Soy protein without isoflavones reduces aortic total and cholesterol ester concentrations greater than soy protein with isoflavones compared流星飞
with cain in hypercholesterolemic hamsters ☆
Thomas A.Wilson a,⁎,Robert J.Nicolosi a ,Timothy Kotyla a ,Brent Fleckinger b
a
Department of Clinical Laboratory and Nutritional Sciences,Center for Health and Dia Rearch,University of Massachutts Lowell,
Lowell,MA 01854,USA
b
Archer Daniels Midland Company,Randall Rearch Center,Decatur,IL 62526,USA
Received 11September 2006;revid 23April 2007;accepted 7May 2007
Abstract
The prent study was designed to investigate the effects of replacing cain with soy protein either containing isoflavones or not on plasma lipids and lipoprotein cholesterol concentrations and early aortic atherosclerosis (measured as aortic cholesterol concentration)in hypercholesterolemic hamsters.Thirty,8-week-old,male F 1B Golden Syrian hamsters were individually houd and fed a mipurified hypercholesterolemic diet (HCD)containing 22%cain,20%coconut oil,2%corn oil,and 0.12%cholesterol (wt/wt)for a period of 2weeks.The hamsters were then divided into 3groups of 10bad on similar mean fasting plasma cholesterol concentrations and placed on experimental diets for 6weeks.Group 1continued on the HCD (cain).Group 2was fed the HCD with 22%water-washed soybean protein isolate (soy +I)(1.76mg of isoflavones per gram of protein)in place of cain.Group 3was fed the HCD with 22%alcohol-washed soybean protein isolate (soy −I)(0.08mg isoflavones per gram of protein)in place of cain.Compared with hamsters fed cain,hamsters fed both soy diets had significant decreas in their plasma total and non –high-density lipoprotein (very low and low-density lipoprotein)–cholesterol concentrations by week 2,which continued across weeks 4and 6.However,the hamsters fed the soy −I diet accumulated significantly less aortic total cholesterol compared with the hamsters fed cain and the soy +I diet.In addition,the hamsters fed the soy −I diet accumulated significantly less aortic cholesterol ester compared with the hamsters fed cain only.In conclusion,dietary replacement of cain with soy protein either containing isoflavones
or not in hypercholesterolemic hamsters lowers plasma total and non –high-density lipoprotein –cholesterol concentrations;however,only the soy protein without isoflavones reduced aortic cholesterol accumulation.©2007Elvier Inc.All rights rerved.
Keywords:
Soy protein;Cain;Plasma cholesterol;Aortic cholesterol;Hamster
中考新政1.Introduction
There have been studies in human beings [1,2]and various animal models [3-10]that have shown that substitution of soybean protein for cain lowers plasma cholesterol concentrations.However,the extent to which plasma cholesterol is reduced when cain is replaced by soybean protein is highly variable and may depend on at least 3factors:the intake of dietary cholesterol,the degree
of
Nutrition Rearch 27(2007)498–
504
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This work was supported by Archer Daniels Midland Company,Randall Rearch Center,Decatur,Ill (RJN).
⁎Corresponding author.Tel.:+19789344509;fax:+19789343025.E-mail address:thomas_wilson@uml.
edu (T.A.Wilson).0271-5317/$–e front matter ©2007Elvier Inc.All rights rerved.doi:10.1016/j.nutres.2007.05.005
hypercholesterolemia,and the level of protein fed.Previous studies have shown that the differential cholesterolemic effect of dietary cain and soybean protein becomes evident only when high-cholesterol diets are fed[7].In human beings,substitution of soybean protein for cain in the diet resulted in a decrea in plasma cholesterol levels when the diets contained cholesterol[1]or when fed to hypercholes-terolemic patients[2].The type of protein had little effect when low-cholesterol diets were ud or when fed to normolipidemic subjects[11,12].Similar results have been found in experimental animals.The feeding of cholesterol-containing diets to rats[4],pigs[5],monkeys[6,10],and hamsters[8,9]has shown a clear differential cholesterolemic effect of dietary cain and soybean protein.In contrast,the feeding of a cholesterol-free diet did not show a differential respon to the type of dietary protein[13,14].
