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The ovarian hormone deficiency-induced hypercholesterolemia is reversed by soy protein and the synthetic isoflavone, ipriflavone
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Nutrition Research, Vol. 17, No. 5, pp. 885-894, 1997 Copyright © 1997 Elsevier Science Inc. Printed in the USA. All fights reserved 0271-5317/97 $17.00 + .00
ELSEVIER
PII S0271-5317(97)00055-9
THE OVARIAN HORMONE DEFICIENCY-INDUCED HYPERCHOLESTEROLEMIA IS REVERSED BY SOY PROTEIN AND THE SYNTHETIC ISOFLAVONE, IPRIFLAVONE
Bahrain H. Arjmandi, Ph.D., R D . t, Dilshad A. Khan, M.D. t, Shanil S. Juma, M,S. t, Alvar Svanborg, M.D., Ph.D. tDepartment of Human Nutrition and Dietetics, and Department of Medicine University of Illinois at Chicago, IL 60612
ABSTRACT The purpose of this study was to compare the effects of soy protein isolate with normal isoflavone content (soy) and with reduced isoflavone content (soy-), ipriflavone (IP), a synthetic isoflavone; and 17B-estradiol (E2) on lipid metabolism in ovariectomized (ovx) rats. Seventy-two 95-day old Sprague-Dawley rats were assigned to six groups: sham operated (sham), ovx, ovx+soy, ovx+soy-, ovx+IP, and ovx+~. Rats in the sham, ovx, ovx+IP, and ovx+E 2 groups were fed a casein-based diet, whereas the soy and soy- groups were fed diets in which casein was replaced with soy or soy-. Animals were pair-fed to the mean food intake of ovx+E 2 for 35 days. At the end of the study, animals were sacrificed in a nonfasted state and blood was collected via abdominal aorta. The ovx-induced increase in serum total cholesterol was reversed by all the treatments including ipriflavone and soy. Serum triglyceride levels were not significantly affected by any of the treatments. Liver cholesterol (lamol/g) in animals receiving IP or fed soy were significantly (p <0.01) lower than the ovx, ovx+soy-, and ovx+E 2 groups. Liver lipids (mg/g) were significantly (p<0.05) lower in the animals that received E 2 or fed soy, but not those which were fed soy- or given IP. The ovx-induced increase in abdominal fat was completely reversed by soy and E 2 treatments but not by soy- or ipriflavone treatments. Soy, soy-, and IP had no uterotrophic activity as compared to E2. Ovariectomy significantly increased body weight gains which were not suppressed by any of the treatments except E 2. These data indicate that ipriflavone is effective in preventing the unfavorable changes in serum and liver cholesterol associated with ovarian hormone deficiency in this animal model. Moreover, the consumption of synthetic or natural isoflavones may offer a potential alternative therapy in the treatment of hypercholesterolemia in ovarian hormone-deficient women. @1997Elsevi~SciemccInc.
KEY WORDS: Estrogen, cholesterol, Iprifiavone, Isoflavones, Ovariectomy, Rats Address reprint requests to: Bahram H. Arjmandi, Department of Human Nutrition and Dietetics, 1919 West Taylor Street (MC/517), University of Illinois at Chicago, IL 60612-7256. Fax: (312)413-0319; e-mail: [email protected]
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886
B.H. ARJMANDI et al.
INTRODUCTION Women experience a lower rate of coronary heart disease (CHD) before menopause in comparison to men. However, after the onset of menopause, the risk of CHD increases drastically due to ovarian hormone deficiency (1,2). With regards to cardiovascular disease, high blood cholesterol has been implicated as a major risk factor (3). Estrogen replacement therapy (ERT) has shown potential for the reduction or prevention of CHD (4) and is also considered the most effective method to reduce the rate o f postmenopausal bone loss (5) in women who have experienced naturally or surgically induced menopause. The benefits of ERT have been exhibited primarily through favorable alterations in lipid and lipoprotein metabolism (6) and suppression of bone resorption. Unfortunately, ERT may be accompanied by side effects such as endometrial and breast cancer (7,8) and is therefore recommended only for women who have no contraindications. One alternative may be the use of estrogen-like substances (phytoestrogens) that are found either in plants, e.g. isoflavones, or made synthetically (ipriflavone). Until the turn of this century it was assumed that estrogens were exclusively produced by animals. However, the principle that plants also can produce phytoestrogens was established by 1966 (9). Now it is well known that certain plants and plant products contain these phytoestrogens. One group of such compounds which are reported to have estrogenic activity is fiavonoids (10). This group of compounds include isoflavones which are found in a limited number of plants and plant products including soy protein isolate which is a rich source of isoflavones: genistein and daidzein. The hypocholesterolemic properties of soy protein has received much attention lately (11). From the review of the literature it is not clear whether the cholesterol lowering effect of soy protein is due to its amino acid composition, nonprotein constituents such as saponins, phytic acid, isoflavones, or a combination of these factors. However, in recent years, there has been rising interest regarding the soy isoflavones and their influence not only on sex hormone metabolism, but also other biological activities including cholesterol-lowering properties (12). Genistein and related isoflavones, found predominantly in soy, have long been recognized as naturally occurring phytoestrogens (13). Both in vitro and in vivo studies have shown that genistein exerts a weak estrogenic effect, approximately 1 x 10"s to 1 x 10-s that of estradiol (14). Since the hypocholesterolemic effect of soy protein, in part, has been linked to its isoflavone content (11), we decided to use a synthetic isoflavone, ipriflavone, and compare its effects on serum and liver cholesterol and lipid with those of soy protein and 17B-estradiol in ovariectomized rats. Ipriflavone, 7-isopropoxy-3-phenyl-4H-l-benzopyran-4-one, a synthetic isoflavone, has been shown to have beneficial effects on bone in both postmenopausal women (15,16) and ovariectomized animal models (17). However, there are no studies in which the effect of this compound on lipid and cholesterol metabolism has been evaluated. Ipriflavone is very appealing because of its potential acceptability and its lack of demonstrated toxicity by human subjects (18). The purpose of this study was to compare the effects of soy protein isolate with normal isoflavone content and with reduced isoflavone content, ipriflavone, a synthetic isoflavone; and 17B-estradiol on lipid metabolism in ovariectomized rats.
