Effects of Almond Dietary Supplementationon Coronary Heart Disease Lipid Risk Factors

and Serum Lipid Oxidation Parametersin Men with Mild Hyperlipidemia

Beman-Ali Jalali-Khanabadi, PhD,1 Hassan Mozaffari-Khosravi, PhD,2 and Nayereh Parsaeyan, MSc1

Abstract

Objectives: Oxidation and level of plasma lipids are closely implicated in the development of coronary heartdisease (CHD). Dietary almond supplementation may participate in beneficial effects on CHD lipid risk factorlevels and their susceptibility to oxidative modification. The aim of this study was to evaluate the effects ofdietary supplementation with almond on serum lipid levels and their relation to lipid oxidation parameters inmen with mild hyperlipidemia.Design: Thirty (30) healthy volunteer men (age 45.57� 7.14 years and body–mass index 24.29� 2.15 kg/m2) withmild hyperlipidemia received 60 g almond daily for 4 weeks.Outcome measures: Overnight fasting blood samples were obtained before and after supplementation. Serumlevels of lipids, lipoproteins, and apolipoproteins and copper-induced serum lipid oxidation were determined.Lipid oxidation was followed by monitoring of the change of conjugated dienes in diluted serum after additionof Cu2þ. A number of quantitative parameters including lag-time, maximal rate of oxidation (V-max), andmaximal amount of lipid peroxide products (OD-max) were evaluated.Results: After 4 weeks, almond supplementation significantly decreased low-density lipoprotein cholesterol,total cholesterol (TC), and apolipoprotein B100 (apo-B100). At baseline, there was little correlation between lipidrisk factors and lipid oxidation parameters, but a positive correlation was observed between TC and lag-time(r¼ 0.6, p¼ 0.001), negative correlation between TC with V-max and OD-max (r¼�0.65, p< 0.001 and r¼�0.61,p¼ 0.001), and also positive correlation between apo-B100 with V-max and OD-max (r¼ 0.48, p¼ 0.01 andr¼ 0.54, p¼ 0.003) after almond supplementation.Conclusions: These results demonstrated that almond supplementation, in addition to lowering effects on serumlevels of CHD lipid risk factors, may contribute to a dramatic change in the relation of lipid risk factors andsusceptibility of serum lipids to oxidative modification. This may be due to the distribution of different almondphenolic antioxidants in different components of serum including nonlipoprotein molecules such as serum albumin.

Introduction

Coronary heart disease (CHD) is the leading cause ofdeath in many populations.1 One of the considered

strategies to control CHD is to identify and modify the mostserious and modifiable local risk factors. There are severalgroups of risk factors for atherosclerosis and cardiovasculardiseases.2 High plasma level of lipids and lipid peroxidationare closely implicated in the development and progression ofatherosclerosis.3

During the last decades, considerable interest has beendirected toward investigation of plasma lipids susceptibilityto oxidation in healthy and different diseased conditions.4–6

Another area of interest is evaluation of the protective effects ofdifferent medications or specific diet supplementation on thesusceptibility of isolated and whole serum lipids to oxidation.7–9

Epidemiologic and clinical studies have shown that con-sumption of nuts is associated with favorable plasma lipidprofiles and reduced cardiovascular risk.10,11 Almonds havebeen used in a wide variety of food formulations, and some

1Department of Biochemistry, Faculty of Health, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.2Department of Nutrition, Faculty of Health, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.

THE JOURNAL OF ALTERNATIVE AND COMPLEMENTARY MEDICINEVolume 16, Number 12, 2010, pp. 1279–1283ª Mary Ann Liebert, Inc.DOI: 10.1089/acm.2009.0693

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research indicated its plasma lipids-lowering effect on iso-lated low density lipoprotein (LDL) oxidation.12,13

Whether the monounsaturated fatty acid or nonfat (pro-tein, fiber, flavonoids) components of almonds are responsi-ble for the beneficial effect of the nut on cardiovascular risk isnot fully elucidated. On the other hand, there are littleavailable data on the effect of dietary almond supplementa-tion on the serum lipid oxidation parameters and their rela-tion to lipid risk factors for CHD in unfractionated serum.