Most of the dietary protein studies including our previous communications on plasma lipids and lipoproteins in hamsters[8]and monkeys[6,10]ud soybean protein isolate as the source of soy protein.Despite the composi-tional differences,in more recent publications[15,16]we demonstrated that,relative to cain,both soybean protein isolate and soybean protein concentrate fed at the20%or
40%level significantly reduced plasma total cholesterol (TC)and low-density lipoprotein–cholesterol(LDL-C).
大口径狙击步枪
The cholesterol-lowering mechanism of soy protein is still far from clear.This effect on blood cholesterol concentrations is thought to be due largely to the amino acid composition of soy protein.However,studies in which amino acids patterned after soy protein or cain were fed to experimental animals have suggested that other nonprotein components prent in soy may be partially responsible for the hypocholesterolemic effect[17-19].There are a number of non–amino acid effects found in soy products that have been associated with lowering blood lipids[20].The include soybean oil[21],soy lecithin [21],saponins[22],protein digestibility[23],protein phos-phorylation[24],and isoflavones/phytoestrogens[25-27].
Although most studies have shown that the replacement of cain with soy protein lowers blood cholesterol levels, there still exists some controversy over whether soy protein containing isoflavones lowers it better than soy protein not containing isoflavones and whether there is a difference in early atherosclerosis development on the diets.Therefore, the prent study was designed to investigate the effects of replacing cain as the sole protein source with water-washed soy protein,which contains isoflavones,vs alcohol-washed soy protein,which does not contain isoflav
ones,on the accumulation of cholesterol in the aortic arch of hypercholesterolemic hamsters.
2.Methods and materials
2.1.Experimental animals and diets
Thirty,8-week-old,male F1B Golden Syrian hamsters (Mesocricetus auratus)(BioBreeders,Inc,Watertown,Mass)were individually houd in hanging stainless steel wire-bottomed cages in an environmentally controlled room with a12-hour light/dark cycle and were fed a nonpurified standard diet(Purina Rodent Chow,Purina,St Louis,Mo)ad libitum for1week to get acclimated to the facility.The hamsters were then fed a control mipurified hypercholes-terolemic diet(HCD)(Rearch Diets,New Brunswick,NJ) containing22%cain,20%coconut oil,2%corn oil,and 0.12%cholesterol,by weight,for a period of2weeks.After an overnight fast(16hours),the hamsters were bled and plasma cholesterol concentrations were measured(week0). The hamsters were then divided into3groups of10bad on similar mean plasma cholesterol concentrations and placed on the different diets for an additional6weeks.Group1 continued on the control HCD(cain).Group2was fed the HCD with22%water-washed soybean protein isolate with isoflavones(1.76mg of isoflavones per gram of protein)in place of cain as the sole protein source in the diet(soy+I). Group3was fed th
e HCD with22%alcohol-washed soybean protein isolate without isoflavones(0.08mg isoflavones per gram of protein)in place of cain as the sole protein source in the diet(soy−I).The complete composition of the diets is shown in Table1.Food disappearance and body weights were monitored on a weekly basis throughout the study.Hamsters were main-tained in accordance with the guidelines of the Committee on Animals of the University of Massachutts Lowell Rearch Foundation and the guidelines prepared by the Committee on Table1
Composition of diets fed to hamsters for10weeks a
Ingredient(g/kg diet)Cain Soy+I Soy−I Cain217.0––Water-washed soy protein b–217.0–
Alcohol-washed soy protein c––217.0 L-Methionine 3.0 3.0 3.0 Lodex10125.0125.0125.0 Corn starch225.0225.0225.0 Cellulo151.8151.8151.8 Coconut oil(hydrogenated)200.0200.0200.0 Corn oil20.020.020.0 Mineral mix d10.010.010.0 Calcium phosphate,dibasic13.013.013.0 Calcium carbonate 5.5 5.5 5.5 Potassium citrate16.516.516.5 Vitamin mix e10.010.010.0 Choline bitartrate 2.0 2.0 2.0 Cholesterol 1.2 1.2 1.2 Protein(%energy)18.018.018.0 Carbohydrate(%energy)34.034.034.0 Fat(%energy)47.047.047.0
a Semipurified diets were supplied by Rearch Diets,Inc.
b Soy protein supplied by Archer Daniels Midland Company and contains1.04mg genistein and0.71mg of daidzein per gram of soy protein.
c Soy protein supplie
d by Archer Daniels Midland Company and contains0.03mg genistein and0.01mg of daidzein per gram of soy protein.
d S10026=mineral mix(rd-96salt mix without calcium,phosphorus, and potassium).
e V10001=vitamin mix for AIN-76A rodent diet.