IPRIFLAVONE: A HYPOCHOLESTEROLEMIC AGENT
887
MATERIALS AND METHODS Animals and diets
Seventy-two 90 day-old female Sprague-Dawley rats, were purchased from Harlan Sprague-Dawley (Indianapolis, IN) and used for this study when they were 95 days old. On arrival, animals were housed in an environmentally controlled laboratory. Rats were acclimated to a standard laboratory non-purified diet for 5 days. After acclimation, using a randomized complete block design, rats were divided into six groups of 12 rats each and placed in pairs of two per cage: sham operated (sham), ovariectomized (ovx), ovx+soy, ovx+soy-, ovx+IP, and ovx+E2. For surgical purposes, rats were anesthetized with an intraperitoneal injection of ketamine hydrochloride and xylazine at doses of 100 mg/kg body weight and 5 mg/kg body weight, respectively. Bilateral ovariectomy were performed in the ovx, ovx+soy, ovx+soy-, ovx+IP, and ovx+F~ groups, and animals in the sham group went through the stress of surgery without removal of their ovaries. Following surgery, the animals were carefully watched and a heat lamp was used to keep the rats warm as they came out of anesthesia. Postoperatively, no additional analgesics or antibiotic were given to the animals. Known quantity of E2 was dissolved in a small amount of absolute ethanol and the volume was adjusted with sesame oil to a final concentration of 10 ~tg E2/ml. The solvent vehicle contained similar quantities of ethanol and sesame oil. Rats in the ovx+E2 group received 1 ml of the E2 solution/kg body weight, whereas rats in all other treatment groups received 1 ml of solvent vehicle/kg body weight subcutaneously on a daily basis. Sites of injection in each animal were varied daily in order to avoid possible complications such as accumulation of fluid under the skin. Rats in the sham, ovx, ovx+IP, and ovx+E2 groups were fed a powdered casein-based Teldad diet #88190 (Madison, WI) (Table 1). The soy-fed groups (soy and soy-) received similar diets in which casein was replaced with soy protein isolate without removal of its normal isoflavone content (soy) or soy protein isolate with reduced isoflavone content (soy-; containing less than 10% of the amount normally found in soy protein isolate). Ovx+IP animals received 100 mg/kg body weight/day of ipriflavone orally via gavaging in less than 2 ml deionized water containing 1% hydroxypropyl cellulose as an emulsifying agent (17). Rats which received E 2 consumed food ad libitum and their precise food intakes were measured every three days. Before each feeding, the food remaining was weighed and the amount ingested calculated. Rats in the other groups were pair-fed to the mean intake of the ovx+E2 animals and had free access to deionized water. Thirty-five days from the date of surgery, animals in a nonfasted state were anesthetized with a mixture of ketamine hydrochloride and xylazine as described above. After anesthesia, rats were sacrificed by exsanguination via bleeding from abdominal aorta. Blood samples were collected in nonheparinized tubes and serum was separated by centrifugation at 1500 x g for 20 minutes at 4°C. Aliquots of serum were frozen and kept at -20°C for later analyses. The liver was immediately removed, rinsed with ice-cold 0.4 mmol/L NaC1 solution, kept in sealed containers and stored at 70°C for lipid and cholesterol analyses. Guidelines for the ethical care and treatment of animals from the'Animal Care Committee of the University of Illinois at Chicago were strictly followed.
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B.H. AIqJMANDI et al. TABLE 1 Composition of Casein and Soy Protein Diets
Ingredients
Casein Diet ~
Soy Protein Diet
~/100 Protein Casein Soy Protein Isolate 2 Carbohydrate Sucrose Corn Starch Fiber Source - Cellulose~ Fat - Corn Oil Vitamin Mixture 4 Total cholecalciferol Mineral Mixture (Ca-P Deficient)5 Calcium Carbonate Sodium Phosphate, monobasic Potassium Phosphate, monobasic Potassium Citrate, monohydrate
22.70 22.70 41.76 20.00 5.60 5.70 1.0 0.00016 1.34 0.988 0.388 0.238 0.090
41,76 20,00 5.60 5.70 1.0 0.00016 1.34 0.988 0.388 0.238 0.090
~Teklad diet #88190 (Harlan Teklad, Madison, WI). :Soy protein isolate obtained from Protein Technologies International (St. Louis, MO). Soy protein isolate contained (mg/kg) of the following isoflavones: Genistin, 1462; genistein, 25.1; daidzin, 590; daidzein 11.3. 3Alphacel obtained from ICN Biochemicals (Costa Mesa, CA). 4Vitamin mixture (g/kg diet; TD 40060) obtained from Harlan Teklad (Madison, WI): p-aminobenzoic acid, 0.1101; ascorbic acid, 1.0166; biotin, 0.00044; vitamin B-12 (0.1% trituration), 0.0297; calcium pantothenate, 0.0661, choline dihydrogen citrate, 3.4969; folic acid, 0.00198; inositol, 0. I 101; menadione, 0.0495; niacin, 0.0991; pyridoxine HC1,0.0220; riboflavin, 0.0220; thiamin HC1, 0.0220; dry retinyl palmitate, 0.0044; dry d,1a-tocopheryl acetate, 0.2423; corn starch (diluent), 4.6669. 5Mineral mixture (TD 79055) obtained from Harlan Teklad (Madison, WI). This mixture is a modification of AIN 76.
