Effects of soy protein supplemented with methionine on blood lipids and adiposity of rats
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BASIC NUTRITIONAL INVESTIGATION Effects of Soy Protein Supplemented With Methionine on Blood Lipids and Adiposity of Rats Mark Kern, PhD, RD, Danielle Ellison, BS, Yessenia Marroquin, BS, Marie Ambrose, MS, and Kelly Mosier, MS From the Department of Exercise and Nutritional Sciences, San Diego State University, San Diego, California, USA OBJECTIVE: The effects of soy protein isolate (SPI) versus casein on blood lipids and adiposity were investigated in rats fed methionine-equivalent diets. METHODS: Twenty-eight male Sprague-Dawley rats (230 to 250 g) were assigned in equal numbers to groups consuming SPI- or casein-based diets (20%) supplemented with L-methionine. After 28 d, blood was collected for triacylglycerol, total cholesterol, and high-density lipoprotein cholesterol assessment and epididymal fat pads were weighed. RESULTS: Food intake (519 90 versus 490 115 g), weight gain (144 35 versus 133 28 g), food efficiency ratio (0.29 0.09 versus 0.28 0.06), epididymal fat pad weights (5.409 2.076 versus 4.768 1.867 g), and serum concentrations of triacylglycerol (96.3 41.8 versus 93.4 37.4 mg/dL) and high-density lipoprotein cholesterol (32.6 7.4 versus 33.8 4.4 mg/dL) were similar between the casein and SPI groups, respectively. However, total cholesterol (73.8 17.8 versus 59.3 11.9 mg/dL) concentration was higher for the casein-fed rats than for the SPI-fed rats, respectively (P 0.05). CONCLUSIONS: These results suggest that methionine supplementation may eliminate the decreased fat deposition previously ascribed to soy protein; however, methionine did not abolish the commonly observed hypocholesterolemic effects of soy. Nutrition 2002;18:654 – 656. ©Elsevier Science Inc. 2002 KEY WORDS: soy, casein, protein, methionine, lipid, cholesterol, fat INTRODUCTION Diets rich in soy protein improve the blood lipid profiles of animals 1 and humans. 2 The mechanisms for these changes have been studied extensively, with the conclusion that the hypocholes- terolemic effects of soy may be due to its amino acid profile or other non-protein components in soy. 3 Isoflavones have been iden- tified as possible non-protein contributors to soy’s hypocholester- olemic effects. 4 However, the amino acid composition of soy cannot be dismissed as a potential explanation. In fact, a major difference in the amino acid profiles of soy protein versus casein is the methionine content, which is typically present in soy protein at less than half the amount found in casein. Because methionine has been demonstrated to elevate serum cholesterol concentration, 5 the methionine difference of the two proteins offers a plausible expla- nation for differences in cholesterol observed between the two proteins. The role of soy protein in the maintenance of energy balance also has been examined. When part of a starch-based diet, soy as opposed to casein appears to reduce body fat gain of rats. 6 Baba et al. 1 also observed greater fat deposition in rats fed casein versus soy. This effect, however, may not be exclusive for soy protein but rather an effect of plant proteins in general. Kayashita et al. 7 fed buckwheat protein extract to rats, resulting in lower gains in epididymal fat pad weight in comparison to casein. This finding may be explained by the induction of lipogenic enzymes 7 or simply may be due to the quantity of essential amino acids in the proteins consumed. 