政治责任感499
T.A.Wilson et al./Nutrition Rearch27(2007)498–504
Care in U of Laboratory Animals Resources,National Rearch Council(Department of Health,Education,and Welfare publication no.85-23,revid1985).
2.2.Plasma lipoprotein cholesterol and triacylglycerol measurements
Blood samples were collected after an overnight fast (16hours)and measured for plasma lipid and lipoprotein cholesterol concentrations at weeks0,2,4,and6.Blood from fasted hamsters,anesthetized with ultrapure50:50CO2: O2(Northeast Airgas,Salem,NH),was collected via the retroorbital sinus into heparinized tubes;and plasma was harvested after low-speed centrifugation at2500g for 15minutes at room temperature.Plasma was analyzed for plasma TC,high-density lipoprotein–cholesterol(HDL-C), very low density lipoprotein–cholesterol(VLDL-C)and LDL-C(non–HDL-C),and triacylglycerol(TG).Plasma TC [28]and TG[29]were measured enzymatically using the Infinity Cholesterol Reagent(procedure401)and Triacyl-glycerol(GPO-Trinder)Reagent(procedure337)from Sigma Diagnostics(Sigma-Aldrich,St Louis,Mo)with a Cobas Mira Plus Clinical Chemistry Autoanalyzer(Roche, Bal,Switzerland);and after the apolipoprotein B–contain-ing lipoproteins VLDL and LDL were precipitated with phosphotungstate reagent[30],the supernatant was assayed for HDL-C by the same method stated for TC.Plasma non–HDL-C was calculated from the difference between TC and HDL-C.Accuracy and precisions of plasma lipid and lipoprotein determinations are maintained by participation in the Center for Dia Control–National Heart,Lung,and Blood Institute Standardization Program.
2.3.Aortic cholesterol measurements
At the end of week6,hamsters were anesthetized with an intraperitoneal injection of sodium pentobarbital(62.5mg/ mL at a dosage of0.2-0.25mL/200g of body weight)(Henry Schein,Port Washington,NY);and aortic tissue was obtained for determination of cholesterol concentration [31].Specimens were rind with phosphate-buffered saline and cleaned of blood and connective tissue;subquently, they were stored in vials at−80°C for subquent analysis. To measure cholesterol concentrations in the aortic arch,a piece of aortic tissue extending from as clo to the heart as possible to the branch of the left subclavian artery was ud (approximately20-40mg).The tissue was cleaned,weighed, and placed in a vial containing4mL of methanol and10mL of chloroform.The sample was mixed vigorously and left at room temperature for48hours before extraction.The solution was then placed in a37°C water bath under N2. When approximately half of the solution was evaporated, 1mL of chloroform with1%Triton-100was added,mixed, and evaporated to dryness at37°C under N2.Two hundred fifty microliters of distilled water was added to the samples, vortexed,and placed in a shaking water bath at37°C for 20minutes to solubilize the lipid.After incubation,aortic total and free cholesterol concentrations were determined in triplicate using25μL of sample enzymatically(Wako Chemicals,Richmond,Va)using an microplate reader. Aortic cholesterol ester concentration was determined as the difference between the total and the free cholesterol concentrations.A pilot study was conducted to evaluate the extent to which this proc
edure removed tissue choles-terol.Aortic cholesterol concentrations were determined after tissue was placed in solvent(4mL of methanol and 10mL of chloroform)overnight with frequent vigorous mixing and compared with the concentrations obtained after tissue mincing or homogenization as reported pre-viously[32].No significant differences in aortic cholesterol content were obrved between the different cholesterol extraction procedures.