Abdominal fat Post-euthanasia, all the organs within the abdominal cavity, including the adrenal glands, intestines, kidneys, liver and spleen were removed. Visceral fat and any visible fat adhering to the organs or abdominal wall were collected, blotted, and weighed to the nearest milligram.
Serum tri21vceride and total cholesterol, liver lipids and cholesterol Serum triglyceride and total cholesterol were determined enzymatically (Sigma Diagnostics, St. Louis, MO). Portions o f livers were homogenized, then extracted with a 2:1 (v/v) chloroform: methanol mixture. After addition of 7.3 g/L NaC1 solution to the extraction and separation o f phases, aliquots of the organic phase were analyzed for liver cholesterol. Liver cholesterol was determined
IPRIFLAVONE: A HYPOCHOLESTEROLEMIC AGENT
889
using a color reagent of glacial acetic acid-FeSO4-H2SO4 (19). Liver lipids were determined using the Foich gravimetric method (20). The remainder of the organic phase was evaporated, dried to constant weight, and weighed to measure liver lipids. Liver cholesterol and lipids were calculated and reported both per g and whole liver weight. Statistical analyses
Data analysis involved computation of means and standard deviations (SD) for each of the treatment groups (21). Analysis of variance (ANOVA) was performed to determine whether there were statistically significant (p< 0. 05) differences by treatment group. When an ANOVA indicated any significant difference among the means, the Tukey-Kramer follow-up multiple comparison test was used to determine which means were significantly different. GraphPad Instat Software (version 2.0, 1993; San Diego, CA) was used for all statistical analysis (21,22)
RESULTS Body and orean weiehts
The data on body and organ weights are presented in Table 2. The rats were pair-fed to the mean food intake of the E 2 group and hence all the groups consumed the same amount of food (14.5+1.8 g/day). Although the mean initial body weights did not differ among the treatment groups, all the ovx groups, except the E2-administered group had significantly higher mean final body weight and weight gain in comparison to sham. Ovariectomy caused atrophy of uterine tissue, and this atrophy was prevented by E 2 administration but not by soy, soy- or ipriflavone treatments. The liver weights did not differ among the six treatment groups. The ovx-induced increase in abdominal fat was prevented by soy and E 2 treatments, but not by soy- or ipriflavone. Serum total cholesterol and triglycerides
Serum total cholesterol concentrations were significantly (p<0.05) higher in ovx control animals in comparison with sham. This ovx-induced rise in serum total cholesterol was prevented by soy, soy-, IP, and E2 treatments (Table 3). Serum triglyceride levels were not significantly affected by any of the treatments (Table 3). Liver cholesterol and lipid concentrations
When liver cholesterol concentrations were expressed per gram of liver (liver cholesterol), animals which were fed soy or received ipriflavone had significantly (p<0.05) lower liver cholesterol concentrations in comparison with ovx controls (Table 3). Liver total cholesterol concentrations were significantly (p<0.05) lower in (soy)-fed and E2-treated animals in comparison with ovx controls. When liver lipids were assessed per gram liver, the ovariectomy-induced rise in liver lipid levels were prevented by soy and E2 but not by soy- or IP treatments (Table 3). Liver total lipid concentrations were significantly (p<0.05) lower in soy, soy-, and E 2 groups compared with ovx control group.
