Biochemical and Molecular Action of Nutrients
Ethanol-Extracted Soy Protein Isolate Does Not Modulate Serum Cholesterol in Golden Syrian Hamsters: A Model of Postmenopausal Hypercholesterolemia1 Edralin A. Lucas, Dania A. Khalil, Bruce P. Daggy and Bahram H. Arjmandi2 Department of Nutritional Sciences, Oklahoma State University, Stillwater, Oklahoma 74078
KEY WORDS:
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lipids
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triglycerides
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casein
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ovariectomy
Menopause, whether natural or surgical, is associated with elevations in circulating total and low density lipoprotein (LDL)3 cholesterol concentrations, placing postmenopausal women at greater risk for coronary heart disease (CHD) (Bruschi et al. 1996, Fukami et al. 1995, Goldstein and Stampfer 1995, Sullivan 1996). These changes are a consequence of reductions in the levels of circulating estrogens, which is the basis for estrogen replacement therapy (ERT). Estrogen replacement therapy reduces the risk of CHD in part through the modulation of serum cholesterol (Godsland et al. 1987, Knight and Eden 1996). However, ERT and other cholesterol-lowering pharmacological agents may be accompanied by side effects (U.S. Department of Health and Human Services 1993) and therefore are recommended only for women without known contraindications. Moreover, many women chose not to comply with a recommendation for ERT because of safety concerns (Kessel 1998). Therefore, other
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isoflavones
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ABSTRACT Soy protein consumption has been linked to reduction in hypercholesterolemia, a risk for coronary heart disease. However, to what extent soy protein itself or its non-nutritive components, e.g., isoflavones and saponins, exert this cholesterol-lowering effect requires further investigation. To evaluate the effect of the protein component alone on lipid variables, ethanol-extracted, isoflavone-depleted soy protein isolate (SPe) was studied in ovarian hormone-deficient hamsters. Forty-eight 6-month-old female Golden Syrian hamsters were either sham-operated or ovariectomized and fed casein-based or SPe-based diets for 70 d. Ovariectomy, but not protein source, significantly (P ⬍ 0.05) increased serum phospholipids and total, non– high density lipoprotein, free and esterified cholesterol concentrations. Serum HDL cholesterol concentrations were not altered with either treatment. No significant differences were observed in liver total lipids or liver total cholesterol among the groups. Soy protein isolate, however, lowered serum triglyceride concentrations in both sham-operated and ovariectomized hamsters. These findings confirm the ovariectomized hamster as a model of postmenopausal hypercholesterolemia. The results are consistent with earlier observations that isoflavones or other nonprotein components, perhaps in combination with soy protein, play an important role in exerting this hypocholesterolemic effect. Further studies are needed to investigate whether isolated nonprotein components of soy would be able to prevent the ovarian hormone deficiency–associated rise in serum cholesterol regardless of dietary protein source. J. Nutr. 131: 211–214, 2001.
means that present no known side effects for the treatment of postmenopausal hypercholesterolemia are preferred. Recently, the Food and Drug Administration approved a CHD risk reduction claim for soy protein (U.S. Food and Drug Administration 1999); the health claim does not specify a requirement for the presence or quantity of the nonprotein constituents such as saponins, phytic acid, trypsin inhibitors and isoflavones known to influence cholesterol. The amount of these compounds, in a given soy protein preparation, can be affected by processing. For instance, isolates prepared through extraction with water and ethanol can be substantially depleted in their nonprotein components (Anderson and Wolf 1995). Hence, variable results can be obtained from the use of different soy protein preparations. Another issue of importance is to investigate whether the cholesterol-lowering efficacy of nonprotein constituents of soy, such as isoflavones, differ when given with soy or other protein sources. The findings from some human (Crouse et al. 1999, Wong et al. 1998) and animal (Anthony et al. 1996 and 1997, Arjmandi et al. 1997, Kirk et al. 1998) studies suggest the importance of isoflavones in reducing total and LDL cholesterol in the context of soy protein. However, when isoflavonerich extract of soy has been fed to cynomolgus monkeys in the absence of soy protein, it has not produced any cholesterollowering effects (Greaves et al. 1999). The question remains
1 Supported by Oklahoma Center for the Advancement of Science and Technology project HR 98-38. 2 To whom correspondence should be addressed at Department of Nutritional Sciences, 416 Human Environmental Sciences; Oklahoma State University, Stillwater, OK 74078. E-mail:
[email protected] 3 Abbreviations used: CHD, coronary heart disease; ERT, estrogen replacement therapy; HDL, high density lipoprotein(s); LDL, low density lipoprotein(s); ovx, ovariectomy; SPe, ethanol-extracted soy protein isolate; TG, triglycerides.