2.4.Statistical analys
SigmaStat software(Jandel Scientific,San Rafael,Calif) was ud for all statistical evaluations.A repeated-measures 2-way analysis of variance(ANOVA)was ud to analyze body weights and plasma lipid and lipoprotein cholesterol concentrations between the3diet groups and within each group for the4time points.A1-way ANOV A was ud to analyze food consumption data and aortic cholesterol concentrations between the3diet groups.When statistical significance was found by either ANOVA,the Student-Newman-Keuls paration of means was ud to determine group differences.All values are expresd as mean±SEM, and statistical significance was t at P b.05[33].
3.Results
The hamsters adapted well to all diets,and all hamsters survived the dietary treatments.No differences were obrved between dietary treatment groups for food consumption over the cour of the6-week study(Table2).All animals for each dietary group incread their body weight significantly by the end of the6weeks(Table2).The body weights for the hamsters during the dietary treatment period were not significantly different from each other,although the hamsters fed the cain and water-washed soy protein gained slightly more weight than the hamsters fed the alcohol-washed soy protein(Table2).
Total plasma cholesterol concentrations in hamsters consuming the HCD were approximately the same in all groups after a2-week lead-in diet(Table3).After the lead-in Table2
Initial and final body weights and food composition of hamsters after consuming dietary treatments for6weeks
Diet Cain Soy+I Soy−I
Initial body weight,g88.9±1.7792.6±2.9187.5±2.51 Final body weight,g104.1±2.71103.5±2.1498.4±2.09 Food consumption,g/d8.19±0.307.92±0.377.81±0.32
Values are means±SEM;n=10.
500T.A.Wilson et al./Nutrition Rearch27(2007)498–504
period,hamsters were placed on the indicated diets.
Compared with hamsters fed the cain HCD,hamsters fed
the water-and alcohol-washed soy protein–containing diets
had significant decreas in their plasma TC concentrations
by week2(−43%and−38%,respectively),which continued
across week4(−28%and−31%,respectively)and week6
(−32%and−27%,respectively)(Table3).No differences
were obrved between the hamsters fed the water-washed or
alcohol-washed soy protein diets at any time point for plasma
央组词组
TC concentrations.
Plasma LDL-C concentrations in hamsters consuming the
HCD were approximately the same in all groups after a
2-week lead-in diet(Table4).Compared with hamsters fed
the cain HCD,hamsters fed the water-and alcohol-washed
soy protein–containing diets had significant decreas in
their plasma LDL-C concentrations by week2(−60and −53%,respectively),which continued across week4(−48% and−57%,respectively)and week6(−48%and−42%,
respectively)(Table4).No differences were obrved
between the hamsters fed the water-washed or alcohol-
补肾方法washed soy protein diets at any time point for plasma
LDL-C concentrations.
Plasma HDL-C concentrations in hamsters consuming the
HCD were approximately the same in all groups after a
2-week lead-in(Table5).Hamsters fed the water-washed soy
protein diet had a significant increa in their plasma HDL-C
concentrations at week2compared with the hamsters fed the
cain HCD(17%)(Table5).No differences were obrved
between any of the dietary treatment groups for plasma
消防安全教育培训HDL-C concentrations at week-4or week-6time points.
Plasma TG concentrations in hamsters consuming the
HCD were approximately the same in all groups after a
2-week lead-in(Table6).Compared with hamsters fed the
cain HCD,hamsters fed the water-and alcohol-washed soy protein–containing diets had a significa
nt decrea in their plasma TG concentrations at week2(−77%and−68%, respectively)(Table6).No differences were obrved between any of the dietary treatment groups for plasma TG concentrations at week-4or week-6time points.
The hamsters fed the alcohol-washed soy protein diet accumulated significantly less aortic TC compared with the hamsters fed the cain HCD(−57%)and the water-washed soy protein diet(−55%)(Table7).Furthermore,the hamsters fed the alcohol-washed soy protein diet accumulated significantly less aortic cholesterol esters compared with the hamsters fed the cain HCD(−66%)(Table7).No differences were obrved between dietary treatments for aortic free cholesterol accumulation.
