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Nutrición Hospitalaria

versión On-line ISSN 1699-5198versión impresa ISSN 0212-1611

Nutr. Hosp. vol.25 no.5 Madrid sep./oct. 2010




The effect of consuming meat enriched in walnut paste on platelet aggregation and thrombogenesis varies in volunteers with different apolipoprotein A4 genotype

El efecto del consumo de carne enriquecida en pasta de nuez sobre la agregacion plaquetaria y la trombogenesis varia en voluntarios con diferente genotipo para la apolipoproteina A4



A. Canales1, J. Benedi2, S. Bastida1, D. Corella3, M. Guillen3, J. Librelotto1, M. Nus1 y F. J. Sánchez-Muniz1

1Departamento de Nutrición y Bromatología I (Nutrición).
2Departamento de Farmacología. Facultad de Farmacia. Universidad Complutense de Madrid. E-28040 Madrid (Spain).
1,2Facultad de Farmacia. Universidad Complutense de Madrid. E-28040 Madrid (Spain).
3Department of Preventive Medicine. Universidad de Valencia and CIBER Fisiopatología de la Obesidad y Nutrición, ISCIII, Spain.

Funds for this study were granted by the Spanish Ministerio de Educación y Ciencia, Project AGL 2001-2398-C03 and AGL 2005-07204-C02-01/ALI and Consolider-Ingenio 2010, reference CSD2007-00016.





Background and aim: Low-fat meat (LM) has been considered adequate under a cardiovascular disease point of view. Meat enriched in walnut paste (WM) consumption produces beneficial antithrombogenic effects but with striking inter-individual variability that may be related to gene polymorphism. Variants in the APOA4 gene (APOA4) polymorphism are known to affect the cardiovascular risk. This study aimed to compare the effects of consumption of WM and LM on platelet aggregation, production of thromboxane A2 (TXA2) and prostacyclin I2 (PGI2), and the TXA2/PGI2 ratio in 22 volunteers with different APOA4 polymorphism.
Subjects and Methods: Six volunteers carried the Gln allele (APOA4-2) while 16 were homozygous for the His allele (APOA4-1). Platelet aggregation, TXA2 (measured as TXB2), PGI2 (measured as 6-keto-PGF1α), and the thrombogenic ratio (TXB2/6-keto-PGF1α) were determined at baseline and at weeks 3 and 5 for the WM and LM dietary periods.
Results: Platelet aggregation decreased significantly (P<0.05) more in APOA4-1 than in APOA4-2 volunteers at 3-wk WM period, while TXB2 levels dropped more in APOA4-2 than in APOA4-1 volunteers at 5-wk WM period. TXB2 levels and the TXB2/6-keto-PGF1α ratio decreased significantly more (P<0.05) after 5 wk treatment in APOA4-2 than in APOA4-1 carriers on the WM diet than on the LM counterpart. However, 6-keto-PGF1α levels increased more (P<0.05) in APOA4-1 than in APOA4-2 volunteers after the 5-wk WM period than after the 5-wk LM diet.
Conclusions: Present results suggest that consumption of WM with respect to LM decrease the thrombogenic risk more in Gln carriers than in His/His.

Key words: APOA4 polymorphism. Meat enriched in walnut paste. Low-fat meat. Platelet aggregation. Prostacyclin. Thromboxane. Thrombogenic ratio. Functional food.