890
B.H. ARJMANDI et al. TABLE 2
Effects o f Ovariectomy (ovx), Soy with Normal Isoflavone Content (Soy), Soy with Reduced Isoflavone Content (Soy-), Ipriflavone (IP), and 1713-Estradiol (Fq) on Body and O r g a n Weights in Rats 1'2
Measures
Sham
Ovx
Ovx+Soy
Ovx+Soy-
Ovx+IP
Ovx+E2
Initial
212-46.7
2114-6.0
212-46.4
2124-6.4
2124-6.3
2124-6.2
Final
265=1:14.5b
299~12.6"
2954-16.6"
288:t:8.4"
2944-15.1"
249A27.5 b
Weight Gain
534-10.4 b
864-9.1'
844-15.7"
774-10.4 i
824-15.0"
37+7.5 b
Uterus
0.4974-0.14'
0.127:K).03 b
0.134+0.05 b
0.113+0.02 b
0.1184-0.02 b
0.5304-0.11 '
Liver
8.25+1.6
8.594-1.9
8.854-1.2
8.05:83.88
9.45~-0.95
7.694-0.66
Abdominal Fat
7.83+2.0 b
9.474-1.9"
7.68-41.3 b
8.20+2.0 "b
8.244-1.64~b
5.974-0.93 b
Body Weights (g)
Organ Weights (g)
1Values are m e a n s ± SD, n = 12 in each group, 2 Within a row, values that do not share the s a m e superscript letters are significantly (p <0.05) different fIom each other. TABLE 3 Effects o f Ovariectomy (Ovx), Soy with Normal Isoflavone Content (Soy), Soy with Reduced Isoflavone Content (Soy-), Ipriflavone (IP), and 17~3-Eslxadiol (E2) on Liver Cholesterol, Liver Lipids, Serum Total Cholesterol, and Serum Triglyceride Levels m R a t s ) ,2
Measures
Sham
Ovx
Ovx+Soy
Ovx+Soy-
Ovx+IP
Ovx+E2
total cholesterol
1.95~q3.25b
2.744-0.75"
1.62:1:0.25 b
1.8040.68 b
1.414-0.36 b
1.964-0.33 b
triglyeerides
0.61:83.19
0.66-40.10
0.54:1--0.15
0.54±0.19
0.594-0.10
0.714-0.18
Cholesterol (•mol/g liver)
7.7340.23 "b
8.35:L-0.85"
6.9341.00 b
8.20±1.73"
6.85+0.31 ~
8.2840.34"
Total cholesterol (~mol/whole liver)
62.64_9.4 ,b
71.74-7.2 °
58.7q-8.2 b
67.8:t:4.0 "b
63.5=1:4.9 "b
60.4:1:5.5b
Lipids (mg/g liveO
47.04-8.5 "b
48.5:t:5.2'
41.4±5.0 b
46.04-3.3 'b
47.044.4 "b
42.04-5.4 b
Total lipids (rag/whole liver)
3804-22 b
4234-17"
365q-31 b~
366+21 ~
4214-36"
3274-29 c
Serum (mmol/L)
Liver
1Values are m e a n s :t: SD, n = 8. 2 Within a row, values that do not share the same superscript letters are significantly (p<0.05) different fxom each other.
IPRIFLAVONE: A HYPOCHOLESTEROLEMIC AGENT
891
DISCUSSION The higher final mean body weights and weight gain in ovx animals suggest a shift in energy metabolism due to ovarian hormone deficiency. Other investigators have also reported increased body weights due to ovarian hormone deficiency in animals (23) and humans (24). Human studies have reported that hormone replacement therapy in postmenopausai women prevents increases in body fat (25), particularly centrally distributed fat (26) as was the case in this ovx animal model. Whereas ipriflavone did not have a significant effect on abdominal fat, both E 2 and soy protein with normal isoflavone content prevented the ovx-induced increase in abdominal fat. These findings suggest that the effect of the isoflavones present in soy protein on body fat may differ from that of ipriflavone. Hence, soy isoflavones may direct triglycerides to other tissues and away from adipose tissue for catabolism, similar to the proposed mechanism of action for estrogen on adipose tissue (27). In this study, the cholesterol lowering efficacy of soy protein isolate with reduced isoflavone content was somewhat diminished in comparison with that of soy protein isolate with normal isoflavone content. These observations suggest that some of the beneficial effects of soy protein on lipid metabolism are partially due to its isoflavones or other nonprotein components such as saponins which have been removed in processing. In support of our observations, a recent study by Anthony et al. (28) has shown that feeding soy protein rich in isoflavones to female Rhesus monkeys was more effective in lowering serum cholesterol than feeding a soy protein diet poor in isoflavone content. Balmir et al. (29) also observed that feeding a casein-based diet with added isoflavones (isolated from soy protein) to male rats resulted in a significant decline in serum total cholesterol concentrations. The hypocholesterolemic role ofisoflavones in the diet can be further supported by our findings that ipriflavone administration was effective in lowering serum total cholesterol in the absence of dietary soy protein. Since ipriflavone, a synthetic isoflavone, effectively prevented the ovx-induced rise in serum and liver cholesterol, this suggests that isofiavones, in general, play an important role in lipid metabolism. This is the first reported observation that ipriflavone potently suppresses the ovarian hormone deficiencyinduced rise in serum total cholesterol as much as 50%. These findings would be of great benefit to postmenopausal women, since ipriflavone is also reported to prevent bone loss associated with ovarian hormone deficiency (15,16,17), another major risk factor in this population. Additionally, it has been reported that ipriflavone is devoid of any known toxic side effects and is well tolerated by human subjects (18). The hypocholesterolemic properties of soy protein have been linked to enhanced fecal excretion of acidic and neutral sterols (30). In this regard, soy protein, or its other active components such as isoflavones, may behave in a similar fashion to certain soluble fibers, which enhance fecal sterol excretion causing cholesterol depletion from the body (31). Enhanced activity of hydroxymethylglutaryl CoA reductase, the major enzyme regulating hepatic cholesterol synthesis, in soy prote~n-fed animals has been reported by Huffand Carroll (32). These and other mechanisms (33) working alone or in combination, seem to contribute to the cholesterol lowering effects of soy protein or its active constituents. Additional studies are needed to further demonstrate their efficacy and illustrate their mode of action in humans.
892
B.H. ARJMANDI et al.
In conclusion, the findings of the present study suggest that ipriflavone, similar to estrogen and soy protein isolate, can effectively prevent the ovarian hormone deficiency-associated rise in serum cholesterol. If long term studies in humans demonstrate that ipriflavone is free of undesirable side effects, then it might be considered a desirable alternative for dealing with postmenopausal women's increased risks of coronary heart disease and osteoporosis.
ACKNOWLEDGMENTS The assistance of Daxa Amin, M,S., Mary Jane Getlinger, M.S., Noopur Goyal, M.S., and James Artwohl, D.V.M. in animal care, participation in specimen collection and technical procedures is gratefully acknowledged.