0022-3166/01 $3.00 © 2001 American Society for Nutritional Sciences. Manuscript received 27 June 2000. Initial review completed 23 August 2000. Revision accepted 27 October 2000. 211
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whether it is the isoflavone or other components of soy, soy protein itself or their combination that lower cholesterol. A previous hamster study in this laboratory has shown hypercholesterolemic changes after ovariectomy (Sohn et al. 1999a). Hence, in the present study, we used this animal model to address the question of whether soy protein depleted of its nonprotein constituents such as isoflavones would be able to suppress the ovariectomy-induced rise in serum cholesterol. MATERIALS AND METHODS
TABLE 1 Composition of experimental diets Ingredient
Casein based
SPe based
RESULTS
g/kg Carbohydrates Rice flour1 Fiber (total) Wheat bran2 Cellulose3 Protein (total) Casein3 Soy protein isolate4 Fat (total) Hydrogenated coconut oil3 Safflower oil3 Soybean oil3 Choline chloride3 Potassium bicarbonate5 Vitamin mix6 Mineral mixture7 1 2 3 4
395 395 144 72 72 240 240 — 154 96 19 39 3 20 10 34
395 395 144 72 72 240 — 240 154 96 19 39 3 20 10 34
California Natural Products (Lathrop, CA). Natural Ovens of Manitowoc (Manitowoc, WI). Harlan-Teklad (Madison, WI). Supro670 (Protein Technologies Inc., St. Louis, MO), which has been further processed via ethanol extraction. Contains 93.4% protein, 3.92% ash and 0.08 mg isoflavones/g. 5 Sigma Chemical Co. (St. Louis, MO). 6 Vitamin mixture (TD 40060; Harlan-Teklad; g/kg): p-aminobenzoic acid, 11.0132; ascorbic acid, coated (97.5%), 101.664; biotin, 0.0441; folic acid, 0.1982; vitamin B-12 (0.1% in mannitol), 2.9736; calcium panthothenate, 6.6079; choline dihydrogen citrate, 349.6916; inositol, 11.0132; menadione, 4.9559; niacin, 9.9119; pyridoxine HCl, 2.2026; riboflavin, 2.2026; thiamine HCl, 2.2026; dry vitamin A palmitate (500,000 U/g); 3.9648; dry vitamin D-3 (500,000 U/g), 0.4405; dry vitamin E acetate (500 U/g), 24.2291; and corn starch, 466.6878. 7 Mineral mixture (TD 170911; Harlan-Teklad; g/kg): Ca, 6.232; P, 3.993; K, 3.829; Na, 2.109; Cl, 3.472; S, 0.644; Mg, 0.465; I, 0.0006; Fe, 0.0252; Cu, 0.0052; Mn, 0.0501; and Zn, 0.0118.