8 We investigated the effects of soy protein isolate (SPI) versus those of casein on blood lipids and adipose deposition in rats provided with diets supplemented to supply equal amounts of methionine. MATERIALS AND METHODS Animals and Diets Twenty-eight male Sprague-Dawley rats (230 to 250 g) were housed individually in wire-bottomed cages on a 12-h light– dark cycle. Temperature and humidity were controlled at approximately 20°C to 24°C and 40% to 45% humidity, respectively. Water was available ad libitum. All procedures for use of animals were reviewed and approved by the animal subjects committee at San Diego State University. Rats were assigned in equal numbers to groups consuming diets (Table I) consisting of 20% by weight of protein from either SPI or casein for 28 d. L-methionine was adjusted to be equivalent in both diets. Experimental Design and Procedures Rats were provided free access to their food for 28 d. At the conclusion of the study, food was removed from the cages 12 h before rats were killed with carbon dioxide. Carcasses were de- capitated and exsanguinated. Blood was collected into test tubes, allowed to clot at room temperature, and centrifuged at 1200g for 15 min at 2°C to 8°C. Serum was stored at 70°C until time of analysis. Epididymal fat pads were dissected and weighed. Serum total cholesterol, high-density lipoprotein cholesterol, and triacyl- glycerols were assessed colorimetrically with kits from Sigma Diagnostics (St. Louis, MO, USA). All data are expressed as mean standard deviation. Differ- Correspondence to: Mark Kern, PhD, RD, Department of Exercise and Nutritional Sciences, San Diego State University, 5500 Campanile Drive, San Diego, CA 92182-7251, USA. E-mail: [email protected] Nutrition 18:654 – 656, 2002 0899-9007/02/$22.00 ©Elsevier Science Inc., 2002. Printed in the United States. All rights reserved. PII S0899-9007(02)00783-9
Transcript of Effects of soy protein supplemented with methionine on blood lipids and adiposity of rats
BASIC NUTRITIONAL INVESTIGATION
Effects of Soy Protein Supplemented WithMethionine on Blood Lipids and Adiposity of RatsMark Kern, PhD, RD, Danielle Ellison, BS, Yessenia Marroquin, BS, Marie Ambrose, MS,
and Kelly Mosier, MSFrom the Department of Exercise and Nutritional Sciences, San Diego State University,
San Diego, California, USA
OBJECTIVE: The effects of soy protein isolate (SPI) versus casein on blood lipids and adiposity wereinvestigated in rats fed methionine-equivalent diets.METHODS: Twenty-eight male Sprague-Dawley rats (230 to 250 g) were assigned in equal numbers togroups consuming SPI- or casein-based diets (20%) supplemented with L-methionine. After 28 d, bloodwas collected for triacylglycerol, total cholesterol, and high-density lipoprotein cholesterol assessmentand epididymal fat pads were weighed.RESULTS: Food intake (519 � 90 versus 490 � 115 g), weight gain (144 � 35 versus 133 � 28 g), foodefficiency ratio (0.29 � 0.09 versus 0.28 � 0.06), epididymal fat pad weights (5.409 � 2.076 versus4.768 � 1.867 g), and serum concentrations of triacylglycerol (96.3 � 41.8 versus 93.4 � 37.4 mg/dL)and high-density lipoprotein cholesterol (32.6 � 7.4 versus 33.8 � 4.4 mg/dL) were similar between thecasein and SPI groups, respectively. However, total cholesterol (73.8 � 17.8 versus 59.3 � 11.9 mg/dL)concentration was higher for the casein-fed rats than for the SPI-fed rats, respectively (P � 0.05).CONCLUSIONS: These results suggest that methionine supplementation may eliminate the decreased fatdeposition previously ascribed to soy protein; however, methionine did not abolish the commonlyobserved hypocholesterolemic effects of soy. Nutrition 2002;18:654–656. ©Elsevier Science Inc. 2002
KEY WORDS: soy, casein, protein, methionine, lipid, cholesterol, fat
INTRODUCTION
Diets rich in soy protein improve the blood lipid profiles ofanimals1 and humans.2 The mechanisms for these changes havebeen studied extensively, with the conclusion that the hypocholes-terolemic effects of soy may be due to its amino acid profile orother non-protein components in soy.3 Isoflavones have been iden-tified as possible non-protein contributors to soy’s hypocholester-olemic effects.4 However, the amino acid composition of soycannot be dismissed as a potential explanation. In fact, a majordifference in the amino acid profiles of soy protein versus casein isthe methionine content, which is typically present in soy protein atless than half the amount found in casein. Because methionine hasbeen demonstrated to elevate serum cholesterol concentration,5 themethionine difference of the two proteins offers a plausible expla-nation for differences in cholesterol observed between the twoproteins.