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Benvenuti S, Tanini A, Frediani U, Bianchi S, Masi L, Casano R, Bufalino L, Serio M, Brandi ML. Effects ofipriflavone and its metabolites on clonal osteoblastic cell line. J Bone Min Res 1991; 6:987-996.
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Accepted for publication January 10, 1997.
Nutrition Research, Vol. 17, No. 5, pp. 885-894, 1997 Copyright © 1997 Elsevier Science Inc. Printed in the USA. All fights reserved 0271-5317/97 $17.00 + .00
ELSEVIER
PII S0271-5317(97)00055-9
THE OVARIAN HORMONE DEFICIENCY-INDUCED HYPERCHOLESTEROLEMIA IS REVERSED BY SOY PROTEIN AND THE SYNTHETIC ISOFLAVONE, IPRIFLAVONE
Bahrain H. Arjmandi, Ph.D., R D . t, Dilshad A. Khan, M.D. t, Shanil S. Juma, M,S. t, Alvar Svanborg, M.D., Ph.D. tDepartment of Human Nutrition and Dietetics, and Department of Medicine University of Illinois at Chicago, IL 60612
ABSTRACT The purpose of this study was to compare the effects of soy protein isolate with normal isoflavone content (soy) and with reduced isoflavone content (soy-), ipriflavone (IP), a synthetic isoflavone; and 17B-estradiol (E2) on lipid metabolism in ovariectomized (ovx) rats. Seventy-two 95-day old Sprague-Dawley rats were assigned to six groups: sham operated (sham), ovx, ovx+soy, ovx+soy-, ovx+IP, and ovx+~. Rats in the sham, ovx, ovx+IP, and ovx+E 2 groups were fed a casein-based diet, whereas the soy and soy- groups were fed diets in which casein was replaced with soy or soy-. Animals were pair-fed to the mean food intake of ovx+E 2 for 35 days. At the end of the study, animals were sacrificed in a nonfasted state and blood was collected via abdominal aorta. The ovx-induced increase in serum total cholesterol was reversed by all the treatments including ipriflavone and soy. Serum triglyceride levels were not significantly affected by any of the treatments. Liver cholesterol (lamol/g) in animals receiving IP or fed soy were significantly (p <0.01) lower than the ovx, ovx+soy-, and ovx+E 2 groups. Liver lipids (mg/g) were significantly (p<0.05) lower in the animals that received E 2 or fed soy, but not those which were fed soy- or given IP. The ovx-induced increase in abdominal fat was completely reversed by soy and E 2 treatments but not by soy- or ipriflavone treatments. Soy, soy-, and IP had no uterotrophic activity as compared to E2. Ovariectomy significantly increased body weight gains which were not suppressed by any of the treatments except E 2. These data indicate that ipriflavone is effective in preventing the unfavorable changes in serum and liver cholesterol associated with ovarian hormone deficiency in this animal model. Moreover, the consumption of synthetic or natural isoflavones may offer a potential alternative therapy in the treatment of hypercholesterolemia in ovarian hormone-deficient women. @1997Elsevi~SciemccInc.
KEY WORDS: Estrogen, cholesterol, Iprifiavone, Isoflavones, Ovariectomy, Rats Address reprint requests to: Bahram H. Arjmandi, Department of Human Nutrition and Dietetics, 1919 West Taylor Street (MC/517), University of Illinois at Chicago, IL 60612-7256. Fax: (312)413-0319; e-mail: [email protected]
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B.H. ARJMANDI et al.
INTRODUCTION Women experience a lower rate of coronary heart disease (CHD) before menopause in comparison to men. However, after the onset of menopause, the risk of CHD increases drastically due to ovarian hormone deficiency (1,2). With regards to cardiovascular disease, high blood cholesterol has been implicated as a major risk factor (3). Estrogen replacement therapy (ERT) has shown potential for the reduction or prevention of CHD (4) and is also considered the most effective method to reduce the rate o f postmenopausal bone loss (5) in women who have experienced naturally or surgically induced menopause. The benefits of ERT have been exhibited primarily through favorable alterations in lipid and lipoprotein metabolism (6) and suppression of bone resorption. Unfortunately, ERT may be accompanied by side effects such as endometrial and breast cancer (7,8) and is therefore recommended only for women who have no contraindications. One alternative may be the use of estrogen-like substances (phytoestrogens) that are found either in plants, e.g. isoflavones, or made synthetically (ipriflavone). Until the turn of this century it was assumed that estrogens were exclusively produced by animals. However, the principle that plants also can produce phytoestrogens was established by 1966 (9). Now it is well known that certain plants and plant products contain these phytoestrogens. One group of such compounds which are reported to have estrogenic activity is fiavonoids (10). This group of compounds include isoflavones which are found in a limited number of plants and plant products including soy protein isolate which is a rich source of isoflavones: genistein and daidzein. The hypocholesterolemic properties of soy protein has received much attention lately (11). From the review of the literature it is not clear whether the cholesterol lowering effect of soy protein is due to its amino acid composition, nonprotein constituents such as saponins, phytic acid, isoflavones, or a combination of these factors. However, in recent years, there has been rising interest regarding the soy isoflavones and their influence not only on sex hormone metabolism, but also other biological activities including cholesterol-lowering properties (12). Genistein and related isoflavones, found predominantly in soy, have long been recognized as naturally occurring phytoestrogens (13). Both in vitro and in vivo studies have shown that genistein exerts a weak estrogenic effect, approximately 1 x 10"s to 1 x 10-s that of estradiol (14). Since the hypocholesterolemic effect of soy protein, in part, has been linked to its isoflavone content (11), we decided to use a synthetic isoflavone, ipriflavone, and compare its effects on serum and liver cholesterol and lipid with those of soy protein and 17B-estradiol in ovariectomized rats. Ipriflavone, 7-isopropoxy-3-phenyl-4H-l-benzopyran-4-one, a synthetic isoflavone, has been shown to have beneficial effects on bone in both postmenopausal women (15,16) and ovariectomized animal models (17). However, there are no studies in which the effect of this compound on lipid and cholesterol metabolism has been evaluated. Ipriflavone is very appealing because of its potential acceptability and its lack of demonstrated toxicity by human subjects (18). The purpose of this study was to compare the effects of soy protein isolate with normal isoflavone content and with reduced isoflavone content, ipriflavone, a synthetic isoflavone; and 17B-estradiol on lipid metabolism in ovariectomized rats.