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Animals and diets. Forty-eight 6-mo-old female Golden Syrian hamsters (Harlan Sprague-Dawley, Indianapolis, IN) were individually housed and kept in an environmentally controlled laboratory. Guidelines for the ethical care and treatment of animals from the Animal Care and Use Committee at Oklahoma State University were strictly followed. After 3 d of acclimation, hamsters were either sham-operated (sham) or ovariectomized (ovx) and divided into four groups of 12 hamsters each as follows: sham ⫹ casein, ovx ⫹ casein, sham ⫹ SPe and ovx ⫹ SPe. Animals were fed for 70 d a semipurified cholesterol-free powdered diet that was either casein or SPe based (Table 1). Diet composition was a modification of the formulation by Terpstra et al (1991). Ovariectomized hamsters were pair-fed to the sham group; deionized water access was unrestricted. Food intake was determined daily. Seventy days after surgery, hamsters were anesthetized with a mixture of ketamine hydrochloride (100 mg/kg body) and xylazine (5
mg/kg body) and exsanguinated from the abdominal aorta. Blood samples were collected, and serum was separated via centrifugation at 1500 ⫻ g for 20 min at 4°C. Aliquots of serum were kept at ⫺20°C for later analyses. The liver was immediately removed, rinsed with ice-cold saline, weighed, kept in a sealed container and stored at ⫺20°C until analyzed. The uterus and small intestine were collected, blotted and weighed. Serum triglycerides (TG); phospholipids; total, free, esterified and high density lipoprotein (HDL) cholesterol and 17-estradiol. Serum TG and total cholesterol concentrations were determined enzymatically with Sigma Diagnostics kits (St. Louis, MO). Serum HDL cholesterol was determined with a precipitation technique (Sohn et al. 1999a, Sjoblom and Elklund 1989). Serum phospholipids and free cholesterol concentrations were determined through colorimetric methods with commercially available kits (Wako, Richmond, VA). These tests were performed with a Cobas-Fara II Clinical Analyzer (Montclair, NJ). Non-HDL cholesterol was calculated by subtracting HDL cholesterol from total cholesterol. Esterified cholesterol was calculated by subtracting free cholesterol from total cholesterol. Serum 17-estradiol was determined with a radioimmunoassay kit (ICN Biomedical, Costa Mesa, CA). Liver total lipids and cholesterol. Portions of the liver were homogenized and then extracted with a 2:1 (v/v) chloroform/methanol mixture. After the addition of 0.12 NaCl mol/L solution to the extraction solution and the separation of phases, aliquots of the organic phase were analyzed for liver total cholesterol. Liver total cholesterol was determined with a color reagent of glacial acetic acid/FeSO4/H2SO4 (Searcy and Bergquist 1960). Total liver lipids were determined with the Folch gravimetric method (Folch et al. 1957). Statistical analyses. The data were analyzed as a 2 ⫻ 2 factorial arrangement in a completely randomized design with SAS software, Version 6.11 (SAS Institute, Cary, NC). Analysis of variance and least squares means were calculated using the general linear model procedure. Data are reported as least squares means ⫾ SEM; unless otherwise indicated, P ⬍ 0.05 is regarded as significant.
Food intake and body and organ weights. Daily food consumption did not differ among the groups (Table 2). Hamsters in all groups had similar initial and final mean body weights. Ovariectomy caused atrophy of uterine tissue, indicating the success of the surgical procedure. There was no difference in relative liver weight among the treatments. The small intestine relative weight was significantly (P ⬍ 0.05) lower in the ovx groups than in the sham groups. Serum and liver variables. As expected, ovariectomy significantly (P ⬍ 0.001) reduced circulating serum levels of 17-estradiol (Table 3), confirming our earlier observations in this model (Sohn et al. 1999a) ovariectomy significantly increased serum total- and non-HDL cholesterol concentrations regardless of dietary protein source. Serum phospholipid and free and esterified cholesterol concentrations were also significantly elevated in ovx hamsters compared with sham hamsters regardless of whether they were fed SPe- or casein-based diets. Regardless of ovarian hormone status, hamsters that were fed the casein-based diet had significantly higher TG concentrations than did those fed SPe-based diet. Neither liver total cholesterol nor total lipid concentrations were significantly affected by either surgical or dietary treatments. DISCUSSION This study further confirms that ovariectomy increases serum total and non-HDL cholesterol concentrations of Golden Syrian hamsters, similar to observations made in postmenopausal women (Bruschi et al. 1996). Our previous study in this model (Sohn et al. 1999a) reported a significant rise in total
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TABLE 2 Effects of ovariectomy (ovx) and source of protein in the diet on food intake, body and relative organ weights in hamsters1 P-values Sham ⫹ casein
Ovx ⫹ casein
Sham ⫹ SPe2
Ovx ⫹ SPe
7.1 ⫾ 0.2
7.0 ⫾ 0.2
7.4 ⫾ 0.1
7.3 ⫾ 0.2
172 ⫾ 4 164 ⫾ 3
173 ⫾ 3 159 ⫾ 3
171 ⫾ 3 161 ⫾ 3
171 ⫾ 4 161 ⫾ 3
0.60 ⫾ 0.04 6.7 ⫾ 0.3 2.4 ⫾ 0.1
0.12 ⫾ 0.04 6.5 ⫾ 0.3 2.1 ⫾ 0.1
0.65 ⫾ 0.04 6.5 ⫾ 0.3 2.4 ⫾ 0.1
0.13 ⫾ 0.04 6.1 ⫾ 0.3 2.3 ⫾ 0.1
Measures Food intake, g/d Body weight, g Initial Final Organ weight, g/100 g body Uterus Liver Small intestine
