NMR analysis of lipoprotein particle size does notincrease sensitivity to the effect of soy protein onCVD risk when compared with the traditional lipidprofile

Antonio S. Santo, Ariana M. Cunningham, Sofiya Alhassan, Richard W. Browne,Harold Burton, John J. Leddy, Peter W. Grandjean, Steven M. Horvath, andPeter J. Horvath

Abstract: The traditional lipid profile compared with nuclear magnetic resonance (NMR) may underestimate the risk forcardiovascular disease and may explain some of the discrepancies in results between studies analyzing the salubrious effectsof soy. Our purpose was to compare the traditional lipid profile with NMR quantification of the number of lipoprotein par-ticles, subclasses, and diameters or sizes in 30 sedentary males, between 18 and 30 years of age, consuming 1 of the follow-ing 3 supplements daily for 28 days: milk protein (Milk), isoflavone-poor soy protein (Soy–), or isoflavone-rich soy protein(Soy+). The study used a double-blind, parallel-arm design with random assignment to 1 of the 3 protein supplement groups.Fasting EDTA blood samples were collected at baseline and after 28 days of supplementation and analyzed for the numberand size of very low-density lipoprotein (VLDL), low-density lipoprotein (LDL), and high-density lipoprotein (HDL) par-ticles, respectively. Fasting serum samples were analyzed for concentrations of total cholesterol (TC), LDL cholesterol(LDL-C), total HDL cholesterol (HDL-C), HDL2-C, HDL3-C, triglycerides (TGs), free fatty acids (FFAs), and glucose. Fast-ing heparin blood samples were collected at baseline and after supplementation and analyzed for apolipoproteins A-I, A-II,B, C-II, C-III, and E, as well as hepatic and lipoprotein lipase concentrations. HDL3-C increased by 47.2% after Soy+ sup-plementation and hepatic lipase decreased 19.2% after Soy– supplementation (p < 0.05). HDL-C and apolipoproteins A-Iand A-II were found to increase in all 3 groups (p < 0.05). Results support that NMR analysis of lipoprotein particle numberand size are not more sensitive to the effect of soy protein on CVD risk compared with the traditional lipid profile. Further-more, the lack of isoflavones in soy protein seems to have a deleterious effect on hepatic lipase.

Key words: soy protein, lipoprotein subclasses, lipoprotein particle number, lipoprotein particle size, NMR analysis, cardio-vascular disease risk, lipid profile.

Resume : Comparativement aux observations faites par la resonance magnetique nucleaire (NMR), le profil lipidique clas-sique peut sous-estimer le risque de maladie cardiovasculaire et expliquer les divergences notees a propos des effets dusoya sur la sante. Le but de l’etude est de comparer le profil lipidique classique aux resultats obtenus par la quantificationdes observations faites par NMR sur le nombre de particules, de sous-classes et de dimension des lipoproteines chez trentesujets masculins sedentaires ages de 18 a 30 ans qui devront consommer quotidiennement durant vingt-huit jours un destrois supplements suivants : proteines du lait (Milk), des proteines de soya pauvres en isoflavones (Soy–) ou des proteinesde soya riches en isoflavones (Soy+). Les sujets sont repartis aleatoirement dans les trois groupes experimentaux en fonc-tion d’un devis experimental a double insu avec des groupes paralleles. Au tout debut et apres 28 jours de supplementation,on preleve des echantillons de sang des sujets a jeun dans des tubes contenant de l’acide ethylene-diamine-tetraace-tique (EDTA) et on en analyse le nombre et la taille des particules de VLDL, de LDL et de HDL. Dans les preleve-

Received 30 August 2007. Accepted 25 January 2008. Published on the NRC Research Press Web site at apnm.nrc.ca on 29 April 2008.

A.S. Santo.1 Division of Healthful Living and Sports Studies, School of Health Sciences, Lenoir-Rhyne College, Moretz 104 – ExercisePhysiology Laboratory, Hickory, NC 28603, USA.A.M. Cunningham. The Center for Diabetes Self-Management Care, Frye Regional Medical Center, 420 North Center St., Hickory,NC 28601, USA.S. Alhassan. Stanford Prevention Research Center, Stanford University School of Medicine, Hoover Pavilion, N229, 211 Quarry Rd.,Stanford, CA 94305, USA.R.W. Browne. Biotechnical and Clinical Laboratory Sciences, University at Buffalo, State University of New York, 26 Cary Hall,Buffalo, NY 14214, USA.H. Burton, J.J. Leddy, S.M. Horvath,2 and P.J. Horvath. Department of Exercise and Nutrition Sciences, University at Buffalo, StateUniversity of New York, Kimball Tower 108, Buffalo, NY 14214, USA.P.W. Grandjean. Department of Health and Human Performance, Auburn University, 2070 Beard–Eaves Memorial Coliseum, Auburn,AL 36849, USA.

1Corresponding author (e-mail: santoa@lrc.edu).2Deceased.

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ments seriques des sujets a jeun, on determine les concentrations de cholesterol total (TC), de LDL-cholesterol (LDL-C), de HDL-cholesterol total (HDL-C), de HDL2-C, HDL3-C, de triglycerides (TG), d’acides gras libres (FFA) et deglucose. Dans les prelevements heparines des sujets a jeun obtenus au tout debut et apres vingt-huit jours de supple-mentation, on determine les concentrations des apolipoproteines A-I, A-II, B, C-II, C-III et E et des lipases hepatiqueset de lipoproteines. La supplementation en Soy+ augmente de 47,2 % la concentration de HDL3-C et la supplementa-tion en Soy– diminue de 19,2 % la concentration de lipase hepatique (p < 0,05). Dans tous les groupes, on observeune augmentation des concentrations de HDL-C et des apolipoproteines A-I et A-II (p < 0,05). D’apres ces observa-tions, l’analyse par NMR du nombre et de la taille des particules de lipoproteines n’est pas plus sensible a l’effet desproteines de soya sur le risque de maladie cardiovasculaire que ne le revele le profil lipidique classique. En outre, lacarence d’isoflavones dans les proteines de soya semble nefaste pour la lipase hepatique.

Mots-cles : proteines de soya, sous-classes de lipoproteines, nombre de particules de lipoproteines, taille des particules delipoproteines, analyse par resonance magnetique nucleaire, risque de maladie cardiovasculaire, profil lipidique.

[Traduit par la Redaction]

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IntroductionThe salubrious effect of consuming soy products on cardio-

vascular disease risk reduction is controversial and has re-cently been an area of contention in the literature (Ma etal. 2005; Sacks et al. 2006). The Anderson et al. (1995)meta-analysis is often cited as a key article demonstratingthe beneficial effects of soy protein intake and is the basisfor the US Food and Drug Administration’s health claimregarding soy protein consumption (Department of Healthand Human Services 1999). They reported that total cho-lesterol (TC), low-density lipoprotein cholesterol (LDL-C),and triglycerides (TGs) decreased by 23.2 mg�dL–1,21.7 mg�dL–1, and 13.3 mg�dL–1, respectively, and thathigh-density lipoprotein cholesterol (HDL-C) increased by1.2 mg�dL–1 in normo- and hyper-cholesterolemic subjectsingesting soy protein with varying isoflavone quantities. Ameta-analysis by Zhan and Ho (2005) in normo- and hyper-cholesterolemic subjects ingesting only isolfavone-rich soyprotein (Soy+) showed weaker effects, with TC, LDL-C,and TGs decreasing by only 8.51 mg�dL–1, 8.13 mg�dL–1,and 3.87 mg�dL–1, respectively, whereas HDL-C increasedslightly more by 1.55 mg�dL–1. A more recent meta-analysisin normo- and hyper-cholesterolemic subjects ingesting onlyisolated soy protein with varying isoflavone moieties reportedeven smaller changes in concentrations of TC, LDL-C, TGs,and HDL-C (–5.26 mg�dL–1, –4.25 mg�dL–1, –6.26 mg�dL–