IPRIFLAVONE: A HYPOCHOLESTEROLEMIC AGENT
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MATERIALS AND METHODS Animals and diets
Seventy-two 90 day-old female Sprague-Dawley rats, were purchased from Harlan Sprague-Dawley (Indianapolis, IN) and used for this study when they were 95 days old. On arrival, animals were housed in an environmentally controlled laboratory. Rats were acclimated to a standard laboratory non-purified diet for 5 days. After acclimation, using a randomized complete block design, rats were divided into six groups of 12 rats each and placed in pairs of two per cage: sham operated (sham), ovariectomized (ovx), ovx+soy, ovx+soy-, ovx+IP, and ovx+E2. For surgical purposes, rats were anesthetized with an intraperitoneal injection of ketamine hydrochloride and xylazine at doses of 100 mg/kg body weight and 5 mg/kg body weight, respectively. Bilateral ovariectomy were performed in the ovx, ovx+soy, ovx+soy-, ovx+IP, and ovx+F~ groups, and animals in the sham group went through the stress of surgery without removal of their ovaries. Following surgery, the animals were carefully watched and a heat lamp was used to keep the rats warm as they came out of anesthesia. Postoperatively, no additional analgesics or antibiotic were given to the animals. Known quantity of E2 was dissolved in a small amount of absolute ethanol and the volume was adjusted with sesame oil to a final concentration of 10 ~tg E2/ml. The solvent vehicle contained similar quantities of ethanol and sesame oil. Rats in the ovx+E2 group received 1 ml of the E2 solution/kg body weight, whereas rats in all other treatment groups received 1 ml of solvent vehicle/kg body weight subcutaneously on a daily basis. Sites of injection in each animal were varied daily in order to avoid possible complications such as accumulation of fluid under the skin. Rats in the sham, ovx, ovx+IP, and ovx+E2 groups were fed a powdered casein-based Teldad diet #88190 (Madison, WI) (Table 1). The soy-fed groups (soy and soy-) received similar diets in which casein was replaced with soy protein isolate without removal of its normal isoflavone content (soy) or soy protein isolate with reduced isoflavone content (soy-; containing less than 10% of the amount normally found in soy protein isolate). Ovx+IP animals received 100 mg/kg body weight/day of ipriflavone orally via gavaging in less than 2 ml deionized water containing 1% hydroxypropyl cellulose as an emulsifying agent (17). Rats which received E 2 consumed food ad libitum and their precise food intakes were measured every three days. Before each feeding, the food remaining was weighed and the amount ingested calculated. Rats in the other groups were pair-fed to the mean intake of the ovx+E2 animals and had free access to deionized water. Thirty-five days from the date of surgery, animals in a nonfasted state were anesthetized with a mixture of ketamine hydrochloride and xylazine as described above. After anesthesia, rats were sacrificed by exsanguination via bleeding from abdominal aorta. Blood samples were collected in nonheparinized tubes and serum was separated by centrifugation at 1500 x g for 20 minutes at 4°C. Aliquots of serum were frozen and kept at -20°C for later analyses. The liver was immediately removed, rinsed with ice-cold 0.4 mmol/L NaC1 solution, kept in sealed containers and stored at 70°C for lipid and cholesterol analyses. Guidelines for the ethical care and treatment of animals from the'Animal Care Committee of the University of Illinois at Chicago were strictly followed.
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B.H. AIqJMANDI et al. TABLE 1 Composition of Casein and Soy Protein Diets
Ingredients
Casein Diet ~
Soy Protein Diet
~/100 Protein Casein Soy Protein Isolate 2 Carbohydrate Sucrose Corn Starch Fiber Source - Cellulose~ Fat - Corn Oil Vitamin Mixture 4 Total cholecalciferol Mineral Mixture (Ca-P Deficient)5 Calcium Carbonate Sodium Phosphate, monobasic Potassium Phosphate, monobasic Potassium Citrate, monohydrate
22.70 22.70 41.76 20.00 5.60 5.70 1.0 0.00016 1.34 0.988 0.388 0.238 0.090
41,76 20,00 5.60 5.70 1.0 0.00016 1.34 0.988 0.388 0.238 0.090
~Teklad diet #88190 (Harlan Teklad, Madison, WI). :Soy protein isolate obtained from Protein Technologies International (St. Louis, MO). Soy protein isolate contained (mg/kg) of the following isoflavones: Genistin, 1462; genistein, 25.1; daidzin, 590; daidzein 11.3. 3Alphacel obtained from ICN Biochemicals (Costa Mesa, CA). 4Vitamin mixture (g/kg diet; TD 40060) obtained from Harlan Teklad (Madison, WI): p-aminobenzoic acid, 0.1101; ascorbic acid, 1.0166; biotin, 0.00044; vitamin B-12 (0.1% trituration), 0.0297; calcium pantothenate, 0.0661, choline dihydrogen citrate, 3.4969; folic acid, 0.00198; inositol, 0. I 101; menadione, 0.0495; niacin, 0.0991; pyridoxine HC1,0.0220; riboflavin, 0.0220; thiamin HC1, 0.0220; dry retinyl palmitate, 0.0044; dry d,1a-tocopheryl acetate, 0.2423; corn starch (diluent), 4.6669. 5Mineral mixture (TD 79055) obtained from Harlan Teklad (Madison, WI). This mixture is a modification of AIN 76.