Diet
Ovx ⫻ diet
0.6710
0.0762
0.9043
0.7731 0.3987
0.7325 0.7935
0.8178 0.3361
⬍0.0001 0.2648 0.0256
0.5356 0.2130 0.2804
0.5919 0.7243 0.2330
Ovx
1 Values are means ⫾ SEM, n ⫽ 12. 2 SPe, ethanol-extracted soy protein.
ovariectomy reduced the weight of the small intestine compared with that of intact hamsters. The atrophy of the intestine, a tissue that expresses estrogen receptors (Arjmandi et al. 1993), suggests that the intestine may be a target tissue for estrogen, and hence the impact of estrogen deficiency on the function of this important organ is meritorious of further study. The inability of the SPe to prevent the ovx-induced rise in circulating total, non-HDL, esterified and free cholesterol concentrations may further indicate the importance of soy isoflavones or other nonprotein components in modulating lipid metabolism. A study by Balmir et al. (1996) of male hamsters confirmed the importance of isoflavones in reducing cholesterol. Their results showed that hamsters that were fed soy protein with normal isoflavone content or casein with added isoflavones had lower serum total cholesterol concentrations than those fed casein alone. A study by Greaves et al. (1999) concluded that soy components other than isoflavones are responsible for its cholesterol-lowering properties in ovx monkeys; however, their findings neither confirmed nor rejected the observations made in the present study. Their results suggested that isoflavone-rich extract of soy alone fed in conjunction with protein other than
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plasma cholesterol and a trend (P ⫽ 0.073) toward higher concentrations of non-HDL cholesterol. In the present study, however, the increase in the non-HDL cholesterol was significant. In agreement with our earlier observations (Arjmandi et al. 1997, Sohn et al. 1999a), ovariectomy did not alter liver weight, lipids or cholesterol concentrations. Although ovariectomy has been reported to reduce the activity of hepatic hydroxymethylglutaryl (HMG)-coenzyme A (CoA) reductase in rats (Abul-Hajj 1981, Carlson et al. 1999), this may not be the case in ovx hamsters. We previously observed no change in hepatic rates of sterol synthesis due to ovariectomy in this animal model (Sohn et al. 1999a). Hence, altered de novo cholesterol synthesis cannot be a mechanism by which ovariectomy causes hypercholesterolemia in hamsters. A reduction in the conversion of cholesterol to bile acids may in part explain the ovariectomy-induced rise in serum cholesterol, as ovarian hormone deficiency has been shown to reduce both hepatic 7␣-hydroxylase mRNA expression and activity (Colvin et al. 1998, Kushawa and Born 1991, Sohn et al. 1999b). Similar to our earlier observations (Sohn et al. 1999a),
TABLE 3 Effects of ovariectomy (ovx) and source of protein in the diet on serum and liver variables in hamsters1 P-values Measure Serum 17-Estradiol, pmol/L Cholesterol, mmol/L Total HDL Non-HDL2 Free Esterified3 Triglycerides, mmol/L Phospholipids, mmol/L Liver Total lipids, mg/g Total cholesterol, mol/g
Sham ⫹ casein
Ovx ⫹ casein
Sham ⫹ SPe
Ovx ⫹ SPe
Ovx
Diet
Ovx ⫻ diet
705 ⫾ 117
106 ⫾ 11
492 ⫾ 103
117 ⫾ 7
⬍0.0001
0.2046
0.0650
6.2 ⫾ 0.3 3.3 ⫾ 0.2 2.9 ⫾ 0.2 1.3 ⫾ 0.1 4.9 ⫾ 0.2 3.2 ⫾ 0.3 4.5 ⫾ 0.2
6.6 ⫾ 0.3 3.3 ⫾ 0.2 3.3 ⫾ 0.2 1.4 ⫾ 0.1 5.2 ⫾ 0.2 3.2 ⫾ 0.3 5.1 ⫾ 0.2