1, +0.77 mg�dL–1, respectively; Reynolds et al. 2006). Themost current meta-analysis in normo- and hyper-cholestero-lemic subjects ingesting Soy+ observed a decrease of3.9 mg�dL–1 and 5.0 mg�dL–1 for TC and LDL-C, respec-tively, but found no changes in TG or HDL-C concentra-tions when compared with an isoflavone-poor soy protein(Soy–) (Taku et al. 2007). Discrepancies among these stud-ies may be related to the effect of soy protein extractionand processing techniques on the bioavailability of soyconstituents (e.g., isoflavones, fiber, saponins, and fat),study design (e.g., parallel vs. crossover), populationstudied (e.g., normo-cholesterolemic vs. hypercholesterole-mic; ethnicity), geographic area of cultivation, and lengthof the treatment (Caldwell et al. 2005; Coward et al.1998; Friedman and Brandon 2001; Lu and Anderson1998; Potter 1998; Slavin et al. 1998; Taku et al. 2007).In addition, the isoflavone daidzein is converted by gastro-intestinal microflora to a more potent isoform, equol,

which can vary greatly with respect to bioavailability andpotency (e.g., responders vs. non-responders) (Lu and An-derson 1998; Setchell 1998; Slavin et al. 1998).

The traditional lipid profile (i.e., TC, LDL-C, HDL-C,and TGs) may underestimate the risk for cardiovascular dis-ease, which may partially explain the disparities regardingthe beneficial effects of soy. The standard LDL-C andHDL-C measurements do not take into account the distribu-tion of cholesterol over the number of LDL and HDLparticles. Evidence suggests that small, dense LDL (19.0–20.5 nm) and HDL (7.2–8.2 nm) particles are potentiallymore atherogenic than larger, less dense LDL and HDL par-ticles; therefore, a greater number of small, dense LDL andHDL particles may be a better indicator of CVD risk(Berneis and Krauss 2002; Otvos et al. 2002; Sacks andCampos 2003). Thus, studies involving soy ingestion and itseffect on the traditional lipid profile may be missing a morefavorable lipoprotein remodeling and constituent distribu-tion. The first study to evaluate lipoprotein number and sizewith respect to soy protein consumption found that in hyper-cholesterolemic postmenopausal women, LDL particle num-ber and LDL-C significantly decreased after 6 weeks andremained at this level for 12 weeks, whereas LDL and HDLsize were unaltered, suggesting a more favorable distributionof cholesterol (Allen et al. 2007). Others have also sup-ported the use of LDL particle number as a means of evalu-ating the effectiveness of treatment interventions on CVDrisk (El Harchaoui et al. 2007; Otvos et al. 2006; Pischon etal. 2005). However, recent data from the EPIC NorfolkProspective Population Study suggests that after adjustingfor HDL-C and triglycerides, LDL particle number may notbe a better indicator for CVD risk than LDL-C, and it re-quires further evaluation before implementation as an addi-tional screening tool (El Harchaoui et al. 2007).

Several mechanisms have been postulated regarding thecholesterol-lowering effects of soy. Research suggests thatthe storage proteins glycinin and conglycinin, about 38% ofthe soybean weight, are responsible for the lowering of fast-ing total and LDL cholesterol concentrations in mild tomoderate hypercholesterolemic individuals (Bakhit et al.1994; Teixeira et al. 2000). Specifically, Soy– may enhancethe uptake of LDL-C through increased LDL receptor activity,which may be indirectly implicated through the measurementof apolipoprotein B-100 (Ho et al. 1989; Vessby et al.

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1982). In addition, Soy– may also reduce the number ofsmall, dense LDL particles through alterations in lipopro-tein and hepatic lipase activities (Carroll and Kurowska1995). McCarty (1999) discussed how a diet higher innon-essential amino acids (e.g., Soy– compared with milkprotein) increases the glucagon–insulin ratio, which down-regulates the synthesis of several enzymes required for denovo lipogenesis and cholesterol synthesis (i.e., citratelyase, acetyl-CoA carboxylase, fatty acid synthetase, andHMG-CoA reductase), while up-regulating gluconeogenicand fatty acid oxidation enzymes, LDL receptors, andIGFBP-1 (an antagonist to IGF-1). Other researchers havesuggested that soy phytoestrogens (i.e., the isoflavones gen-istein and daidzein) may be responsible for the cholesterol-lowering effect of soy products (Goodman-Gruen andKritz-Silverstein 2001; Merz-Demlow et al. 2000; Wash-burn et al. 1999). However, several well-controlled studieshave found no alterations in fasting serum lipids in pri-mates receiving casein protein containing extracted soy iso-flavones or in humans receiving a 55 mg tablet of soyisoflavones for 8 weeks (Greaves et al. 2000; Hodgson etal. 1998). Clarkson (2002) suggested that the contentiousfindings of these studies comparing soy isoflavone extractswith Soy+ may be due to an existing interaction betweenthe protein and isoflavone constituents. Other researcherssuggest that soy fiber, saponins, and phytosterols may de-crease intestinal bile acid and cholesterol absorption, thuspartly explaining the cholesterol-lowering effect of soyproducts (Lo et al. 1986; Nguyen 1999). In addition, thephytoestrogens found in soy may exhibit antioxidant proper-ties and activities (Arora et al. 1998; Potter 1998).

Based on the foregoing, the primary purpose of this studywas to determine if measuring lipoprotein particle size andnumber was more sensitive than the traditional lipid profilefor detecting any beneficial effects of consuming soy proteinwith and without isoflavones. A secondary purpose was toevaluate apolipoprotein and lipase concentrations as poten-tial mechanisms responsible for any beneficial effects. Sincemost soy studies have focused on women, in particular post-menopausal subjects, we sought to describe the effects ofSoy– and Soy+ in normocholesterolemic, sedentary males.

Materials and methods

ParticipantsThis study was approved by the University at Buffalo’s

Human Subject’s Review Committee. Thirty-five men aged18–30 years were recruited to voluntarily participate in astudy investigating the effects of soy on CVD risk factorsfrom the Western New York community. Participants wererecruited by flyers and advertisements in local newspapers.Informed consent was obtained prior to participation in thescreening process. Participants were included in the study ifthey met the following criteria: not taking cholesterol-reducing medications, body mass index (BMI) between 18and 26 kg�m2, non-habitual soy consumers, non-extremistdietary practitioners (i.e., high protein and (or) fat intakes),and non-smokers. Participants were also screened for fastinglipid (TC, HDL-C, LDL-C, and TG) and glucose concentra-tions using the Cholestech L.D.X1 (Cholestech Corp., Hay-ward, Calif.) and Accu-Chek Advantage Analyzers (Roche

Diagnostics Corp., Indianapolis, Ind.), respectively. Due tothe heparin injections used for lipase measurements, partici-pants could not be allergic to pig products, have had recentsurgery (within 1 month of participating), or manifest anyactive gastrointestinal bleeding (e.g., active peptic ulcers,etc.). A physician recorded the participant’s medical historyand performed a physical examination to exclude those par-ticipants with a known history of cardiovascular and pulmo-nary diseases.

Anthropometric measurementsHeight was taken with an anthropometer attached to a

Detecto-Medic scale (model No. 3P412; Detecto ScalesInc., Brooklyn, N.Y.) and weight was measured using aTanita1 scale (Model No. TBF-521; Tanita1 Corporationof America, Arlington Heights, Ill.). BMI was calculated asbody mass in kilograms divided by height in meters squared(kg�m2). Circumferences and percent body fat determinedfrom skinfolds were measured as indicators of weight lossor gain by the same investigator to reduce tester variability(ACSM’s guidelines for exercise testing and prescription(American College of Sports Medicine 2006)).