The role of soy protein in the maintenance of energy balancealso has been examined. When part of a starch-based diet, soy asopposed to casein appears to reduce body fat gain of rats.6 Baba etal.1 also observed greater fat deposition in rats fed casein versussoy. This effect, however, may not be exclusive for soy protein butrather an effect of plant proteins in general. Kayashita et al.7 fedbuckwheat protein extract to rats, resulting in lower gains inepididymal fat pad weight in comparison to casein. This findingmay be explained by the induction of lipogenic enzymes7 orsimply may be due to the quantity of essential amino acids in theproteins consumed.8
We investigated the effects of soy protein isolate (SPI) versus
those of casein on blood lipids and adipose deposition in ratsprovided with diets supplemented to supply equal amounts ofmethionine.
MATERIALS AND METHODS
Animals and Diets
Twenty-eight male Sprague-Dawley rats (230 to 250 g) werehoused individually in wire-bottomed cages on a 12-h light–darkcycle. Temperature and humidity were controlled at approximately20°C to 24°C and 40% to 45% humidity, respectively. Water wasavailable ad libitum. All procedures for use of animals werereviewed and approved by the animal subjects committee at SanDiego State University.
Rats were assigned in equal numbers to groups consuming diets(Table I) consisting of 20% by weight of protein from either SPIor casein for 28 d. L-methionine was adjusted to be equivalent inboth diets.
Experimental Design and Procedures
Rats were provided free access to their food for 28 d. At theconclusion of the study, food was removed from the cages 12 hbefore rats were killed with carbon dioxide. Carcasses were de-capitated and exsanguinated. Blood was collected into test tubes,allowed to clot at room temperature, and centrifuged at 1200g for15 min at 2°C to 8°C. Serum was stored at �70°C until time ofanalysis. Epididymal fat pads were dissected and weighed. Serumtotal cholesterol, high-density lipoprotein cholesterol, and triacyl-glycerols were assessed colorimetrically with kits from SigmaDiagnostics (St. Louis, MO, USA).
All data are expressed as mean � standard deviation. Differ-
Correspondence to: Mark Kern, PhD, RD, Department of Exercise andNutritional Sciences, San Diego State University, 5500 Campanile Drive,San Diego, CA 92182-7251, USA. E-mail: [email protected]
Nutrition 18:654–656, 2002 0899-9007/02/$22.00©Elsevier Science Inc., 2002. Printed in the United States. All rights reserved. PII S0899-9007(02)00783-9
ences among groups were assessed with a one-way analysis ofvariance. An � level of P � 0.05 was selected as the criterion forstatistical significance.
RESULTS
Food intake, weight gain, and food efficiency ratio were similarbetween the casein and SPI groups (Table II). Further, fat depo-sition as indicated by epididymal fat pad weight did not differbetween groups.
Figure 1 depicts the effects of the two diets on the blood lipidprofile. Serum concentrations of triacylglyerol (1.09 � 0.47 versus1.06 � 0.42 mM/L) and high-density lipoprotein cholesterol(0.84 � 0.19 versus 0.88 � 0.11 mM/L) were similar (P � 0.05)between the casein and SPI groups, respectively. Total cholesterol(1.91 � 0.46 versus 1.54 � 0.31 mM/L) concentration was higher(P � 0.05) for the casein-fed rats than for the SPI-fed rats,respectively.
DISCUSSION
These results suggest that the low amount of methionine in SPIversus casein may be responsible for its purported benefit ofdecreasing body fat accumulation. In contrast, methionine supple-mentation did not abolish the commonly observed hypocholester-olemic effects of soy protein in relation to casein, which indicatesthat some factor other than methionine is at least partly responsiblefor the cholesterol-lowering effect of soy.
Isoflavones provide a well-established possibility for explain-ing the hypocholesterolemic properties of soy. However, theisoflavone content of the diets used in this study are unknown. Inprevious research, isoflavones were demonstrated to be responsiblefor part, but not all, of the cholesterol-lowering effects of SPI.4 Itis likely that multiple factors are responsible for this phenomena.With regard to the amino acid profiles of these proteins, differ-ences other than methionine content exist between casein and soy.Although this study controlled the quantity of methionine in thediet, other amino acids were not adjusted.