This study was undertaken to assess the effect of almondsupplementation on in vitro copper-induced lipid oxidationparameters in diluted whole serum, and its relation withlipid, lipoprotein, and apolipoprotein levels in men withmild hyperlipidemia.

Materials and Methods

Subjects and study design

The study population consisted of 30 healthy male vol-unteers (age 45.57� 7.14 years and body–mass index24.29� 2.15 kg/m2) with mild hyperlipidemia (serum cho-lesterol and triglycerides 200–300 mg/dL) who received 60 galmonds daily for 4 weeks. The participants were given apacket weekly including 7 sachets containing 60 g of rawalmonds, and they were instructed to use it 2 times daily as asnack for the next week.

During the study, subjects followed their own normaldiets, to which was added the supplement. None of thesubjects had clinical or biochemical evidence of diabetes orcardiovascular, liver, or renal disease, and no one was takingmedications, antioxidant supplements, or smoking ciga-rettes. During the study, subjects were asked not to consumeany additional nuts or nut products or alter consumption ofdietary fiber, and also to maintain their level of physicalactivity.

Measures and biochemical analyses

Overnight fasting blood samples and body weight wereobtained before and after the supplementation. Serum levelsof lipids, lipoproteins, and apolipoproteins were determined,and the in vitro copper-induced profiles of serum lipids ox-idation were evaluated. Serum total cholesterol (TC) andtriglycerides (TG) concentration were determined by enzy-matic methods: cholesterol oxidase and glycerol oxidase,respectively. High density lipoprotein cholesterol (HDLc) con-centration was determined with dextran-sulfate–magnesiumchloride precipitation of b-lipoproteins, followed by the sameenzymatic method for TC. Low density lipoprotein choles-terol (LDLc) was calculated using the Friedewald formula.14

Apolipoprotein-A1 (apo-A1) was determined by the immu-noturbidimetry method.15 Apolipoprotein-B100 (apo-B100)and lipoprotein(a) [Lp(a)] were determined by the electro-immunoassay method.16

Specific anti-Lp(a), anti-apo-B100, anti-apo-A1 antibodies,primary standards, and controls were from Dako (DK-2600Glostru, Denmark). Copper-induced lipid peroxidation wasestimated in a 60-fold diluted serum in 20 mmol phosphatebuffer containing 150 mmol NaCl and 720 mmol citrate,pH¼ 7.4. The lipid oxidation procedure was conducted at378C and was initiated by the addition of CuCl2 to give afinal concentration of 60mmol/L.

The kinetics of conjugated dienes formation were moni-tored spectrophotometrically (Perkin-Elmer UV.VIS Doublebeam spectrophotometer 505S) by measuring absorbance in a1-cm quartz cuvette at 245 nm every 10 minutes for 300minutes. Microsoft Excel software was used for plotting ofthe kinetic curves of the accumulation of lipid peroxideproducts (change of absorbance at 245 nm over time inminutes), and a number of quantitative oxidation parametersincluding lag-time (the time interval between the addition ofCuCl2 to the serum and the beginning of extensive oxida-tion), maximal rate of oxidation (V-max), and maximalamount of lipid peroxide products accumulation (OD-max)were evaluated. Before the processing of samples, the se-rum lipid oxidation method was optimized and an inter-individual coefficient of variations (CV) of 6% (for lag-time,n¼ 10), 7.4% (for OD-max, n¼ 10), and 7.5% (for V-max,n¼ 10) was obtained.

Data analysis

All values are reported as mean� standard deviation(SD). Differences between before and after supplementationwere assessed by paired t test. The Pearson correlation testwas used for evaluation of relationship between oxidationparameters and other variables in the study population.P-value <0.05 was considered as significant. All P-valueswere two-tailed. All the tests were performed by the SPSSpackage Version 11 (SPSS Inc., Chicago, IL).