Antecedentes y objetivos: La carne con bajo contenido graso (LM) se considera adecuada bajo el punto de vista cardiovascular. La ingesta de carne enriquecida en pasta de nuez (WM), mejora los efectos antitrombogénicos con una variabilidad inter-individual que puede estar relacionada con el polimorfismo genético. Variaciones en los genes APOA4 (APOA4) del polimorfismo afectan el riesgo cardiovascular. Este estudio compara los efectos de la ingesta de WM y LM sobre la agregación plaquetaria, la producción de tromboxano A2 (TXA2) y prostaciclina I2 (PGI2), y el cociente TXA2/PGI2 ratio en 22 voluntarios con diferentes polimorfismos APOA4.
Material y métodos: Seis voluntarios portaban el alelo Gln (APOA4-2) frente a los 16 homozigotos para el alelo His (APOA4-1). La agregación plaquetaria, el TXA2 (medido como TXB2), la PGI2 (medida como 6-keto-PGF1α), y el cociente trombogenético (TXB2/6-keto-PGF1α) se determinaron al comienzo y en las semanas 3 y 5 de los periodos de WM y LM.
Resultados: La agregación plaquetaria disminuyó significativamente más (P <0.05) en los voluntarios APOA4-1 que en los APOA4-2 en la semana 3 del periodo WM. El descenso de los niveles de TXB2 fue mayor para los voluntarios APOA4-2 que para los APOA4-1 en la semana 5 del periodo WM. Después de 5 semanas con dieta WM, la concentración de TXB2 y el cociente TXB2/6-keto- PGF1α disminuyeron significativamente más (P<0.05) en los individuos APOA4-2 que en los APOA4-1 que con la dieta LM. Sin embargo, después de 5 semanas, la dieta WM con respecto a la LM incrementó más (P <0.05) los niveles de 6-keto-PGF1α en los voluntarios APOA4-1 que en los APOA4-2.
Conclusiones: Estos resultados sugieren que la ingesta de WM en comparación d LM, disminuye más el riesgo trombogénico en los voluntarios portadores de Gln que en los que His/His.

Palabras clave: Polimorfismo APOA4. Carne enriquecida en pasta de nuez. Carne baja en gras. Agregación plaquetaria. Prostaciclina. Tromboxano. Índice trombogénico. Alimento funcional.



Coronary heart disease (CHD) is a multifactorial disorder in which genetics and diet are both known to play major roles.1,2 While no consensus has yet been reached, dietary fatty acid composition appears to influence the pathogenesis of thrombosis and atherosclerosis. 3,4 Platelet synthesis of TXA2 and endothelial PGI2 production decrease with diets containing high concentrations of fish oils or n-3 fatty acids.5,6 However, in men, the effects of diets with increased amount of linolenic acid with respect to that of linoleic acid decreases TXB2 but increases the PGI2 productions, thus, differing from diets containing fish n-3 long fatty acids.7 Results from earlier studies by our research group indicate that the dietary oil exchange affects platelet aggregation and thrombogenesis in postmenopausal women.4,8 In addition, our group has very recently observed that a 5-time per wk consumption of walnut paste-enriched meat had a more positive effect on volunteers at high risk of cardiovascular disease than that of meat with a low fat content.9

Irrespective of such risk factors as age, excess body weight, hypertension, smoking and lack of physical activity, an inverse correlation exists between myocardial infarction or mortality and habitual walnut consumption. 10,12 Epidemiological studies suggest that the Mediterranean diet enriched with nuts may be a useful tool in metabolic syndrome management.12 Our research group has studied the effect of adding walnut paste, as a functional ingredient, to restructured meat and has found that consumption of this restructured meat significantly increases the concentration of certain endogenous antioxidants.13,14 Moreover, a related study15 found that volunteers who consumed walnut-enriched meat displayed significantly lower LDL-cholesterol levels than those who ate low-fat meat. However, the interindividual variability of the findings was striking 9,13-15 and genetic factors may have influenced the results.

APOA4 is thought to have a broad range of physiological functions. Thus, it is involved in intestinal cholesterol absorption16 and in chylomicrons assembly17 and composition.18 Intestinal APOA4 synthesis is specifically stimulated by absorption of long-chain fatty acids.19 Variants in the APOA4 gene (APOA4) polymorphism are known to affect the cholesterol absorption.18 APOA4-2 (His Gln) encodes a Q360H substitution near the C terminus of APOA4 and alters its biological properties.20 Although modifications in dietary cholesterol and saturated fat affect plasma lipids differently in carriers of each APOA4 variant,21,22 up to date there have been no studies on the effect of a APOA4 polymorphism on thrombogenesis. Moreover, no data are available on the effect of APOA4 polymorphism and the varied response to walnut consumption on individuals at increased risk of cardiovascular disease.