6.0 ⫾ 0.3 3.3 ⫾ 0.2 2.7 ⫾ 0.2 1.2 ⫾ 0.1 4.8 ⫾ 0.2 2.1 ⫾ 0.3 4.7 ⫾ 0.2
7.3 ⫾ 0.3 3.7 ⫾ 0.2 3.6 ⫾ 0.2 1.6 ⫾ 0.1 5.7 ⫾ 0.2 2.8 ⫾ 0.3 5.5 ⫾ 0.2
0.0114 0.3757 0.0006 0.0405 0.0193 0.1995 0.0024
0.4392 0.3218 0.7551 0.7712 0.3928 0.0192 0.0904
0.2030 0.3861 0.1861 0.2190 0.2797 0.2672 0.6266
58.2 ⫾ 2.8 4.1 ⫾ 0.2
60.2 ⫾ 2.6 4.4 ⫾ 0.2
57.9 ⫾ 2.7 3.7 ⫾ 0.2
63.8 ⫾ 2.8 4.1 ⫾ 0.2
0.1595 0.1317
0.5430 0.1461
0.4796 0.5989
1 Values are means ⫾ SEM; n ⫽ 12. 2 Non– high density lipoprotein (HDL) cholesterol ⫽ total cholesterol ⫺ HDL cholesterol. 3 Esterified cholesterol ⫽ total cholesterol ⫺ free cholesterol.
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ACKNOWLEDGMENTS The authors thank Douglas Lange for his technical assistance. Gratitude is also extended to the California Natural Products (Lathrop, CA) for providing rice flour and the Natural Ovens of Manitowoc (Manitowoc, WI) for providing wheat bran for diet formulation.
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(1996) Lipoprotein(a) and other lipids after oophorectomy and estrogen replacement therapy. Obstet. Gynecol. 88: 950 –954. Carlson, S. E., Mitchell, A. D., Carter, M. L., and Goldfarb, S. (1980) Evidence that physiologic levels of circulating estrogens and neonatal sex-imprinting modify postpubertal hepatic microsomal 3-hydroxy-3-methylglutaryl coenzyme A reductase activity. Biochim. Biophys. Acta 633: 154 –161. Colvin, P. L., Jr., Wagner, J. D., Adams, M. R. & Sorci-Thomas, M. G. (1998) Sex steroids increase cholesterol 7␣-hydroxylase mRNA in nonhuman primates. Metabolism 47: 391–395. Crouse, J. R. 3rd, Morgan, T., Terry, J. G., Ellis, J., Vitolins, M. & Burke, G. L. (1999) A randomized trial comparing the effect of casein with that of soy protein containing varying amounts of isoflavones on plasma concentrations of lipids and lipoproteins. Arch. Intern. Med. 159: 2070 –2076. Folch, J., Lees, M. & Sloane-Stanley, G. H. (1957) A simple method for isolation and purification of total lipids from animal tissue. J. Biol. Chem. 226: 497–509. Fukami, K., Koike, K., Hirota, K., Yoshikawa, H. & Miyake, A. (1995) Perimenopausal changes in serum lipids and lipoproteins: A 7-year study. Maturitas 22: 193–197. Godsland, I. F., Wynn, V., Crook, D. & Miller, N. E. (1987) Sex, plasma lipoproteins, and atherosclerosis: Prevailing assumptions and outstanding questions. Am. Heart J. 114: 1467–1503. Goldstein, F. & Stampfer, M. (1995) The epidemiology of coronary heart disease and estrogen replacement in postmenopausal women. Prog. Cardiovasc. Dis. 38: 99 –210. Greaves, K. A., Parks, J. S., Williams, J. K. & Wagner, J. D. (1999) Intact dietary soy protein, but not adding an isoflavone-rich soy extract to casein, improves plasma lipids in ovariectomized cynomolgus monkeys. J. Nutr. 129: 1585– 1592. Iritani, N., Hosomi, H., Fukuda, H., Tada, K. & Ikeda, H. (1996) Soybean protein suppresses hepatic lipogenic enzyme gene expression in Wistar fatty rats. J. Nutr. 126: 380 –388. Kessel, B. (1998) Alternatives to estrogen for menopausal women. Proc. Soc. Exp. Biol. Med. 217: 38 – 44. Kirk, E. A., Sutherland, P., Wang, S. A., Chait, A. & LeBoeuf, R. C. (1998) Dietary isoflavones reduce plasma cholesterol and atherosclerosis in C57BL/6 mice but not LDL receptor-deficient mice. J. Nutr. 128: 954 –959. Knight, D. C. & Eden, J. A. (1996) A review of the clinical effects of phytoestrogens. Obstet. Gynecol. 