Ethical considerations

An informed consent was obtained from each participant.They could quit the study freely, whenever they liked. Allthe patients were continuing their usual diet. The ResearchEthics Committee of the Shahid Sadoughi University ofMedical Sciences approved the research proposal of thestudy.

Results

Almond supplementation significantly decreased serumlevels of LDLc, TC, and apo-B100. Comparison of serumlipids, lipoproteins, and apolipoproteins before and aftersupplementation are summarized in Table 1.

The kinetics analysis of conjugated diene production re-vealed that almond supplementation prolonged the lag-timeof conjugated diene formation (from 60.5� 34 minutes to79� 50 minutes), but this and other changes in oxidationparameters were not statistically significant.

Serum lipid oxidation parameters before and after inter-vention are compared in Table 2. At baseline, a significantassociation was found between TC and lag-time, but afteralmond supplementation, a significant positive correlationbetween these two variables was observed.

Table 3 shows the relationship between some cardiovas-cular lipid risk factors and lipid oxidation parameters of se-rum in the study population before and after theintervention. No significant changes were found for Lp(a)and apo-A1 following the almond supplementation.

Discussion

Consumption of 60 g almonds daily as a supplement in thediet of men with mild hyperlipidemia reduced LDLc and

1280 JALALI-KHANABADI ET AL.

apo-B100. These data suggest a favorable effect of almonds inmodifying lipid risk factors for CHD. These benefit effectshave been demonstrated by other authors over the last de-cade and may be due to the unsaturated fat contents ofalmonds.17–19

A noticeable finding of this study was a significant changein the association of lipid CHD risk factors with lipid oxi-dation parameters of serum in the study population follow-ing the almond supplementation. The most effective changeshave been observed on TC and related parameters such asTC/HDLc ratio following the supplementation. TC indicateda negative correlation with lag-time before intervention,while there was seen a strong positive correlation betweenTC and lag-time after intervention. Lag-time is an index forresistance of serum lipid to oxidation, and longer lag-timeindicated more resistance of lipid to initiation of oxidation.Therefore, in subjects with normal diet, increased serumlevels of TC have been associated with increased suscepti-bility of serum lipids to oxidation, while following almondsupplementation serum lipids are more resistant to initiationof copper-induced oxidation. Minimal changes also wereobserved on LDLc and LDLc/HDLc ratio following the

supplementation. This is predominantly related to lag-time,and there is a shift from negative correlation of these lipidrisk factors with lag-time toward a more positive correlationafter supplementation. Association of serum TC and LDLcwith susceptibility of serum to lipid oxidation also has beenreported by others.20,21 The current results show that almondsupplementation reversed this effect, and in subjects withthis intervention, increased serum levels of TC and LDLc areassociated with decreased susceptibility of whole serumlipids to oxidation.

On the other hand, TC had little or no correlation withother oxidation parameters (OD-max and V-max) before theintervention, while an extensive negative correlation betweenTC with OD-max and V-max was seen after the supplemen-tation. V-max and OD-max are indexes for the rate and finalextent of lipid oxidation products accumulation in the courseof in vitro copper-induced profile of serum lipid oxidation,respectively. Therefore, in subjects with supplementation, in-creased serum levels of TC are associated with decreased rateand extent of accumulation of lipid oxidation products in lipidoxidation profile in the experimental condition.

As compared to TC and LDLc, reverse changes have beenobserved on apo-B100 and related parameters following thesupplementation. However, apo-B100 had no statisticallysignificant correlation with lipid oxidation parameters beforeintervention, but a weak negative correlation of apo-B100with lag-time was seen, and a strong positive correlation wasseen between apo-B100 with OD-max and V-max after in-tervention. These results indicated that in subjects aftersupplementation, higher serum levels of apo-B100 are asso-ciated with more susceptibility to initiation and higher rateand extent of lipid oxidation in the course of copper-inducedserum lipid oxidation. Apparently, supplementation causesmore effectiveness of apo-B100 in the process of serum lipidoxidation in this experimental condition. Thus, following thealmond supplementation, serum lipids susceptibility to oxi-dation is more sensitive to the variation of serum apo-B100concentration. Chen et al., in an in vitro study on isolatedLDL and apo-B100, demonstrated that phenolic compoundsfrom almond reduced the oxidative modification of apo-B100and stabilized LDL conformation.22 In this study, we as-sessed unfractionated serum for lipid susceptibility to oxi-dation after supplementation and observed a shift to morenegative association of apo-B100 and lag-time. Therefore, inour experimental condition some almond phenolic com-pounds with antioxidant properties may bind on moleculesother than lipoproteins, including serum albumin. Therefore,this reversed effect on apo-B100 in comparison to TC that weobserved after supplementation may be due to this profile ofantioxidant serum distribution.