Dietary procedures and analysesA double-blind, parallel-arm design was used to compare

the effects of a milk protein isolate (Milk, as the controlgroup; atherogenic equivalent), an isoflavone-poor soy pro-tein isolate (Soy–), and an isoflavone-rich soy protein isolate(Soy+) on fasting lipid concentrations and various fasting li-pase activities. Each participant was randomly assigned by atrained research assistant to 1 of the 3 protein supplementgroups: Milk, Soy–, or Soy+. Protein packets were placed inlarge envelopes and labeled 1, 2, or 3 without the presenceof the principle investigator to ensure that the study wasblinded. The supplements were donated by The Solae Com-pany (St. Louis, Mo.) and consisted of 25 g of powderedprotein isolate packaged in a vacuum-sealed packet (Table 1).The protein powder was mixed with a beverage of the partic-ipant’s choice (including milk) and taken daily for 28 days.All unused and (or) empty packets were returned in the largeenvelopes to the laboratory as an indicator of compliance.

A registered dietitian collected 3 day diet records duringthe screening process and at 14 and 28 days during the studyto ensure consistency in the participant’s diets. Participantswere instructed on how to properly complete the 3 day dietrecords (i.e., type, portion size, and preparation of the food).In addition to completing the 3 day diet records on 3 separateoccasions, subjects met with the registered dietitian to verifythe types, portion sizes, and preparation of the foods theyconsumed. Nutritionist Pro Software (First Databank, SanBruno, Calif.) was used to analyze the 3 day diet records.

Blood draws: days 0 and 28Fasting blood samples were taken at baseline and approx-

imately 28 days later, and accounted for 2 of the 4 blooddraw visits (Fig. 1). Participants visited the laboratory aftera 12 h fast and a registered nurse placed an IV-catheter inan antecubital vein. A 7 mm microbore extension was con-nected to the catheter and secured with sterile TegadermTM

(3M Health Care, St. Paul, Minn.). Fasting blood sampleswere taken in a 10 mL syringe that was connected to the

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microbore extension and approximately 10 mL of bloodwere drawn. An 18-gauge needle was attached to the syringeand approximately 3 mL of blood were placed into one ofthree 5 mL Vacutainer tubes in the following order: serum(allowed to clot at room temperature), EDTA, and heparin.Heparin and EDTA blood samples were immediately placedon ice and centrifuged within 30 min of collection at3000 r�min–1 (1875g) for 30 min at 4 8C (Sorvall RT6000BRefrigerated Centrifuge, Newton, Conn.). Plasma and serumsamples were stored at –80 8C until analysis.

Lipid profile and glucose analysisPlasma collected from days 0 and 28 were analyzed for

TC, TG, free fatty acids (FFAs), HDL-C, HDL2-C, HDL3-C,and glucose using commercially available kits from WakoChemicals USA, Inc. (Richmond, Va.) and techniques previ-ously described (Rifai et al. 2000). In addition, all pro-cedures were followed as directed in the applicationparameters obtained from Wako for the Cobas Fara Centrifu-gal Analyzer for Automated Chemistry Analysis (model No.

22-2081, Roche Diagnostic Systems, Nutley, N.J.). All as-says for each subject were run in duplicate on the same daywith the same reagent batch. External calibrators were in-cluded on all assays on every run, and the range of concen-trations in the calibration curves encompassed the range ofexpected sample values. Two controls were used, Accutroland Cardiolipid, which were run 10 times throughout the du-ration of each test to ensure intra-assay reproducibility.

Lipoprotein number and particle sizeFasting EDTA plasma samples were shipped on dry ice

and analyzed by nuclear magnetic resonance (NMR)spectroscopy for VLDL, LDL, and HDL total particulatesand subclasses and VLDL, LDL, and HDL size by Lipo-Science (Raleigh, N.C.). Detailed procedures for this techni-que have previously been published (Otvos 2002).

ApolipoproteinsFasting sodium heparin plasma samples were shipped on

dry ice and analyzed by LINCO Research (St. Charles,Mo.), using a proprietary LINCOplex panel: a multiplexedbiomarker immunoassay for Luminex Instrumentation, forapolipoproteins A-I, A-II, B, C-II, C-III, and E.

Blood draws: days 3 and 31Participants were analyzed for fasting hepatic and lipo-

protein lipases on a separate laboratory visit, not more than3 days after the previously described fasting blood draws.The participants were weighed and their heparin dose wascalculated as 30 U�kg–1 body mass. The heparin sodiumused in this study was commercially prepared from porcineintestinal mucosa. The microbore extension – catheter setupused for previous fasting blood draws was used for fastingand post-heparin sodium blood draws. A 10 mL syringewas connected to the microbore extension and 10 mL ofblood were collected from an antecubital vein. An 18-gaugeneedle was attached to the syringe and approximately 3 mLof blood were placed into three 5 mL Vacutainer tubescontaining EDTA. The heparin solution was thenadministered as a continuous injection through the micro-bore extension – catheter setup, followed by 3 mL of 0.9%sterile saline. At exactly 15 min post-heparin sodium injec-tion, a second 10 mL blood sample was drawn and distrib-uted among three 5 mL Vacutainer tubes containingheparin. All blood samples were immediately placed on iceand centrifuged within 30 min of collection at 3000 r�min–1

(1875g) for 30 min at 4 8C (Sorval RT6000B RefrigeratedCentrifuge). Samples were stored at –80 8C until analysis.

Table 1. Daily protein packet composition (36.5 g each).

Nutrient Milk Soy– Soy+Kilocalories 130.0 130.0 130.0Total fat (g) 0.5–1.0 0.5–1.0 0.5–1.0

Cholesterol (mg) 0–5 0–5 0–5Total carbohydrate (g) 6.0 6.0 6.0

Sugars (g) 3.0 3.0 3.0Protein (g) 25.7 24.9 25.8Vitamins

A (IU) 625.0 625.0 625.0B12 (mcg) 1.0 1.0 1.0D3 (IU) 125.0 125.0 125.0Folate (mg) 35–75 35–75 35–75Riboflavin (mg) 0.5 0.5 0.5

Minerals (mg)Calcium 996.5 912.5 949.0Iron 1–4 1–4 1–4Magnesium 50–140 50–140 50–140Phosphorous 726.4 759.2 722.7Potasium 5.9 227.0 299.7Sodium 134.7 250.0 276.3

Isoflavones (mg)Total - all forms NA* 1.5 96.4

Daidzein-containingcompounds

NA* 0.4 37.6

Genistein-containingcompounds

NA* 0.7 54.0

Glycitein-containingcompounds

NA* 0.4 5.1

Total aglyconecomponents

NA* 0.7 56.2

Daidzein NA* 0.4 21.9Genistein NA* 0.4 31.4Glycitein NA* 0.4 2.9

Ash (g) 3.4 3.8 4.2Moisture (g) 1.2 1.0 1.0

Note: Only significant levels listed. Information provided by TheSolae Company, St. Louis, Mo. Soy–, isoflavone-poor soy protein;Soy+, isoflavone-rich soy protein.*Contains no isoflavones.

Fig. 1. Study design timeline.

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Lipase analysisFasting sodium heparin plasma samples collected on days

3 and 31 were transported on dry ice to Auburn University(Auburn, Ala.) and analyzed for total, hepatic, and lipo-protein lipase activities using a previously publishedmethodology (Rifai et al. 2000).