4.Discussion
Dietary soy protein has well-documented beneficial effects on plasma lipid and lipoprotein concentrations. Numerous studies in human beings[1,2,25,34]and our studies in hamsters and monkeys[8,10,15,16],along with other studies in swine and rabbits[35-39],have shown that substitution of soybean protein for cain lowers plasma cholesterol concentrations.However,the extent to which plasma cholesterol is reduced when cain is replaced by soybean protein is highly v
天蝎座女明星
ariable.Still,the beneficial effects of soy protein on plasma lipoprotein concentrations culminated recently in the US Food and Drug Adminis-tration's approval of a health claim that“25g of soy protein a day,as part of a diet low in saturated fat and cholesterol,may reduce the risk of heart dia”[40].Although the cholesterol-lowering effect of dietary soy protein in regulat-ing cholesterolemia has been established,postulated bio-chemical mechanisms include upregulation of LDL receptors
Table3
Plasma TC concentrations(millimoles per liter)of hamsters during the6-week treatment period
Diet Cain Soy+I Soy−I
Wk08.68±0.658.71±0.558.43±0.43 Wk211.64±0.90a 6.69±0.22b7.21±0.51b Wk49.50±0.92a 6.83±0.23b 6.52±0.22b Wk69.00±0.48a 6.11±0.25b 6.57±0.33b Values are means±SEM;n=10.Means in each row with different superscripts are significantly different(P b.05;Student-Newman-Keuls test).
Table4
Plasma non–HDL-C concentrations(millimoles per liter)of hamsters during the6-week treatment period
Diet Cain Soy+I Soy−I
Wk0 6.17±0.65 5.91±0.53 5.91±0.50 Wk29.01±0.95a 3.61±0.16b 4.22±0.50b Wk4 6.25±1.06a 3.25±0.21b 2.68±0.24b Wk6 5.42±0.56a 2.84±0.17b 3.17±0.32b Values are means±SEM;n=10.Means in each row with different superscripts are significantly different(P b.05;Student-Newman-Keuls test).Table5
Plasma HDL-C concentrations(millimoles per liter)of hamsters during the 6-week treatment period
Diet Cain Soy+I Soy−I
Wk0 2.51±0.11 2.80±0.07 2.51±0.15 Wk2 2.63±0.11a 3.08±0.11b 2.99±0.14ab Wk4 3.25±0.19 3.58±0.09 3.84±0.12 Wk6 3.59±0.15 3.26±0.12 3.40±0.14 Values are means±SEM;n=10.Means in each row with different superscripts are significantly different(P b.05;Student-Newman-Keuls test).
Table6
Plasma TG concentrations(millimoles per liter)of hamsters during the6-week treatment period
Diet Cain Soy+I Soy−I
Wk0 1.16±0.16 1.57±0.270.91±0.07 Wk29.63±1.58a 2.21±1.52b 3.10±0.47b Wk4 3.30±0.73 1.74±0.12 1.37±0.08 Wk6 2.84±0.29 2.61±0.20 2.03±0.09 Values are means±SEM;n=10.Means in each row with different superscripts are significantly different(P b.05;Student-Newman-Keuls test).
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T.A.Wilson et al./Nutrition Rearch27(2007)498–504
[35,36],incread excretion of cholesterol and bile acids [37,38],and decread cholesterol absorption[39].Turnover studies with cain-and soy protein–fed rabbits indicate an impaired VLDL and LDL catabolism consistent with downregulation of LDL receptors only in the cain-fed animals[35,36].In swine and rabbits fed cain compared with soybean protein,the increa in plasma cholesterol in the former group was also associated with decread excretion of cholesterol and bile acids[37,38].Thus,it is possible that the changes in lipoprotein cholesterol with dietary protein feeding could be condary to the alterations in whole body cholesterol metabolism.
The lowering of plasma TC and LDL-C concentrations from week2through week6by the soy protein
diets in hamsters compared with cain-fed hamsters in the prent study is in agreement with previous obrvations that have shown that replacing cain with soy protein in the diet significantly reduces plasma cholesterol concentrations [1,6,16,41-44].We also obrved that hamsters fed the soy protein diets had slight increas over time in their plasma HDL-C concentrations,which is consistent with previous work that showed either no effect on blood HDL-C concentrations or even an increa in blood HDL-C concentrations[16,43,44].