In this study, we assessed the effects of almond supple-mentation on serum levels of CHD lipid risk factors and alsotheir relationship with lipid oxidation parameters. However,almond supplementation had beneficial effects on CHD lipidrisk factor levels, but its effect on the relation of lipid riskfactors to lipid oxidation parameters was controversial. Aftersupplementation, the most effective change in the relation oflipid risk factor and parameters of serum lipid oxidation wereobserved in TC, moderate change for LDLc, and apo-B100.This controversy may be due to the effects of different com-ponents of almonds and its distribution in serum. The bene-ficial effect of almonds on the serum levels of CHD risk

Table 1. Comparison of Serum Lipids, Lipoproteins,

and Apolipoproteins Levels in Study Population

Before and After Almond Supplementation

Serumparameters

BeforeMean� SD

AfterMean� SD p

TC (mmol/L) 6.61� 0.68 5.99� 1.03 0.01TG (mmol/L) 3.15� 0.99 2.96� 1.15 0.38HDLc (mmol/L) 1.01� 0.21 1.01� 0.27 0.96LDLc (mmol/L) 4.38� 0.70 3.76� 0.66 <0.001Lipoprotein(a) (g/L) 0.255� 0.225 0.239� 0.215 0.36apo-A1(g/L) 1.33� 0.190 1.35� 0.167 0.56apo-B100 (g/L) 1.26� 0.305 1.11� 0.246 0.009

SD, standard deviation; TC, total cholesterol; TG, triglycerides;HDLc, high-density lipoprotein cholesterol; LDLc, low-densitylipoprotein cholesterol; apo-A1, apolipoprotein A1; apo-B100, apo-lipoprotein B100.

Table 2. Comparison of Parameters of Serum

Lipid Oxidation in Study Population Before

and After Almond Supplementation

Oxidation parametersBefore

Mean� SDAfter

Mean� SD p

Lag-time (min) 60.5� 34 79� 50 0.21OD-max 153.5� 70.7 160.8� 73.6 0.57V-max 0.92� 0.41 0.90� 0.37 0.93T-max (min) 149.5� 73 160� 71 0.87

SD, standard deviation; lag-time, the time needed (in minutes) toinitiation of lipid oxidation products accumulation during the lipidoxidation course after addition of CuCl2; OD-max, maximal amountof lipids peroxide products accumulation in mmol/L of conjugateddienes per mmol of low-density lipoprotein cholesterol (LDLc)during the lipid oxidation course; V-max, maximal rate of oxidationin mmol/L of conjugated dienes production per mmol of LDLc perminute in during the lipid oxidation course; T-max, time needed (inminutes) to gained the maximal rate of lipid peroxide productsaccumulation during the lipid oxidation course.

ALMOND, CHD LIPID RISK FACTORS, AND LIPID OXIDATION 1281

factors is mainly related to its unsaturated fatty acids andfiber contents, while its different effects on the relationship oflipid risk factors and lipid oxidation parameters in wholeserum may be due to its phenolic contents and distribution ofthese antioxidant molecules in various components of serum.