StatisticsA general linear model, two-way repeated measures analy-

sis of variance (ANOVA) was used to determine significantdifferences in variables listed in Tables 2–4 after protein sup-plementation. The Bonferroni (all pair-wise) multiple com-parison test was used to determine the main effects of dietcollapsed across time (Milk pre + Milk post vs. Soy– pre +Soy– post vs. Soy+ pre + Soy+ post) and time collapsedacross diet (Milk pre + Soy– pre + Soy+ pre vs. Milk post +Soy– post + Soy+ post) and to determine any diet by time in-teractions (pre vs. post within groups). A p value of less than0.05 was considered statistically significant. The data wereanalyzed with Number Cruncher Statistical Systems software(NCSS 2000, Kaysville, Utah). Protein group identification

was not revealed to the principle investigator until after theyperformed all biochemical and statistical analyses.

Results

ParticipantsOwing to poor study compliance and data outliers, 5 par-

ticipants were eliminated, leaving us with 9, 11, and 10 par-ticipants for the Milk, Soy–, and Soy+ groups, respectively.

AnthropometricsBaseline and post-intervention anthropometric and other

physical data are presented in Table 2. After 4 weeks ofsupplementation with 25 g�d–1 of protein (main effect oftime collapsed across diet), body mass and BMI increasedby 0.7 kg (p = 0.006) and 0.2 kg�m–2 (p = 0.0273), respec-tively. Fat mass (calculated from skinfolds) increased by0.8 kg (p = 0.030) and can primarily be attributed to the ab-domen and triceps skinfold measurements, which increasedby 1.5 (p = 0.027) and 0.9 mm (p 0.025), respectively. Ofall the circumferences, only the waist measurement in-

Table 2. Mean ± SEM values of subject anthropometrics for 30 sedentary males.

Milk (n = 9) Soy– (n = 11) Soy+ (n = 10)

Variable Pre Post Pre Post Pre PostAge (y) 24.0±0.9 24.1±0.9 23.6±0.5 23.7±0.5 25.1±0.8 24.6±1.1Resting heart rate (beats�min–1) 76.4±3.3 71.7±3.1 75.0±3.5 69.5±1.6 67.8±3.2 73.0±3.6Blood pressure (mmHg)

Systolic 111.6±2.3 108.4±3.2 112.5±3.5 110.2±3.5 110.1±3.9 105.1±3.2Diastolic 67.3±2.9 69.1±2.8 72.9±2.3 68.0±2.7 71.4±3.1 66.8±2.5

Height (cm) 177.1±2.3 177.2±2.3 175.4±1.8 175.7±1.8 174.8±1.8 174.7±1.7Mass (kg)* 73.8±4.1 74.6±4.3 78.0±5.6 79.0±5.6 69.9±3.7 70.3±3.7BMI (kg�m–2){ 23.4±1.0 23.6±1.0 25.2±1.4 25.4±1.5 22.8±0.9 23.0±1.0BIA (% fat) 19.7±1.3 19.6±1.9 20.7±2.9 21.4±2.8 18.5±2.1 18.3±2.2

Lean body mass (kg) 59.0±2.7 58.9±1.9 60.3±2.3 59.7±3.5 56.4±1.9 56.8±1.7Fat mass (kg) 14.8±1.6 15.7±2.8 17.7±3.4 19.2±2.8 13.5±2.0 13.4±2.1

Skinfolds (% fat) 15.2±2.0 15.3±2.2 15.9±2.0 16.7±2.2 15.7±1.5 16.7±1.4Lean body mass (kg) 62.0±2.4 61.7±2.2 64.6±3.5 64.9±3.6 58.6±2.5 58.4±2.5Fat mass (kg){ 11.8±2.1 12.9±2.3 13.3±2.3 14.1±2.5 11.3±1.5 11.9±1.5

Skinfolds (mm)Abdomen{ 20.4±3.6 23.3±4.1 24.5±3.4 25.0±3.7 22.6±2.2 23.8±2.4Suprailleum 16.9±2.9 15.7±2.4 17.2±3.0 18.7±3.1 17.7±2.4 18.4±2.3Triceps{ 11.6±1.5 11.4±1.5 12.9±1.9 14.1±1.9 10.9±1.3 12.4±1.0Thigh 18.2±1.8 17.9±2.6 16.9±1.6 17.2±2.2 17.4±1.6 18.3±1.2Sum of skinfolds 67.1±8.6 68.3±9.9 71.5±9.1 75.0±10.4 68.6±6.5 72.9±6.1

Circumferences (cm)Abdomen 81.3±2.7 81.9±2.5 83.9±3.8 84.6±3.7 79.8±3.2 80.1±2.9Arm 28.7±1.0 28.9±1.2 31.4±1.4 31.0±1.4 27.9±1.1 28.0±1.0Calf 35.8±1.1 36.0±1.0 37.4±1.3 37.5±1.3 34.9±1.0 35.1±1.0Forearm 26.6±0.7 26.5±0.8 27.5±0.8 27.5±0.8 25.9±0.6 26.1±0.7Hips/buttocks 98.7±2.8 98.5±2.8 99.4±2.8 100.8±3.1 96.2±2.1 97.4±2.3Hips/thighs 58.7±1.6 57.4±1.9 60.5±2.2 60.5±2.3 58.3±1.5 58.0±1.7Waist* 77.9±2.3 79.1±2.3 81.2±3.7 82.7±3.4 75.4±2.6 76.2±2.4Waist–hip ratio 0.79±0.02 0.80±0.01 0.81±0.02 0.82±0.01 0.78±0.02 0.78±0.02

Note: Soy–, isoflavone-poor soy protein; Soy+, isoflavone-rich soy protein; BMI, body mass index; BIA, bioelctrical impedance (Ta-nita1 scale, model No. TBF-521). Measurements were taken according to the American College of Sports Medicine guidelines (ACSM’sguidelines for exercise testing and prescription (American College of Sports Medicine 2006)).*Values are significantly different after protein supplementation; main effect of time collapsed across diet (p < 0.01).{Values are significantly different after protein supplementation; main effect of time collapsed across diet (p < 0.05).

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creased by 1.1 cm (p = 0.009) after protein supplementation(main effect of time collapsed across diet).

Dietary analysesDifficulty in collecting thoroughly completed dietary re-

cords limited our dietary analysis to 22 participants andmay not reflect the eating patterns of all participants. Base-line and post-intervention dietary data of 22 participants arepresented in Table 3. Dietary ingestion of protein, calculatedas a percentage of the amount of energy consumed, in-creased 2.5% after protein supplementation (p = 0.003;main effect of time collapsed across diet). Saturated fat,calculated as a percentage of the total identifiable lipid,increased 4.0% after protein supplementation (p = 0.023;main effect of time collapsed across diet) and 14.2% afterSoy– supplementation (p = 0.034; pre vs. post within group),and increased by 1.5% and 3.0%, respectively, if calculatedas a percentage of total kilocalories. Polyunsaturated fat,calculated as a percentage of the total identifiable lipid, de-creased 2.8% after protein supplementation (p = 0.031; maineffect of time collapsed across diet), increased 6.3% afterMilk supplementation (p = 0.013; pre vs. post within group),and decreased 11.4% after Soy– supplementation (p = 0.013;pre vs. post within group).

