁽⁺⁾, and 166.93% for O⁽⁺⁾.

These results are well supported by the literature. Chen et al.⁴⁷ suggested that increases in cholesterol and bile acid excretion in feces can decrease the reabsorption of bile acid by the enterohepatic circulation, thereby decreasing the concentration of the bile acid pool and promoting the activity of CYP7A in the liver. These investigators suggested that the increase of bile acid synthesis stimulates cholesterol metabolism in vivo; cholesterol utilization in the liver is increased, and the serum cholesterol concentration is lowered. In rabbits, the measurement of intestinal cholesterol absorption provides evidence that these animals, which are susceptible to hypercholesterolemia, absorb cholesterol more readily when fed casein compared with soy protein. They also excrete less neutral steroids and produce less bile acid than animals fed soy protein. In addition, rabbits fed casein excreted mainly cholesterol, whereas those fed soy protein excreted coprostanol, which is not absorbable and increases the amount of cholesterol excreted, suggesting that soy protein favors the bacterial conversion of cholesterol to coprostanol.⁴⁸

Based on the results of our study, it can be inferred that both soybean cultivars acted to lower serum and hepatic cholesterol in a similar way. This probably occurred by increasing hepatic cholesterol conversion to steroids, raising their fecal output (primarily as bile acids) and reducing liver cholesterol biosynthesis so that the blood levels remained low. Although several studies have reported an increase of cholesterol biosynthesis by soy protein, our study found a reduction, represented by a lower abundance of HMGR mRNA. It is important to clarify that most research studies assessing soy protein effects on cholesterol biosynthesis have tested purified soy protein forms, which excludes the influences of non-protein components. For example, in a recent study, Ronis et al.⁴⁹ demonstrated that feeding prepubertal rats with soy protein isolate containing 3 mg/kg isoflavones resulted in increased expression of hepatic genes regulated by the promiscuous nuclear receptors peroxisome proliferator-activated receptor α, peroxisome proliferator-activated receptor γ, and liver X-receptor α but decreased expression of genes regulated by sterol regulatory element binding protein-1c, which may partially explain the cholesterol-lowering effects of soy. All these transcription factors are related to lipid homeostasis. They also demonstrated that feeding soy protein isolate (3 mg/kg any isoflavone) or isoflavones in the diet had different effects compared with studies accomplished with purified isoflavones in in vitro cell culture models. So, studies of purified soy components in vitro may produce misleading results and need to be interpreted with caution relative to results of feeding whole diets.

Moreover, additional micronutrients, such as phytosterols, trypsin inhibitors, phytic acid, saponins,^37,45,50 ratio of unsaturated to saturated fatty acids,⁵¹ type and amount of carbohydrates,⁵² and the Zn and Cu concentrations,^53,54 are known to have some influence on cholesterol metabolism, having additive or synergistic effects. Still, soluble fiber degradation by intestinal microorganisms produces short-chain fatty acids in the colon, which can inhibit hepatic cholesterol synthesis⁵⁵ and hence reduce blood cholesterol. Therefore, the synergistic effect of these components from the soy flours should be considered on their cholesterol-lowering effects.

In conclusion, our study indicates that both soy cultivars acted similarly in lowering serum and hepatic cholesterol. Therefore, breeding did not affect the hypocholesterolemic effect of the new cultivar. In addition, removal of lipoxygenase 2 and 3 in this cultivar may improve soybean consumption by reducing the beany flavors of soy products and promote health benefits related to heart diseases.

Footnotes

Acknowledgments

This work was supported by FINEP and CAPES-Brazil and the Foods and Nutrition Department, Purdue University, West Lafayette, IN, USA. The authors would like to acknowledge Carol Spahr and Tanya Lodics for their assistance and Dr. Maurílio Alves Moreira for donation of the soybean cultivars.

Author Disclosure Statement

No competing financial interests exist.