The cholesterol-lowering mechanism of soy protein is still far from clear.This effect on blood cholesterol concentrations is thought to be due largely to the amino acid composition of soy protein.Early rearch focud on the amino acid composition of soy protein.However,Huff et al[17]obrved that in hypercholesterolemic rabbits, intact soy protein produced a greater cholesterol-lowering effect than did a diet compod of a mixture of amino acids that was identical to the composition of the intact soy protein.This study[17]provided evidence that there were components of the intact soy protein other than the amino acids that independently lowered plasma cholesterol con-centrations or interacted with the protein moiety to affect lipoprotein metabolism favorably.
Interest in the potential role of isoflavones as an important hypocholesterolemic agent in soy product
s has been raid as a result of their estrogenic potential[45].Isoflavones have structural similarities with estrogens,which have hypocho-lesterolemic property[46].Estrogen may affect the metabo-lism of apolipoproteins,which are constituents of HDL and LDL[47].A number of studies have sought to determine how much of the plasma lipid–lowering properties of soy protein are related to the prence of the isoflavones and how much they relate to effects of the soy peptides.The approach has been to compare soy protein isolate containing isoflavones with alcohol-washed soy protein isolate in which the isoflavones have been removed[43,44,48].In general,the studies have shown greater cardiovascular dia protection with the soy protein isolate containing isoflavones[49].Anthony et al[43,44]showed that alcohol-extracted soy protein,which has been shown to remove phytoestrogens,was less effective at lowering plasma cholesterol levels and preventing the development of atherosclerosis in nonhuman primates relative to non–alcohol-washed soy protein diets.In contrast to the studies [43,44,48,49],the current study showed a decrea in plasma TC and LDL-C concentrations in the hamsters fed the alcohol-washed soy protein,which was similar to the decrea in plasma TC and LDL-C concentrations in the hamsters fed the water-washed soy protein.Previous rearch in human beings has shown decreas in blood cholesterol concentrations when fed alcohol-washed soy protein compared with cain feeding[34,50];however, others have not[51-53].Furthermore,similar to our obrvations of no differe
nces between the water-washed and alcohol-washed soy protein,Adams et al[54]and McVeigh et al[55]recently showed in monkeys and human beings that being fed a soy protein diet low in isoflavones produces similar cholesterol lowering than being fed a soy protein diet high in isoflavones.In contrast to the studies of Anthony et al[43,44],their comparisons were only between animals fed either water-washed or alcohol-washed soy protein and no cain control group was prent.Thus,no interpretation can be made on what extent the alcohol-washed soy protein group might have lowered blood cholesterol concentrations compared with a cain control group.Another important issue in the current study was that the dietary soy protein was not only alcohol washed but also water washed.It is possible that smaller peptides or other components that may be of biological value are removed from the soy product during the water-washing process becau the smaller peptides,among many other consti-tuents,are water soluble.Other previous studies[26,27] have shown that in men and postmenopausal women given isoflavone tablets on top of the regular diet,no significant effect on blood lipids was obrved.This obrvation may show that other constituents of soy protein other than isoflavones,particularly its bioactive peptide components[56-58],may be responsible for soy's cholesterol-lowering effects.
In contrast to previous studies[43,44],the current study showed that only the hamsters fed the alcoho
l-washed soy protein diet accumulated significantly less aortic cholesterol and cholesterol ester compared with the cain-fed hamsters and not the hamsters fed the water-washed soy protein. Although Anthony et al[43,44]have shown greater atherosclerotic protection in animals fed water-washed soy protein compared with that in animals fed alcohol-washed
Table7
Aortic cholesterol concentrations(microgram per milligram of aortic tissue)
in hamsters after the6-week treatment period
Diet Cain Soy+I Soy−I
TC 2.83±0.44a 2.68±0.50a 1.21±0.15b
Free cholesterol0.82±0.09 1.01±0.350.52±0.14
Cholesterol ester 2.01±0.45a 1.68±0.36ab0.68±0.15b
Values are means±SEM;n=10.Means in each row with different
superscripts are significantly different(P b.05;Student-Newman-Keuls test).
502T.A.Wilson et al./Nutrition Rearch27(2007)498–504