Fasting plasma concentrations

Table 4 shows the changes in blood lipid and lipase con-centrations. The number of small HDL particles was lowerby 27.9% (–5.7 mg�dL–1) after Soy+ supplementation com-

pared with post Soy– supplementation (p = 0.027; post vs.post between groups). When expressed as a percentage of to-tal respective particles, large LDL was 50.0% and 45.6%higher in the post Soy+ group compared with the post Milkand Soy– groups, respectively (p = 0.007; post vs. post be-tween groups). When expressed as a percentage of total re-spective particles, total small LDL was 16.3% and 15.9%lower and very small LDL was 16.0% and 15.9% lower inthe post Soy+ group compared with the post Milk and Soy–groups, respectively (p = 0.012 and p = 0.005, respectively;post vs. post between groups). When expressed as a percent-age of total respective particles, small HDL was 24.1% lowerin the post Soy+ group compared with the post Soy– group(p = 0.048; post vs. post between groups). LDL and HDLparticle size was higher by 2.4% and 3.4% (+0.5and +0.36 mg�dL–1) (p = 0.015 and p = 0.034), respectively,after Soy+ supplementation compared with post Soy–supplementation (post vs. post between groups). Apolipo-proteins A-I and A-II increased by 15.0% and 15.5%(140.9 and 39.3 mg�dL–1; p = 0.008 and p = 0.032), re-spectively, after protein supplementation (main effect oftime collapsed across diet). Total HDL-C increased by7.6% (5.5 mg�dL–1) after protein supplementation (p =0.029; main effect of time collapsed across diet). HDL3-Cincreased by 10.9% (6.1 mg�dL–1) after Soy+ supplementa-tion (p = 0.032; pre vs. post within group). Hepatic lipaseconcentrations increased only after Soy– supplementation,by 19.2% (4.2 mmol FFA�mL–1�h–1) (p = 0.018; pre vs.post within group).

Table 3. Mean ± SEM values of daily intakes of selected nutrients and other variables for 22 sedentary males.

Milk (n = 8) Soy– (n = 6) Soy+ (n = 8)Variable Pre Post Pre Post Pre Post

Total food mass (g) 2202±173 2821±398 2538±490 2442±445 2502±163 2423±292Energy (kcal) 2117±187 2710±377 2851±365 2537±404 2238±151 2360±171Protein (g)* 92.3±7.3 130.4±15.8 122.2±15.0 129.4±16.6 86.6±7.0 113.5±8.5Protein (% energy){ 17.6±0.9 19.8±0.8 17.7±2.0 20.9±1.6 15.5±0.9 19.5±1.1Carbohydrate (g) 298.8±22.3 379.2±43.5 412.3±84.8 338.5±59.5 332.9±27.5 331.5±24.5

Lactose* 13.9±4.9 18.8±5.7 4.5±1.3 31.6±12.1 17.7±5.6 24.1±5.8Carbohydrate (% energy) 57.2±2.5 57.4±1.9 55.9±6.0 53.5±4.0 59.3±2.1 56.6±2.0Lipid (g) 58.0±13.3 68.5±17.2 72.3±13.1 67.3±17.7 59.5±6.3 60.1±14.0Lipid (% energy) 23.5±2.6 21.5±2.3 24.6±4.5 23.6±3.1 23.8±1.7 22.6±1.9

% Saturated§,* 50.3±4.44a 45.4±2.14ab 33.7±3.04c 47.9±3.52ab 40.3±3.18bc 43.1±2.69abc% Monounsaturated 33.7±3.0 32.3±2.5 39.9±2.0 37.0±1.9 38.4±1.9 38.8±2.1% Polyunsaturated§,* 16.0±1.97a 22.3±2.74bc 26.5±3.96c 15.1±2.77a 21.3±3.36bc 18.1±2.98ab

Cholesterol (mg) 321.9±55.1 466.0±113.3 505.3±147.4 559.9±209.3 377.2±77.8 290.3±37.2Ethanol (g) 4.3±3.2 8.0±5.8 9.0±7.4 8.6±5.5 3.5±2.4 5.6±3.0Ethanol (% energy) 1.7±1.3 1.4±1.0 1.7±1.3 2.1±1.4 1.1±0.7 1.9±1.0Sodium (mg) 3555±607 3960±850 5002±1213 3925±529 3770±558 3903±937Potassium (mg) 1918±264 2681±392 3037±307 2885±478 2780±253 2940±367Total isoflavones (mg) 0.91±0.61 0.45±0.23 3.97±3.21 1.93±0.23 2.17±1.10 97.48±0.32

Daidzein§ 0.83±0.55a 0.40±0.24a 0.92±0.66a 0.83±0.23a 1.15±0.51a 38.36±0.33bGenistein§ 0.08±0.06a 0.05±0.03a 2.76±2.43a 0.70±0.00a 0.66±0.4a 54.01±0.01bGlycitein§ 0.00±0.00a 0.00±0.00a 0.29±0.13a 0.40±0.00a 0.36±0.22a 5.11±0.01b

Caffeine (mg) 53.0±22.9 62.9±25.3 42.9±17.6 24.9±11.8 42.9±13.5 50.2±16.1

Note: Soy–, isoflavone-poor soy protein; Soy+, isoflavone-rich soy protein.*Values are significantly different after protein supplementation; main effect of time collapsed across diet (p < 0.05).{Values are significantly different after protein supplementation; main effect of time collapsed across diet (p < 0.005).{Values are significantly different after protein supplementation; main effect of time collapsed across diet (p < 0.01).§Values with similar letters are not statistically different (p < 0.05).

494 Appl. Physiol. Nutr. Metab. Vol. 33, 2008

# 2008 NRC Canada

Tab

le4.

Mea

n±

SEM

valu

esfo

rN

MR

anal

ysis

ofpa

rtic

lesi

zean

dnu

mbe

ran

dan

alys

isof

bloo

dlip

id,

gluc

ose,

and

lipas

eco

ncen

trat

ions

in30

sede

ntar

ym

ales

.

Milk

(n=

9)So

y–(n

=11

)So

y+(n

=10

)

Var

iabl

ePr

ePo

stPr

ePo

stPr

ePo

st

No.

oflip

opro

tein

part

icle

sT

otal

VL

DL

part

icle

s(n

mol�L

–1)

74.4

±10

.570

.1±

8.9

60.8

±9.

163

.5±7

.575

.8±1

2.0

68.9

±12.

5L

arge

VL

DL

chyl

omic

rons

2.5±

0.8

2.7±

1.1

3.2±

1.6

2.9±

1.0

3.6±

2.1

2.8±

1.1

%L

arge

VL

DL

chyl

omic

rons

*3.

0±0.

83.

1±1.

15.

6±2.

84.

2±1.

04.

4±2.

13.

7±0.

9M

ediu

mV

LD

L25

.5±

4.4

27.6

±5.

419

.8±

5.2

23.6

±4.8

31.5

±6.1

25.2

±7.1

%M

ediu

mV

LD

L*

34.4

±3.

236

.8±

4.2

30.2

±4.

834

.8±3

.740

.7±3

.434

.9±4

.5Sm

all

VL

DL

46.4

±6.

839

.8±

4.0

37.8

±5.

937

.0±3

.340

.7±8

.841

.0±8

.9%

Smal

lV

LD

L*

62.6

±3.

360

.1±

4.8

64.2

±7.

061

.0±4

.455

.0±5

.261

.4±5

.3T

otal

LD

Lpa

rtic

les

(nm

ol�L

–1)

1169

.0±

148.

911

10.8

±117

.090

9.6±

68.3

1018

.9±

57.7

1103

.0±

149.

610

36.4

±11

1.2

IDL

39.9

±12

.239

.8±

12.0

17.8

±8.

626

.9±6

.941

.4±1

6.4

22.3

±16.

1%

IDL{

2.8±

0.8

3.2±

1.0

1.7±

0.8

2.5±

0.6

3.5±

1.3

1.5±

0.9

Lar

geL

DL

273.

2±40

.326

5.0±

40.0

288.

5±41

.126

3.6±

31.7

306.