References

Gould

, Rossouw

, Santanello

, Heyse

, Furberg

. Cholesterol reduction yields clinical benefit: impact of statin trials. Circulation, 2008; 97:946–952.

Meeker

, Kesten

. Effect of high protein diets on experimental atherosclerosis of rabbits. Arch Pathol, 1941; 31:147–162.

Kurowska

, Carrol

. Hypercholesterolemic responses in rabbits to selected groups of dietary essential amino acids. J Nutr, 1994; 124:364–370.

Lin

, Meijer

, Vermeer

, Trautwein

. Soy protein enhances the cholesterol-lowering effect of plant sterol esters in cholesterol-fed hamsters. J Nutr, 2004; 134:143–148.

Moriyama

, Kishimoto

, Nagai

et al. Soybean beta-conglycinin diet suppresses serum triglyceride levels in normal and genetically obese mice by induction of beta-oxidation, down regulation of fatty acid synthase, and inhibition of triglyceride absorption. Biosci Biotechnol Biochem, 2004; 68:352–359.

Takahashi

, Ide

. Effects of soy protein and isoflavone on hepatic fatty acid synthesis and oxidation and mRNA expression of uncoupling proteins and peroxisome proliferator-activated receptor gamma in adipose tissues of rats. J Nutr Biochem, 2008; 19:682–693.

Teixeira

, Potter

, Weigel

, Hannum

, Erdman

Jr , Hasler

. Effects of feeding 4 levels of soy protein for 3 and 6 wk on blood lipids and apolipoproteins in moderately hypercholesterolemic men. Am J Clin Nutr, 2000; 71:1077–1084.

Puska

, Korpelainen

, Hoie

, Skovlund

, Lahti

, Smerud

. Soy in hypercholesterolaemia: a double-blind, placebo-controlled trial. Eur J Clin Nutr, 2002; 56:352–357.

West

, Hilpery

, Juturu

et al. Effects of including soy protein in a blood cholesterol lowering diet on markers of cardiac risk in men, and postmenopausal women with or without hormone replacement therapy. J Womens Health, 2005; 14:253–262.

10.

Xiao

. Health effects of soy protein and isoflavones in humans. J Nutr, 2008; 138:1244S–1249S.

11.

Anderson

, Johnstone

, Cook-Newell

. Meta-analysis of the effects of soy protein intake on serum lipids. N Engl J Med, 1995; 333:276–282.

12.

Tucker

. Nutritional enhancement of plants. Curr Opin Biotechnol, 2003; 14:221–225.

13.

Reeves

, Nielsen

, Fahey

. AIN-93 purified diets for laboratory rodents: final report of the American Institute of Nutrition Ad Hoc Writing Committee on the reformulation of the AIN-76A rodent diet. J Nutr, 1993; 123:1939–1951.

14.

Institute of Laboratory Animal Resources, Commission on Life Sciences. Guide for the Care and Use of Laboratory Animals. National Academy Press: Washington, DC, 1996.

15.

Rudel

, Morris

. Determination of cholesterol using o-phthalaldehyde. J Lipid Res, 1973; 14:364–366.

16.

Chezem

, Story

. Development of an updated method for fecal bile acid and neutral steroid analysis. Am Clin Lab, 1997; 16:20–21.

17.

Chomczynski

, Sacchi

. Single-step method of RNA isolation by acid guanidium thiocyanate-phenol chloroform extraction. Anal Biochem, 1987; 162:156–159.

18.

Sambrok

, Maniatis

, Fritsch

. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY, 1989.

19.

Tsang

, Yin

, Guzzo-Arkuran

, Jones

, Davison

. Loss of resolution in gel electrophoresis of RNA: a problem associated with the presence of formaldehyde gradients. Biotechniques, 1993; 14:380–381.

20.

Trawick

, Lewis

, Dueland

, Moore

, Simon

, Davis

. Rat hepatoma L35 cells, a liver-differentiated cell line, display resistance to bile acid repression of cholesterol 7α-hydroxylase. J Lipid Res, 1996; 37:588–598.