These results demonstrated that almond supplementation,in addition to beneficial effects on serum levels of CHD lipidrisk factors, may contribute to a dramatic change in the re-lation of lipid risk factors and susceptibility of serum lipidsto oxidative modification. We suggest that different effects ofalmond supplementation on the relation of lipid risk factorlevels and serum lipid susceptibility to oxidation may be dueto the distribution of different almond phenolic antioxidantsin different components of serum including albumin. Furtherstudies are needed to clarify the exact distribution of almondantioxidants in serum and their antioxidant potential againstserum lipid oxidation.

Acknowledgments

We thank the Department of Research Administration,Shahid Sadoughi University of Medical Sciences (SSUMS) forfunding this project. Special thanks to the participants whoparticipated in this study.

Disclosure Statement

No competing financial interests exist.

References

1. Tunstall-Pedoe H, Kuulasmaa K, Amouyel P, et al. Myo-cardial infarction and coronary deaths in the World HealthOrganization MONICA Project: Registration procedures,event rates, and case-fatality rates in 38 populations from 21countries in four continents. Circulation 1994;90:583–612.

2. Acevedo M, Tagle R, Simpfendorfer C. Non-traditional riskfactors for atherosclerosis. Rev Med Chil 2001;129:1212–1221.

3. Massaeli H, Pierce GN. Involvement of lipoproteins, freeradicals, and calcium in cardiovascular disease processes.Cardiovasc Res 1995;29:597–603.

4. Carroll YL, Corridan BM, Morrissey PA. Lipoprotein carot-enoid profiles and the susceptibility of low density lipoprotein

to oxidative modification in healthy elderly volunteers. Eur JClin Nutr 2000;54:500–507.

5. Liu ML, Ylitalo K, Vakkilainen J, et al. Susceptibility of LDLto oxidation in vitro and antioxidant capacity in familialcombined hyperlipidemia: Comparison of patients withdifferent lipid phenotypes. Ann Med 2002;34:48–54.

6. Riemersma RA, Wilson R, Payne JA, Shepherd MJ. Seasonalvariation in copper-mediated low-density lipoprotein oxi-dation in vitro is related to varying plasma concentration ofoxidised lipids in summer and winter. Free Radic Res2003;37:341–347.

7. Yu YM, Chang WC, Liu CS, Tsai CM. Effect of young barleyleaf extract and adlay on plasma lipids and LDL oxidation inhyperlipidemic smokers. Biol Pharm Bull 2004;27:802–805.

8. Atkin MA, Gasper A, Ullegaddi R, Powers HJ. Oxidativesusceptibility of unfractionated serum or plasma: Responseto antioxidants in vitro and to antioxidant supplementation.Clin Chem 2005;51:2138–2144.

9. Upritchard JE, Sutherland WH, Mann JI. Effect of supple-mentation with tomato juice, vitamin E, and vitamin C onLDL oxidation and products of inflammatory activity intype 2 diabetes. Diabetes Care 2000;23:733–738.

10. Kocyigit A, Koylu AA, Keles H. Effects of pistachio nuts con-sumption on plasma lipid profile and oxidative status in heal-thy volunteers. Nutr Metab Cardiovasc Dis 2006;16:202–209.

11. Chisholm A, Mc Auley K, Mann J, et al. Cholesterol lower-ing effects of nuts compared with a canola oil enriched cerealof similar fat composition. Nutr Metab Cardiovasc Dis2005;15:284–292.

12. Jambazian PR, Haddad E, Rajaram S, et al. Almonds in thediet simultaneously improve plasma alpha-tocopherol con-centrations and reduce plasma lipids. J Am Diet Assoc2005;105:449–454.

13. Jenkins DJ, Kendall CW, Marchie A, et al. Dose responseof almonds on coronary heart disease risk factors: Bloodlipids, oxidized low-density lipoproteins, lipoprotein(a),homocysteine, and pulmonary nitric oxide. A random-ized, controlled, crossover trial. Circulation 2002;106:1327–1332.

14. Friedewald WT, Levy RI, Fredrickson DS. Estimation of theconcentration of low-density lipoprotein cholesterol inplasma without use of the preparative ultracentrifuge. ClinChem 1972;18:449–502.