Glycine occurred at almost twice the concentration in the SPI(3.6 g/kg) as in casein (1.9 g/kg) used in this study. Previousresearch suggested that the higher ratio of methionine to glycine incasein may be responsible for elevations of serum cholesterol.9The ratios of methionine to glycine in the present diets were 2.3and 1.2 for casein and SPI, respectively. Glycine added to acasein-based diet fed to rats was shown to lower serum cholesterolconcentration.5 Although amounts of methionine were equal inboth diets, the glycine difference between the sources of proteinmight have been responsible for the differences in serum choles-terol concentration. Further experimentation is required to deter-mine whether this is the case.
TABLE I.
COMPOSITION OF SEMIPURIFIED DIETS*
Ingredients Casein (g/kg) SPI (g/kg)
Casein† 211 —SPI‡ — 217Cornstarch 602 596Cellulose 50 50Coconut oil 70 70Corn oil 10 10Cholesterol 10 10Mineral mix§ 35 35Vitamin mix� 10 10Choline bitartrate 2 2
* L-Methionine was added at the rate of 1.5 g/kg and 3.3 g/kg of pro-tein source for the casein and SPI diets, respectively.† High-nitrogen casein, ICN Biochemicals (Aurora, OH, USA); 95.0%protein. Amino acid composition (g/kg); alanine, 2.8; arginine, 3.7; as-partic acid, 5.8; cystine, 3.6; glutamic acid, 21.1; glycine, 1.9; histidine,2.7; soleucine, 5.9; leucine, 9.0; lysine, 7.3; methionine, 2.9; phenylala-nine, 5.2; proline, 11.0; serine, 5.6; threonine, 4.0; tryptophan, 1.3; ty-rosine, 5.6; valine, 6.9.‡ SPI, ICN Biochemicals (Aurora, OH, USA); 92.0% protein. Aminoacid composition (g/kg): alanine, 3.6; arginine, 6.7; aspartic acid, 10.2;cystine, 1.1; glutamic acid, 17.5; glycine, 3.6; histidine, 2.2; isoleucine,4.3; leucine, 6.8; lysine, 5.6; methionine, 1.1; phenylalanine, 4.7; pro-line, 4.9; serine, 4.5; threonine, 3.2; tryptophan, 1.2; tyrosine, 3.8; va-line, 4.2.§ AIN-76 Mineral Mix (g/kg mixture): calcium phosphate, dibasic,500.0; sodium choloride, 74.0; potassium citrate, 220.0; potassium sul-fate, 52.0; magnesium oxide, 24.0; ferric citrate (16–17% Fe), 6.0; zinccarbonate (70% Zn), 1.6; cupric carbonate (53–55% Cu), 0.3; potassiumiodate, 0.01; sodium selenite, 0.01; chromium potassium sulfate, 0.55;sucrose, 180.� AIN-76 Vitamin Mix (per-kilogram mixture): thiamin HCI, 600 mg;riboflavin, 600 mg; pyridoxine HCI, 700 mg; nicotinic acid, 3 g;D-calcium pantothenate, 1.6 g; folic acid, 200 mg; D-biotin, 20 mg; cya-nocobalamin, 1 mg; retinyl palmitate or acetate, 400 000 IU; dl-�-tocopheryl acetate, 5000 IU; cholecalciferol, 2.5 mg; menaquinone, 5.0mg; sucrose, 972.9.SPI, soy protein isolate.
TABLE II.
FOOD INTAKE, WEIGHT GAIN, FER, AND FAT PAD WEIGHTSOF CASEIN-VERSUS SPI-FED RATS
Variable Casein SPI
Food intake (g) 519 � 90 490 � 115Weight gain (g) 144 � 35 133 � 28FER 0.29 � 0.09 0.28 � 0.06Epididymal fat (g) 5.409 � 2.076 4.768 � 1.867
FER, food efficiency ratio; SPI, soy protein isolate.
FIG. 1. Serum lipid concentrations of rats fed SPI versus casein, withmethionine content adjusted to be equivalent. *Significantly different be-tween groups (P � 0.05). HDL-C, high-density lipoprotein cholesterol;SPI, soy protein isolate; TC, total cholesterol; TG, triacylglycerol.