87: 897–904. Kushawa, R. S. & Born, K. M. (1991) Effect of estrogen and progesterone on the hepatic cholesterol 7-alpha-hydroxylase activity in ovariectomized baboons. Biochim. Biophys. Acta 1084: 300 –302. Searcy, R. L. & Bergquist, L. M. (1960) A new color reaction for the quantitation of serum cholesterol. Clin. Chim. Acta 5: 192–199. Sjoblom, L. & Elklund, A. (1989) Determination of HDL2 cholesterol by precipitation with dextran sulfate and magnesium chloride: establishing optimal conditions for rat plasma. Lipids 24: 532–534. Sohn, E., Daggy, B. P. & Arjmandi, B. H. (1999a) Ovariectomized hamster: A potential model of postmenopausal hypercholesterolemia. J. Nutr. Biochem. 10: 660 – 663. Sohn, E., Kenny, E., Arjmandi, B. H. & Baum C. (1999b) Evidence for the selective estrogenic properties of isoflavones in ovx hamsters. FASEB J. 13: A49. Sullivan, J. M. (1996) Practical aspects of preventing and managing atherosclerotic disease in post-menopausal women. Eur. Heart J. 17(Suppl D): 32–37. Terpstra, A.H.M., Holmes, J. C. & Nicolosi, R. J. (1991) The hypercholesterolemic effect of dietary soybean protein vs casein in hamsters fed cholesterol enriched or cholesterol-free semipurified diets. J. Nutr. 121: 944 –947. U.S. Department of Health and Human Services (1993) Second report of the expert panel on detection, evaluation, and treatment of high blood cholesterol in adults. NIH publication no. 93-3095. U.S. Food and Drug Administration (1999) Food Labeling: Health Claims; Soy Protein and Coronary Heart Disease; Final Rule. Federal Register 64 FR 57699 October 26. Wong, W. W., Smith, E. O., Stuff, J. E., Hachey, D. L., Heird, W. C. & Pownell, H. J. (1998) Cholesterol-lowering effect of soy protein in normocholesterolemic and hypercholesterolemic men. Am. J. Clin. Nutr. 68: 1385S–1389S.
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soy does not produce the desirable hypocholesterolemic effects in ovarian hormone deficiency. Our findings in this and earlier work (Arjmandi et al. 1997) and the majority of the studies by other investigators (Anthony et al. 1996 and 1997, Crouse et al. 1999, Kirk et al. 1998, Wong et al. 1998) imply that the combination of soy protein and its isoflavones is necessary for their effectiveness in lowering serum cholesterol in ovarian hormone deficiency. In the present study, SPe significantly reduced serum TG levels, regardless of ovarian hormone status, compared with casein. The effects of soy on circulating TG concentrations in humans (Ashton and Ball 2000, Crouse et al. 1999) and in rats (Iritani et al. 1996) have been reported previously; however, the data are not conclusive and are sometimes contradictory. Therefore, the effects of soy protein on serum TG, an important risk factor for cardiovascular disease, as well as possible modes of action warrant investigation. Our results suggest that SPe alone is not sufficient to prevent the ovarian hormone deficiency–induced hypercholesterolemia. Ethanol extractable components, either alone or in conjunction with the soy protein, may be needed to lower circulating cholesterol concentrations and therefore reduced the risk of CHD in ovarian hormone deficiency.