6±38

.638

5.6±

36.2

%L

arge

LD

L{,{

25.0

±3.

2a26

.2±

4.0a

35.2

±5.

9ab

27.0

±3.7

a30

.4±4

.1ab

39.3

±3.6

bT

otal

smal

lL

DL{

855.

8±12

4.4a

806.

1±11

8.0a

b60

3.2±

88.8

b72

8.5±

67.2

ab75

5.0±

129.

0ab

628.

4±86

.4ab

%T

otal

smal

lL

DL{,{

72.2

±2.

7a70

.7±

4.0a

63.1

±5.

3ab

70.4

±3.5

a66

.4±3

.6ab

59.2

±3.6

bM

ediu

msm

all

LD

L18

7.6±

27.4

167.

9±22

.013

7.8±

19.6

157.

0±15

.716

0.2±

25.9

133.

4±17

.7%

Med

ium

smal

lL

DL{

15.9

±0.

614

.9±

1.0

14.5

±1.

215

.2±0

.914

.2±1

.012

.7±0

.9V

ery

smal

lL

DL{

668.

3±97

.2a

638.

0±96

.0ab

465.

5±69

.3b

571.

5±51

.8ab

594.

9±10

3.7a

b49

5.0±

69.1

ab%

Ver

ysm

all

LD

L{,{

56.3

±2.

2a55

.8±

3.0a

48.6

±4.

1ab

55.3

±2.6

a52

.2±2

.7ab

46.5

±2.8

bT

otal

HD

Lpa

rtic

les

(mm

ol�L

–1)

28.0

±1.

728

.4±

1.4

27.8

±0.

830

.0±1

.029

.5±1

.228

.6±1

.3L

arge

HD

L4.

7±1.

06.

6±1.

25.

3±0.

95.

2±0.

86.

7±1.

26.

9±1.

0%

Lar

geH

DL

§17

.7±

3.4

23.1

±3.

819

.1±

3.4

17.2

±2.8

22.8

±4.1

24.6

±3.5

Med

ium

HD

L5.

0±1.

03.

1±0.

64.

6±0.

74.

4±0.

84.

1±1.

16.

9±1.

4%

Med

ium

HD

L§

18.1

±3.

811

.0±

2.3

16.9

±2.

715

.3±3

.213

.4±3

.224

.2±5

.4Sm

all

HD

L{

18.3

±2a

b18

.8±

1.3a

b17

.8±

1.4a

b20

.4±1

.2a

18.7

±1.3

ab14

.7±1

.9b

%Sm

all

HD

L{,

§64

.2±

4.1a

b65

.9±

3.0a

b64

.0±

4.7a

b67

.5±2

.4a

63.8

±4.2

ab51

.2±6

.1b

Ave

rage

part

icle

size

(nm

)V

LD

L48

.6±

1.6

47.6

±2.

053

.9±

3.7

50.7

±2.3

50.5

±3.7

50.3

±2.3

LD

L{

20.6

±0.

1a20

.6±

0.2a

21.0

±0.

2ab

20.7

±0.2

a20

.8±0

.2ab

21.2

±0.1

bH

DL{

8.7±

0.1a

8.9±

0.1a

b8.

9±0.

1ab

8.8±

0.1a

8.9±

0.1a

b9.

1±0.

1bA

polip

opro

tein

s(m

g�m

L–1

)A

-I||

878.

1±81

.010

23.6

±47.

098

1.0±

49.0

1155

.4±

36.0

951.

4±46

.010

54.1

±72

.0A

-II}

257.

1±32

.027

9.4±

1824

7.0±

13.0

307.

4±9.

025

8.4±

21.0

293.

7±26

.0B

16.8

±3.

019

.2±

2.0

14.9

±2.

018

.67±

3.0

25.0

±14.

030

.7±8

.0C

-II

41.0

±8.

041

.4±

5.0

36.4

±7.

042

.9±5

.043

.2±5

.050

.1±5

.0C

-III

123.

8±22

.013

1.1±

12.0

114.

5±19

.012

7.6±

9.0

129.

6±17

.013

7.7±

11.0

E40

.2±

6.0

42.4

±3.

039

.3±

4.0

46.3

±2.0

43.5

±4.0

48.8

±4.0

Lip

idpr

ofile

(mg�

dL–1

)T

otal

chol

este

rol

182.

5±15

.016

5.1±

9.7

189.

6±18

.118

9.7±

13.1

166.

9±24

.019

4.0±

13.9

LD

Lch

oles

tero

l11

6.5±

13.2

102.

5±12

.212

7.9±

18.6

113.

1±14

.510

3.9±

25.6

102.

8±23

.3H

DL

chol

este

rol

Tot

alH

DL

chol

este

rol}

46.0

±5.

844

.0±

5.9

38.5

±5.

651

.2±4

.144

.8±5

.272

.1±7

.6H

DL

chol

este

rol 3{

36.0

±3.

5ab

33.8

±3.

6ab

31.1

±4.

5ab

39.9

±3.5

ab32

.6±4

.0a

48.0

±6.1

b%

HD

Lch

oles

tero

l 3**

80.4

±2.

278

.9±

3.0

81.6

±2.

077

.9±2

.173

.7±3

.372

.5±3

.9H

DL

chol

este

rol 2

10.0

±2.

410

.3±

2.6

7.4±

1.4

11.2

±1.3

12.3

±1.7

24.1

±2.3

%H

DL

chol

este

rol 2

**19

.6±

2.2

21.1

±3.

018

.4±

2.0

22.1

±2.1

26.3

±3.3

27.5

±3.9

Santo et al. 495

# 2008 NRC Canada

DiscussionThis study compared the effect of 28 days of Milk supple-

mentation with Soy– and Soy+ supplementation on fastinglipoprotein particle number and size and plasma lipid con-centrations in normocholesterolemic, sedentary males. Ourdata do not support the hypothesis that fasting lipoproteinparticle number and size are more sensitive indicators thanthe traditional lipid profile for detecting favorable changesassociated with soy consumption in this study population,which is in contrast to previous data (Berneis and Krauss2002; Sacks and Campos 2003). Although we did not findany significant changes in the NMR and traditional lipidprofiles, however, Soy+ increased HDL3-C, which wouldtheoretically increase the concentration of HDL3a andsupports previous findings (Goodman-Gruen and Kritz-Silverstein 2001). It is noteworthy that lipoproteins withinthe 8.2–12 nm HDL particle size range (i.e., HDL3a, HDL2a,and HDL2b) are favorable. Collectively, our data at best sug-gest a minimal cardioprotective effect of soy protein con-sumption in the normocholesterolemic, sedentary male,which provides credence to a recent contentious statementby the American Heart Association (Sacks et al. 2006).

Superfluous BMI, body mass, and percent body fat, as wellas an android body shape, are associated with an increasedrisk for CVD (Grundy 1999). In the present study, adding aprotein supplement to the habitual diet of sedentary maleswithin normal anthropometric limits increased BMI, bodymass, and fat mass regardless of the protein type and resultedin new fat mass deposition consistent with the android bodyshape. This suggests that in the short-term, these sedentarymales remained in positive energy balance (kilocalories con-sumed > kilocalories expended) and did not reduce energyintake to compensate for the additional caloric consumption.Interestingly, although all groups gained weight as fat mass,daily caloric intake tended to be higher after Milk and Soy+supplementation and lower after the Soy– supplementation; afat mass gain of 1.1, 0.6, and 0.8 kg were observed, respec-tively. We suspect that the innate limitations of recordingand collecting diet records may account for these counterin-tuitive results in light of the anthropometric data. If an equi-libration to energy balance (kilocalories in = kilocaloriesexpended) is not established, weight gain as fat mass will oc-cur over time and may increase the risk for CVD. However,the amount of weight gained would eventually equal the ad-ditional kilocalorie consumption and would result in the es-tablishment of a new energy balance, assuming no change inthe caloric value of the supplement. It is highly plausible thatthe small increases in BMI, body mass, and fat mass seen inthe present study would have equilibrated in the long term ifconsumption had continued, thus no significant increases inCVD risk would have been observed.