Abdominal fat Post-euthanasia, all the organs within the abdominal cavity, including the adrenal glands, intestines, kidneys, liver and spleen were removed. Visceral fat and any visible fat adhering to the organs or abdominal wall were collected, blotted, and weighed to the nearest milligram.
Serum tri21vceride and total cholesterol, liver lipids and cholesterol Serum triglyceride and total cholesterol were determined enzymatically (Sigma Diagnostics, St. Louis, MO). Portions o f livers were homogenized, then extracted with a 2:1 (v/v) chloroform: methanol mixture. After addition of 7.3 g/L NaC1 solution to the extraction and separation o f phases, aliquots of the organic phase were analyzed for liver cholesterol. Liver cholesterol was determined
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889
using a color reagent of glacial acetic acid-FeSO4-H2SO4 (19). Liver lipids were determined using the Foich gravimetric method (20). The remainder of the organic phase was evaporated, dried to constant weight, and weighed to measure liver lipids. Liver cholesterol and lipids were calculated and reported both per g and whole liver weight. Statistical analyses
Data analysis involved computation of means and standard deviations (SD) for each of the treatment groups (21). Analysis of variance (ANOVA) was performed to determine whether there were statistically significant (p< 0. 05) differences by treatment group. When an ANOVA indicated any significant difference among the means, the Tukey-Kramer follow-up multiple comparison test was used to determine which means were significantly different. GraphPad Instat Software (version 2.0, 1993; San Diego, CA) was used for all statistical analysis (21,22)
RESULTS Body and orean weiehts
The data on body and organ weights are presented in Table 2. The rats were pair-fed to the mean food intake of the E 2 group and hence all the groups consumed the same amount of food (14.5+1.8 g/day). Although the mean initial body weights did not differ among the treatment groups, all the ovx groups, except the E2-administered group had significantly higher mean final body weight and weight gain in comparison to sham. Ovariectomy caused atrophy of uterine tissue, and this atrophy was prevented by E 2 administration but not by soy, soy- or ipriflavone treatments. The liver weights did not differ among the six treatment groups. The ovx-induced increase in abdominal fat was prevented by soy and E 2 treatments, but not by soy- or ipriflavone. Serum total cholesterol and triglycerides
Serum total cholesterol concentrations were significantly (p<0.05) higher in ovx control animals in comparison with sham. This ovx-induced rise in serum total cholesterol was prevented by soy, soy-, IP, and E2 treatments (Table 3). Serum triglyceride levels were not significantly affected by any of the treatments (Table 3). Liver cholesterol and lipid concentrations
When liver cholesterol concentrations were expressed per gram of liver (liver cholesterol), animals which were fed soy or received ipriflavone had significantly (p<0.05) lower liver cholesterol concentrations in comparison with ovx controls (Table 3). Liver total cholesterol concentrations were significantly (p<0.05) lower in (soy)-fed and E2-treated animals in comparison with ovx controls. When liver lipids were assessed per gram liver, the ovariectomy-induced rise in liver lipid levels were prevented by soy and E2 but not by soy- or IP treatments (Table 3). Liver total lipid concentrations were significantly (p<0.05) lower in soy, soy-, and E 2 groups compared with ovx control group.