21.

Donkin

, McNall

, Swencki

, Peters

, Etherton

. The growth hormone dependent decrease in hepatic fatty acid synthase mRNA is the result of a decrease in gene transcription. J Mol Endocrinol, 1996; 16:151–158.

22.

StatSoft. Statistica for Windows: Computer Program Manual [electronic manual] StatSoft, Inc.: Tulsa, OK, 2000.

23.

Carroll

. Review of clinical studies on cholesterol-lowering response to soy protein. J Am Diet Assoc, 1991; 91:820–827.

24.

Kreijkamp-Kaspers

, Kok

, Grobbee

et al. Effect of soy protein containing isoflavones on cognitive function, bone mineral density, and plasma lipids in postmenopausal women: a randomized controlled trial. JAMA, 2004; 292:65–74.

25.

Shukla

, Brandsch

, Bettzieche

, Hirche

, Stangl

, Eder

. Isoflavone-poor soy protein alters the lipid metabolism of rats by SREBP-mediated downregulation of hepatic genes. J Nutr Biochem, 2007; 18:313–321.

26.

Descovich

, Ceredi

, Gaddi

et al. Multicentre study of soybean protein diet for outpatient hyper-cholesterolaemic patients. Lancet, 1980; 2:709–712.

27.

Huff

, Hamilton

RMG

, Carroll

. Plasma cholesterol levels in rabbits fed low fat, cholesterol-free, semipurified diets: effects of dietary proteins, protein hydrolysates and amino acid mixtures. Atherosclerosis, 1977; 28:187–195.

28.

Kritchevsky

, Tepper

, Czarnecki

, Mueller

, Klurfeld

, Story

. Effects of dietary protein on lipid metabolism in rats. J Washington Acad Sci, 1984; 74:1–8.

29.

Torres

, Torre-Villalvazo

, Tovar

. Regulation of lipid metabolism by soy protein and its implication in diseases mediated by lipid disorders. J Nutr Biochem, 2006; 17:365–373.

30.

Song

, Chun

, Hwang

et al. Soy isoflavones as safe functional ingredients. J Med Food, 2007; 10:571–580.

31.

Esteves

, Martino

HSD

, Oliveira

FCE

, Bressan

, Costa

NMB

. Chemical composition of a soybean cultivar lacking lipoxygenases (LOX2 and LOX3) Food Chem, 2010; 122:238–242.

32.

Taku

, Umegaki

, Sato

, Taki

, Endoh

, Watanabe

. Soy isoflavones lower serum total and LDL cholesterol in humans: a meta-analysis of 11 randomized controlled trials. Am J Clin Nutr, 2007; 85:1148–1156.

33.

Sung

, Lee

, Park

, Moon

. Isoflavones inhibit 3-hydroxy-3-methylglutaryl coenzyme A reductase in vitro. Biosci Biotechnol Biochem, 2004; 68:428–432.

34.

Madani

, Lopez

, Blond

, Prost

, Belleville

. Highly purified soybean protein is not hypocholesterolemic in rats but stimulates cholesterol synthesis and excretion and reduces polyunsaturated fatty acid biosynthesis. J Nutr, 1998; 128:1084–1091.

35.

Tasker

, Potter

. Effects of dietary protein source on plasma lipids, HMG CoA reductase activity, and hepatic glutathione levels in gerbils. J Nutr Biochem, 1993; 4:458–462.

36.

Tamaru

, Kurayama

, Sakono

et al. Effects of dietary soybean peptides on hepatic production of ketone bodies and secretion of triglyceride by perfused rat liver. Biosci Biotechnol Biochem, 2007; 71:2451–2457.

37.

Xiao

, Meil

, Wood

. Effect of soy proteins and isoflavones on lipid metabolism and involved gene expression. Front Biosci, 2008; 13:2660–2673.

38.