Dietary intake in free-living participants may limit theinterpretation of a study’s results. Intake of saturated fattyacids (i.e., lauric, myristic, and palmitic acids) has beenshown to elevate blood cholesterol concentrations in all lipo-protein particles including LDL and HDL (Van Horn andErnst 2001). Keys et al. (1970) estimated that for every 1%increase of total energy consumption from saturated fattyacids, there would be a 2.7 mg�dL–1 increase in fastingplasma cholesterol concentration. In the present study, satu-rated fatty acid intake significantly increased after Soy–T

able

4(c

oncl

uded

).

Milk

(n=

9)So

y–(n

=11

)So

y+(n

=10

)

Var

iabl

ePr

ePo

stPr

ePo

stPr

ePo

st{§}{

{{

Tri

glyc

erid

es10

0.0±

23.1

92.4

±16

.411

5.6±

24.5

125.

1±23

.891

.1±

11.9

95.3

±25

.2Fr

eefa

ttyac

ids

0.31

±0.

050.

37±

0.09

0.57

±0.

130.

55±0

.14

0.38

±0.0

80.

36±0

.06

Glu

cose

97.0

±3.

896

.4±

6.0

94.4

±4.

294

.5±

3.1

83.1

±4.

893

.8±

3.1

Lip

ase

conc

entr

atio

ns(m

mol

FFA�m

L–1�h

–1)

Tot

al34

.1±

2.0

34.9

±3.

431

.6±

4.1

37.4

±2.

634

.3±

3.3

34.1

±4.

6H

epat

ic{

24.5

±1.

9ab

23.9

±2.

4ab

21.9

±3.

2a26

.1±

2.7b

24.1

±3.

0ab

23.5

±3.

6ab

Lip

opro

tein

9.6±

0.8

11.0

±1.

39.

7±1.

411

.3±

1.3

9.3±

1.0

11.0

±1.

7

Not

e:So

y–,

isof

lavo

ne-p

oor

soy

prot

ein;

Soy+

,is

ofla

vone

-ric

hso

ypr

otei

n;V

LD

L,

very

low

-den

sity

lipop

rote

in;

LD

L,

low

-den

sity

lipop

rote

in;

IDL

,in

term

edia

te-d

ensi

tylip

opro

tein

;H

DL

,hi

gh-d

ensi

tylip

opro

tein

.*V

alue

sar

eex

pres

sed

asa

perc

enta

geof

tota

lV

LD

Lpa

rtic

les.

{ Val

ues

expr

esse

das

ape

rcen

tage

ofto

tal

LD

Lpa

rtic

les.

{ Val

ues

with

sim

ilar

lette

rsar

eno

tsi

gnif

ican

tlydi

ffer

ent

(p<

0.05).

§ Val

ues

expr

esse

das

ape

rcen

tage

ofto

tal

HD

Lpa

rtic

les.

||V

alue

sar

esi

gnif

ican

tly

diff

eren

tfr

ompr

eva

lues

;m

ain

effe

ctof

time

colla

psed

acro

ssdi

et(p

<0.01).

} Val

ues

are

sign

ific

antly

diff

eren

tfr

ompr

eva

lues

;m

ain

effe

ctof

time

colla

psed

acro

ssdi

et(p<

0.05).

**V

alue

sex

pres

sed

asa

perc

enta

geof

tota

lH

DL

-C.

496 Appl. Physiol. Nutr. Metab. Vol. 33, 2008

# 2008 NRC Canada

supplementation by 3.0% of total energy, which translatesinto an 8.1 mg�dL–1 increase in fasting plasma cholesterolaccording to Keys’ calculations (Keys et al. 1970). Althoughnot statistically significant, fasting serum TC concentrationstended to be higher after Soy+ supplementation, lower afterMilk supplementation, and unchanged after Soy–supplementation. These data suggest that any fluctuationsobserved in saturated fat intake may have had negligibleaffects on fasting TC concentrations in this population ofnormocholesterolemic individuals and (or) highlights thelimitations of using dietary records. Interestingly, totalHDL-C increased non-significantly by 6.0 and 12.0 mg�dL–1

for the Soy– and Soy+ groups, respectively, but did notchange for the Milk group. These data are in agreementwith other studies and suggest that other dietary factors(i.e., soy protein and soy protein – isoflavone interaction)may have played a role in regulating plasma cholesterolconcentrations (Anthony et al. 1996; Anthony et al. 1997).

Several meta-analyses over the past 12 years have re-ported progressively diminutive cardio-protective effects ofsoy protein despite supportive epidemiological mortalityand morbidity data from countries with high soy consump-tion (Anderson et al. 1995; Goodman-Gruen and Kritz-Silverstein 2001; Nagata et al. 1998; Reynolds et al. 2006;Taku et al. 2007; Zhan and Ho 2005). The American HeartAssociation (AHA) recently evaluated the literature andstated in Sacks et al. (2006) that

...the direct cardiovascular health benefit of soy protein orisoflavone supplements is minimal at best; in contrast,soy products such as tofu, soy butter, soy nuts, or somesoy burgers should be beneficial to cardiovascular andoverall health because of their high content of polyunsa-turated fats, fiber, vitamins, and minerals and low contentof saturated fat.

Using the traditional lipid profile, researchers suggest thatdata will show little effect of soy consumption on CVD riskreduction, which would seemingly support the AHA’s state-ment (Berneis and Krauss 2002; Sacks and Campos 2003).However, by utilizing NMR data, the salubrious effects ofsoy may become more transparent by remodeling lipo-proteins to a less atherogenic profile and thus would givecredence to previous epidemiological data with respect to acardio-protective effect. In the first study to use NMR anal-ysis in conjunction with a soy treatment, Allen et al. (2007)reported that LDL particle number and LDL-C decreased by4.3% and 2.6%, respectively, while maintaining LDL size inpostmenopausal women after consuming 20 g of Soy+(160 mg of Isoflavones) for 6 weeks. Since the penetrationof the endothelium by lipoprotein particles is a gradient-driven process, their data suggest that CVD risk is reducedbecause fewer LDL particles are in circulation with less cho-lesterol and thus the potential for oxidation and subsequentdamage is reduced (Weissberg and Rudd 2002). We wereunable to replicate these results in our study population.