Nutrition Volume 18, Numbers 7/8, 2002 655Effects of Soy Protein on Blood Lipids of Rats
Kritchevsky et al.10 suggested that the ratio of lysine to argininemight be a candidate for causing the greater serum cholesterol thatoccurs with casein feeding, potentially due to increased insulinsensitivity. The protein sources in the current study had lysine:arginine ratios of 2.0 and 0.8 for casein and SPI, respectively. Thisnotion was supported by Morita et al.9 who detected a positivecorrelation between serum cholesterol concentration and the ratioof lysine to arginine. In the present study, serum triacylglycerolconcentration did not differ between the two dietary groups. Be-cause increased insulin sensitivity would be expected to lowercholesterol by decreasing liver synthesis and secretion of triacyl-glycerols rich in very–low-density lipoproteins, the similarity oftriacylglycerol concentration does not support this idea. However,neither postprandial triacylglycerol concentration nor insulinemicresponses of the proteins were measured in this study, so thispotential mechanism cannot be completely discounted.
Of note, most previous studies in which amino acids were fedin the same ratios as found in SPI and casein suggested that theamino acid pattern may not be responsible for the positive effectsof soy on blood lipids.11–13 However, because the patterns were fedas individual amino acids as opposed to whole proteins, theseresults may not represent the same results that occur with whole-protein feeding. A difference in bioavailability between free aminoacids and amino acids from whole proteins may offer an explana-tion for the lack of similar effects. In contrast, similarly designedresearch by Morita et al.9 suggested that the pattern of amino acidcompositions in SPI and casein is responsible for the differences incholesterol metabolism. Thus, a consensus regarding this issue isnot available.
Another potential mechanism for the cholesterol-lowering ef-fect of soy is enhanced fecal steroid excretion.14 Greater fecalsteroid excretion may increase bile acid production from choles-terol, thus reducing serum cholesterol concentration. However,some research suggested that increased fecal bile acid secretiondoes not necessarily result in decreased serum cholesterol concen-tration.15 Further, although it is not possible to exclude enhancedsteroid excretion as a potential mechanism, research by Morita etal.9 suggested that this is unlikely to be a primary mechanism inrats.
The effects of soy on body composition have been an area ofless interest; however, research in which methionine content wasnot constant between dietary groups suggests that soy may reducefat deposition in rats.6 In that study, SPI when fed as a componentof a cornstarch-based diet significantly reduced fat gains relative tocasein. The investigators suggested that a potential explanation forthis effect is the difference in the lysine:arginine ratios of theproteins. The lower ratio for SPI may reduce plasma insulinconcentrations and increase insulin sensitivity, thus reducing riskof fat deposition. Further, elegant research by Lavigne et al.16
demonstrated that soy protein improves glucose tolerance andinsulin sensitivity compared to casein in rats. In addition, post-prandial insulinemia is reduced in humans after soy protein ratherthan after casein consumption.17
Although the insulinemic response was not assessed in thisstudy, overall alterations in body weight gains and fat depositionwere not detected. The current study suggested that methioninesupplementation to SPI negates any effects on fat deposition asmeasured by epididymal fat pad weight. A possible explanation forthis finding is that the lower fat deposition and trend for decreasedbody weight gain detected in the SPI-fed rats of the study byHurley et al.6 was simply due to the relative lack of an essentialamino acid. Perhaps the lower fat deposition detected when me-thionine is not added to SPI is merely an indicator of methionine
deficiency. In the present study, the added methionine may havelead to a more optimal growth of all tissues.
This study suggests that methionine supplementation elimi-nates the decreased fat deposition previously ascribed to soyprotein; however, a relative lack of methionine in soy protein maynot be responsible for its commonly observed hypocholesterolemiceffects. Future research examining the cholesterol-lowering effectof soy should include controlling not only methionine content ofthe diet but also the methionine:glycine ratio. Studies of this sortalso should use isoflavone-extracted SPI to reduce the potentialconfounding hypocholesterolemic effects of these compounds.
ACKNOWLEDGMENTS
The authors acknowledge the contributions of the Spring 2000Advanced Nutrition Laboratory students at San Diego State Uni-versity who assisted in conducting and evaluating this research.
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(For an additional perspective, see Editorial Opinions)
656 Kern et al. Nutrition Volume 18, Numbers 7/8, 2002