Borradaile

, Dreu

, Wilcox

, Edwards

, Huff

. Soya phytoestrogens, genistein and daidzein, decrease apolipoprotein B secretion from HepG2 cells through multiple mechanisms. Biochem J, 2002; 366:531–539.

39.

Dyck

JRB

, Kudo

, Barr

, Davies

, Hardie

. Phosphorylation control of cardiac acetyl-CoA carboxylase by cAMP-dependent protein kinase and 5′-AMP activated protein kinase. Eur J Biochem, 1999; 262:184–190.

40.

Tovar

, Murguia

, Cruz

et al. A soy protein diet alters hepatic lipid metabolism gene expression and reduces serum lipids and renal fibrogenic cytokines in rats with chronic nephritic syndrome. J Nutr, 2002; 132:2562–2569.

41.

Davis

, Miyake

, Hui

, Spann

. Regulation of cholesterol-7α-hydroxylase: BAREly missing a SHP. J Lipid Res, 2002; 43:533–543.

42.

Spady

, Cuthbert

, Willard

, Meidell

. Feedback regulation of hepatic cholesterol 7α-hydroxylase expression by bile salts in hamsters. J Biol Chem, 1996; 271:18623–18631.

43.

Horton

, Cuthbert

, Spaky

. Regulation of hepatic 7α-hydroxylase expression and response to dietary cholesterol in the rat and hamster. J Biol Chem, 1995; 270:5381–5387.

44.

Nagata

, Ishiwaki

, Sugano

. Studies on the mechanism of the anti-hypercholesterolemic action of soy protein and soy protein-type amino acid mixtures in relation to their casein counterparts in rats. J Nutr, 1982; 112:1614–1625.

45.

Greaves

, Wilson

, Rudel

, Williams

, Wagner

. Consumption of soy protein reduces cholesterol absorption compared to casein protein alone or supplemented with an isoflavone extract or conjugated equine estrogen in ovariectomized cynomolgus monkeys. J Nutr, 2000; 130:820–826.

46.

Beynen

. Comparison of the mechanism proposed to explain the hypocholesterolemic effect of soybean protein versus casein in experimental animals. J Nutr Sci Vitaminol, 1990; 36:87S–93S.

47.

Chen

, Chiou

, Suetsuna

, Yang

. Lipid metabolism in hypercholesterolemic rats affected by feeding cholesterol-free diets containing different amounts of non-dialyzed soybean protein fraction. Nutrition, 2003; 19:676–680.

48.

Carroll

. Hypercholesterolemia and atherosclerosis: effect of dietary protein. Fed Proc, 1982; 41:2792–2796.

49.

Ronis

, Chen

, Badeaux

, Badger

. Dietary soy protein isolate attenuates metabolic syndrome in rats via effects on PPAR, LXR, and SREBP signaling. J Nutr, 2009; 139:1431–1438.

50.

Xiao

, Mei

, Huang

et al. Dietary soy protein isolate modifies hepatic retinoic acid receptor-beta proteins and inhibits their DNA binding activity in rats. J Nutr, 2007; 137:1–6.

51.

Ramesha

, Paul

, Ganguly

. Effect of dietary unsaturated oils on the biosynthesis of cholesterol, and on biliary and fecal excretion of cholesterol and bile acids in rats. J Nutr, 1980; 110:2149–2158.

52.

Carroll

, Hamilton

RMG

. Effects of dietary protein and carbohydrate on plasma cholesterol levels in relation to atherosclerosis. J Food Sci, 1975; 40:18–23.

53.

Lau

BWC

, Klevay

. Postheparin plasma lipoprotein lipase in copper-deficient rats. J Nutr, 1982; 112:928–933.

54.

Allotta

, Samman

, Roberts

DCK

. The importance of the non-protein components of the diet in the plasma cholesterol response of rabbits to casein. Br J Nutr, 1985; 54:87–94.

55.

Anderson

. Dietary fiber, lipids and atherosclerosis. Am J Cardiol, 1987; 60:17–22.