Table 3. Correlation Coefficient (r) Between Parameters of Serum Lipid Oxidation with Some

Cardiovascular Lipid Risk Factors Before and After Almond Supplementation

Before After

Lag-time OD-max V-max Lag-time OD-max V-max

r p r p r p r p r p r p

TC �0.41 0.04 �0.43 0.03 �0.33 0.11 0.60 0.001 �0.65 <0.001 �0.61 0.001TG �0.03 0.90 �0.13 0.55 �0.15 0.48 0.45 0.02 �0.26 0.19 �0.29 0.14LDLc �0.37 0.07 �0.59 0.002 �0.49 0.01 0.23 0.25 �0.51 0.006 �0.58 0.001apo-B100 0.37 0.06 0.06 0.75 0.25 0.22 �0.30 0.12 0.54 0.003 0.48 0.01apo-B100/apo-A1 0.30 0.14 �0.08 0.72 0.29 0.15 �0.27 0.16 0.59 0.001 0.60 0.001LDLc/HDLc �0.33 0.11 �0.53 0.007 �0.46 0.02 0.41 0.04 0.40 0.04 0.48 0.01TC/HDLc �.029 0.16 �0.44 0.03 �0.37 0.07 0.61 0.001 �0.49 0.009 �053 0.005HDLc 0.04 0.83 0.30 0.14 0.32 0.12 �0.41 0.03 0.22 0.26 0.26 0.18

TC, total cholesterol; TG, triglycerides; LDLc, low-density lipoprotein cholesterol; apo-B100, apolipoprotein B100; apo-B100/apo-A1,apolipoprotein B100 to apolipoprotein A1 ratio; HDLc, high-density lipoprotein cholesterol; LDLc/HDLc, low density lipoprotein cholesterolto high density lipoprotein cholesterol ratio; TC/HDLc, total cholesterol to high density lipoprotein cholesterol ratio.

1282 JALALI-KHANABADI ET AL.

15. Riepponen P, Marniemi J, Rautaoja T. Immunoturbidimetricdetermination of apolipoproteins A-1 and B in serum. ScandJ Clin Lab Invest 1987;47:739–744.

16. Marz W, Gross W. Quantification of human serum lipo-protein Lp(a): Zone immunoelectrophoresis assay, a newsensitive method as compared to electroimmunoassay. ClinChim Acta 1983;134:265–279.

17. Spiller GA, Jenkins DJ, Cragen LN, et al. Effect of a diet highin monounsaturated fat from almonds on plasma cholesteroland lipoproteins. J Am Coll Nutr 1992;11:126–130.

18. Spiller GA, Jenkins DA, Bosello O, et al. Nuts and plasmalipids: An almond-based diet lowers LDL-C while preserv-ing HDL-C. J Am Coll Nutr 1998;17:285–290.

19. Spiller GA, Miller A, Olivera K, et al. Effects of plant-baseddiets high in raw or roasted almonds, or roasted almondbutter on serum lipoproteins in humans. J Am Coll Nutr2003;22:195–200.

20. Yoshida Y, Ito N, Shimakawa S, Niki E. Susceptibility ofplasma lipids to peroxidation. Biochem Biophys Res Com-mun 2003;305:747–753.

21. Yu-Poth S, Etherton TD, Reddy CC, et al. Lowering dietarysaturated fat and total fat reduces the oxidative suscepti-bility of LDL in healthy men and women. J Nutr 2000;130:2228–2237.

22. Chen CY, Milbury PE, Chung SK, Blumberg J. Effect of al-mond skin polyphenolics and quercetin on human LDL andapolipoprotein B-100 oxidation and conformation. J NutrBiochem 2007;18:785–794.

Address correspondence to:Hassan Mozaffari-Khosravi, PhD

Department of NutritionFaculty of Health

Shahid Sadoughi University of Medical SciencesDaneshjoo Boulevard

Yazd 8916278477Iran

E-mail: Mozaffari.kh@gmail.com

ALMOND, CHD LIPID RISK FACTORS, AND LIPID OXIDATION 1283