In addition to the significance of total numbers of circu-lating LDL particles in CVD risk, particle density is also im-portant. Small, dense LDL and HDL particles are formed viareactions involving cholesteryl ester transfer protein (CETP),hepatic lipase (HL), cholesteryl esters, and TGs (Otvos et al.2002). Large LDL particles, with normal amounts of lipids,interact with TG-rich lipoproteins (i.e., chylomicrons and

VLDL), which exchange their core TG, one for one, withcholesteryl esters from the LDL core via CETP. This poten-tially depletes LDL of cholesterol and thus may become la-den with TG, which is then subjected to hydrolysis via HLand results in a small, dense LDL particle. LDL particlescan be quantified using gradient gel electrophoresis into twomajor subclasses based on size (nm): large LDL (pattern A)or small LDL (pattern B), which is inversely associated withCVD risk. In the present study, we did not find pre–post dif-ferences within each of our treatment groups; however, aver-age LDL size was significantly greater by 2.9% and 2.4%after Soy+ supplementation compared with post Milk andSoy– supplementation, respectively, suggesting that iso-flavones may play a small role in LDL remodeling towardslarger particle sizes. In support of this intimation, when thenumber of large LDL particles are expressed as a percentageof total LDL particles, the percentage of LDL particles were33% and 31% higher after Soy+ supplementation whencompared with post Milk and Soy– supplementation, respec-tively (i.e., post vs. post between group differences). In ad-dition, the percent concentration of large LDL particlestended to increase after Soy+ supplementation and decreaseafter Soy– supplementation (i.e., pre vs. post within-groupdifferences). Furthermore, when expressed as a percent oftotal LDL particles, total small LDL particles were lowerby 19% after Soy+ supplementation compared with postMilk and Soy– supplementation, respectively (i.e., post vs.post between-group differences). A non-significant increas-ing trend after Soy– supplementation and a decreasing trendafter Soy+ supplementation were also apparent for totalsmall LDL particles (i.e., pre vs. post within-group differen-ces). We observed similar changes in the percentage of verysmall LDL particles. However, all 3 protein groups showedaverage LDL sizes consistent with the pattern A phenotype,thus no study participant was considered remarkable forCVD risk. Furthermore, LDL size phenotype (pattern A vs.B) seem less informative with respect to CVD riskcompared with the number of small, dense LDL particlesand apo B concentrations (Lamarche et al. 1997). It is note-worthy that differences in quantification of pattern A (>20.5and >25.5 nm) vs. B (£20.5 and <25.5 nm) exist betweenNMR analysis and gradient gel electrophoresis, respectively.Otvos (2002) claims a correlation between these techniques;however, data to support this have not been published, thusadding to the complexity and difficulty of interpretation.

Similarly, HDL2 (which is inversely associated withCVD) can become small and dense by exchanging choles-teryl esters with TG-rich lipoproteins via CETP and sub-sequent TG hydrolysis via HL resulting in HDL3 (neutralassociation with CVD) (Johansson et al. 1991; Miller et al.1981). However, research suggests that these small, denseHDL particles may be converted in a unidirectional mannerto medium or large HDL particles (Colvin et al. 1998;Colvin et al. 1999). Interestingly, and counterintuitive tothis discussion, moderate alcohol consumption, which isnegatively associated with CVD, is actually inversely relatedto HDL3 and not to HDL2, highlighting the complexity ofHDL metabolism and the need for clarification (Haffner etal. 1985; Haskell et al. 1984). In addition, the existence ofother HDL subclasses further complicates the issue: HDL2b(9.7–12 nm), HDL2a (8.8–9.7 nm), HDL3a (8.2–8.8 nm),

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HDL3b (7.8–8.2 nm), and HDL3c (7.2–7.8 nm) (Blanche etal. 1981). Furthermore, NMR analysis infers HDL subclasssize by measuring the total number of methyl groups, whichcollectively reflect the lipoprotein diameter and core andsurface composition, and is reported as the concentration ofthe number of particles: large (HDL2b and HDL2a), medium(HDL3a), and small (HDL3b and HDL3c). In the presentstudy, we demonstrated that HDL3-C significantly increasedby 47.2%, whereas HDL2-C showed a non-significant in-creasing trend of 96.0% after Soy+ supplementation (i.e.,pre vs. post within group differences) and Soy– showed alesser, similar trend (51.4%), which is in agreement withearlier work (Goodman-Gruen and Kritz-Silverstein 2001).It is plausible that this remodeling effect, which makesHDL particles larger because of the greater cholesterol con-centration, would have a cardio-protective effect over a lon-ger period of time than the relatively short intervention inour study. Although we did not measure more finite HDLsubclasses and respective lipid composition directly, basedon NMR subclass size categorization (i.e., particle number)we speculate that there may have been more HDL3a (me-dium HDL) and less HDL3b and HDL3c (small HDLs) afterSoy+ supplementation owing to the decreasing trend of21.4% in the number of small, dense HDL particles and sig-nificantly lower number of small, dense HDL particles andlarger average HDL size in the post Soy+ group comparedwith the post Soy– group (–27.9% and +3.4%, respectively;i.e., post vs. post between-group differences). In addition,the number of medium HDL particles tended to be higherafter Soy+ consumption (i.e., pre vs. post within-group dif-ferences). This intimation is supported by research showingthat HDL3-C concentrations are not associated with thesmall, dense, atherogenic HDL3b (Williams et al. 1992). In-terestingly, the trend for increased HDL2-C after Soy+ sup-plementation was not accompanied by increases in thenumber of large HDL particles as would be expected. SinceNMR analysis groups lipoprotein particles according to sim-ilar but not identical particles with fluctuations in lipid com-position, this may limit concomitant comparison betweenultracentrifugation determination of cholesterol concentra-tion in HDL subclasses and NMR particle number based onlipoprotein particle size (Otvos 2002). We speculate thatalthough there was no increase in the number of large HDLparticles, lipid composition was altered such that there wasa greater concentration of cholesterol being transported onHDL2b and HDL2a particles. It is noteworthy that in thepresent study, average HDL size for all 3 protein groupswas within the 8.2–9.6 nm range, which corresponds withHDL3a and HDL2a.

The aforementioned HDL data suggest that isoflavonesmay play a minor role in lipid remodeling. In support ofthis notion, HL concentrations significantly increased by19.2% and the number of small HDL particles tended to behigher after Soy– supplementation (i.e., pre vs. post withingroup differences), which would theoretically result in moresmall, dense HDL particles. The opposite was true afterSoy+ supplementation. Furthermore, HDL2-C and the per-centage of large HDL particles also tended to increase,which may have ameliorated any potential negative physio-logical significance of increased HDL3-C. Interestingly,when expressed as a percentage of total HDL-C, HDL3-C

tended to decrease and HDL2-C tended to increase afterboth Soy– and Soy+ supplementation (i.e., pre vs. postwithin group differences). Collectively, these data suggestthat the addition of NMR analysis to HDL-C and HDL sub-class analyses may potentially be useful with respect to iso-flavones and HDL-C metabolism. Further research iswarranted to elucidate the clinical significance, if any, ofthese findings.

Apolipoproteins A-I and A-II are the major constituents ofHDL and are key proteins in HDL metabolism. Sixty percentof HDL particles contain both apolipoprotein A-I and A-II,with the remaining 40% containing only apolipoprotein A-I(Cheung and Albers 1982). Apolipoprotein A-I functions asthe preferred acceptor of cell cholesterol, as a cofactor forlecithin:cholesterol acyltransferase (LCAT), as an activatorof reverse cholesterol transport, and is a ligand for the putativeHDL receptor (Sirtori et al. 1999). The function of apolipo-protein A-II is less well described, but may include conferringstability to HDL via lipophilicity and inhibition of LCAT(Pownall and Ehnholm 2006). In the present study, we ob-served a significant increase of 15.0% and 15.5% in apolipo-proteins A-I and A-II, respectively, after protein consumptionregardless of the protein source. It is not clear why consumingmore protein increased these apolipoproteins in this study.

In summary, we have not shown that NMR analysis oflipoprotein particle number and size is a more sensitive indi-cator of CVD risk compared with the traditional lipid profilewith respect to potential salubrious effects of consuming soyprotein for 28 days in sedentary, normocholesterolemicmales. However, our data suggest that Soy isoflavones mayplay a role in HDL-C remodeling and that removing thesephytochemicals in soy could have counterproductive effectson hepatic lipase. Further research is warranted to clarify theuse of NMR analysis as a means of investigating potentialcardio-protective effects of soy protein consumption onHDL-C and subclass metabolism.

AcknowledgementThe authors would like to thank The Solae Company

(St. Louis, Mo.) for donating the protein supplements usedin this study.

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