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B.H. ARJMANDI et al. TABLE 2
Effects o f Ovariectomy (ovx), Soy with Normal Isoflavone Content (Soy), Soy with Reduced Isoflavone Content (Soy-), Ipriflavone (IP), and 1713-Estradiol (Fq) on Body and O r g a n Weights in Rats 1'2
Measures
Sham
Ovx
Ovx+Soy
Ovx+Soy-
Ovx+IP
Ovx+E2
Initial
212-46.7
2114-6.0
212-46.4
2124-6.4
2124-6.3
2124-6.2
Final
265=1:14.5b
299~12.6"
2954-16.6"
288:t:8.4"
2944-15.1"
249A27.5 b
Weight Gain
534-10.4 b
864-9.1'
844-15.7"
774-10.4 i
824-15.0"
37+7.5 b
Uterus
0.4974-0.14'
0.127:K).03 b
0.134+0.05 b
0.113+0.02 b
0.1184-0.02 b
0.5304-0.11 '
Liver
8.25+1.6
8.594-1.9
8.854-1.2
8.05:83.88
9.45~-0.95
7.694-0.66
Abdominal Fat
7.83+2.0 b
9.474-1.9"
7.68-41.3 b
8.20+2.0 "b
8.244-1.64~b
5.974-0.93 b
Body Weights (g)
Organ Weights (g)
1Values are m e a n s ± SD, n = 12 in each group, 2 Within a row, values that do not share the s a m e superscript letters are significantly (p <0.05) different fIom each other. TABLE 3 Effects o f Ovariectomy (Ovx), Soy with Normal Isoflavone Content (Soy), Soy with Reduced Isoflavone Content (Soy-), Ipriflavone (IP), and 17~3-Eslxadiol (E2) on Liver Cholesterol, Liver Lipids, Serum Total Cholesterol, and Serum Triglyceride Levels m R a t s ) ,2
Measures
Sham
Ovx
Ovx+Soy
Ovx+Soy-
Ovx+IP
Ovx+E2
total cholesterol
1.95~q3.25b
2.744-0.75"
1.62:1:0.25 b
1.8040.68 b
1.414-0.36 b
1.964-0.33 b
triglyeerides
0.61:83.19
0.66-40.10
0.54:1--0.15
0.54±0.19
0.594-0.10
0.714-0.18
Cholesterol (•mol/g liver)
7.7340.23 "b
8.35:L-0.85"
6.9341.00 b
8.20±1.73"
6.85+0.31 ~
8.2840.34"
Total cholesterol (~mol/whole liver)
62.64_9.4 ,b
71.74-7.2 °
58.7q-8.2 b
67.8:t:4.0 "b
63.5=1:4.9 "b
60.4:1:5.5b
Lipids (mg/g liveO
47.04-8.5 "b
48.5:t:5.2'
41.4±5.0 b
46.04-3.3 'b
47.044.4 "b
42.04-5.4 b
Total lipids (rag/whole liver)
3804-22 b
4234-17"
365q-31 b~
366+21 ~
4214-36"
3274-29 c
Serum (mmol/L)
Liver
1Values are m e a n s :t: SD, n = 8. 2 Within a row, values that do not share the same superscript letters are significantly (p<0.05) different fxom each other.
IPRIFLAVONE: A HYPOCHOLESTEROLEMIC AGENT
891
DISCUSSION The higher final mean body weights and weight gain in ovx animals suggest a shift in energy metabolism due to ovarian hormone deficiency. Other investigators have also reported increased body weights due to ovarian hormone deficiency in animals (23) and humans (24). Human studies have reported that hormone replacement therapy in postmenopausai women prevents increases in body fat (25), particularly centrally distributed fat (26) as was the case in this ovx animal model. Whereas ipriflavone did not have a significant effect on abdominal fat, both E 2 and soy protein with normal isoflavone content prevented the ovx-induced increase in abdominal fat. These findings suggest that the effect of the isoflavones present in soy protein on body fat may differ from that of ipriflavone. Hence, soy isoflavones may direct triglycerides to other tissues and away from adipose tissue for catabolism, similar to the proposed mechanism of action for estrogen on adipose tissue (27). In this study, the cholesterol lowering efficacy of soy protein isolate with reduced isoflavone content was somewhat diminished in comparison with that of soy protein isolate with normal isoflavone content. These observations suggest that some of the beneficial effects of soy protein on lipid metabolism are partially due to its isoflavones or other nonprotein components such as saponins which have been removed in processing. In support of our observations, a recent study by Anthony et al. (28) has shown that feeding soy protein rich in isoflavones to female Rhesus monkeys was more effective in lowering serum cholesterol than feeding a soy protein diet poor in isoflavone content. Balmir et al. (29) also observed that feeding a casein-based diet with added isoflavones (isolated from soy protein) to male rats resulted in a significant decline in serum total cholesterol concentrations. The hypocholesterolemic role ofisoflavones in the diet can be further supported by our findings that ipriflavone administration was effective in lowering serum total cholesterol in the absence of dietary soy protein. Since ipriflavone, a synthetic isoflavone, effectively prevented the ovx-induced rise in serum and liver cholesterol, this suggests that isofiavones, in general, play an important role in lipid metabolism. This is the first reported observation that ipriflavone potently suppresses the ovarian hormone deficiencyinduced rise in serum total cholesterol as much as 50%. These findings would be of great benefit to postmenopausal women, since ipriflavone is also reported to prevent bone loss associated with ovarian hormone deficiency (15,16,17), another major risk factor in this population. Additionally, it has been reported that ipriflavone is devoid of any known toxic side effects and is well tolerated by human subjects (18). The hypocholesterolemic properties of soy protein have been linked to enhanced fecal excretion of acidic and neutral sterols (30). In this regard, soy protein, or its other active components such as isoflavones, may behave in a similar fashion to certain soluble fibers, which enhance fecal sterol excretion causing cholesterol depletion from the body (31). Enhanced activity of hydroxymethylglutaryl CoA reductase, the major enzyme regulating hepatic cholesterol synthesis, in soy prote~n-fed animals has been reported by Huffand Carroll (32). These and other mechanisms (33) working alone or in combination, seem to contribute to the cholesterol lowering effects of soy protein or its active constituents. Additional studies are needed to further demonstrate their efficacy and illustrate their mode of action in humans.
892
B.H. ARJMANDI et al.
In conclusion, the findings of the present study suggest that ipriflavone, similar to estrogen and soy protein isolate, can effectively prevent the ovarian hormone deficiency-associated rise in serum cholesterol. If long term studies in humans demonstrate that ipriflavone is free of undesirable side effects, then it might be considered a desirable alternative for dealing with postmenopausal women's increased risks of coronary heart disease and osteoporosis.
ACKNOWLEDGMENTS The assistance of Daxa Amin, M,S., Mary Jane Getlinger, M.S., Noopur Goyal, M.S., and James Artwohl, D.V.M. in animal care, participation in specimen collection and technical procedures is gratefully acknowledged.
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Accepted for publication January 10, 1997.