Therefore, it can be hypothesized that volunteers at increased CHD risk having different APOA4 360 polymorphism have differences in response to the intake of restructured beef steaks and sausages containing walnuts on aggregation, TXA2, PGI2 and the thrombogenic ratio (TXA2/PGI2). The present study aims (i) to compare the effects of meat enriched in walnut paste (WM) with those of low-fat meat (LM) on platelet aggregation, TXB2 (a stable metabolite of TXA2), 6-ketoprotaglandin FI1α (6-keto-PGFI1α) (a stable metabolite of PGI2) and the TXB2/6-keto-PGFI1α ratio in volunteers depending on the APOA4 360 polymorphism who consumed both type of meats.


Sample and study design


Of the 144 candidates initially recruited through announcements in the media and in hospitals, twenty five volunteers were chosen for the trial. Eligibility criteria for the study included a) age of men ≥45 years; age of women ≥50 years and postmenopausal) and b) BMI ≥25-<35 kg/m2. In addition, one or more of the following criteria also had to be met: serum total cholesterol ≥5·69 mmol/l; smoking habit (≥10 cigarettes per day); hypertension (systolic pressure ≥140 mmHg and/or diastolic pressure ≥90 mmHg. Candidates with familiar hypercholesterolemia and/or type I diabetes, and those taking any lipid-lowering, anti-hypertensive or anti-inflammatory drugs or hormone therapy were excluded from the trial.

Volunteers who did not frequently consume meat (35 times/wk) were not selected, despite the fact that many displayed three or more risk factors for CHD. Twenty-two candidates were not accepted for other reasons, such as the regular use of therapeutic medication. Three volunteers who did not complete the requisite blood extractions were also excluded from the trial. Procedures followed were in accordance with the standards of the Ethics Committee of the University Hospital of Puerta de Hierro (Madrid, Spain) and the Helsinki Declaration, as indicated in the guidelines of the Scientific Technologic Project AGL 2001-2398-C03. Informed consent was given by all participants prior to the start of the study.

Study design

Volunteers were randomly assigned to follow a non-blinded, cross-over, placebo-controlled study, consisting of two 5-week experimental periods. Participants followed their normal dietary habits during the 4 to 6-wk wash-out interval that separated the two trial periods. This wash-out interval was considered appropriate because according to Keys et al.23 diet induces stables changes in lipoprotein-lipids after 4 weeks and the average lifespan of circulating platelets is less than 2 weeks.24 During the WM period, volunteers consumed four 150 g restructured WM steaks and a 150 g ration of WM sausages per week, all containing 20% walnut paste. During the LM period, volunteers consumed four 150 g restructured LM steaks and a 150 g ration of LM sausages each week. The composition of the two types of meat is presented in table I and additional information is available in Serrano et al.25 Study participants were strongly requested not to include any other meats or meat derivatives in their diet during the trials.


Dietary control and compliance

Frozen LM and WM products were distributed to study participants on a weekly basis. Special emphasis was given to compliance and management of intake with regard to frequency, dates and numbers of steaks consumed. The substitution of conventional meat products by the experimental meat products in the framework of a mixed diet was confirmed and verified by regularly checking the volunteers' dietary records. Food Composition Tables were used to calculate the volunteers' dietary energy and nutrient intakes.26 Participants recorded the amount and kinds of food eaten every day to avoid any possible doubt regarding their diets. In addition, plasma γ-tocopherol concentrations were measured after each trial period to assess compliance.27 Despite the peculiar of WM most volunteers (80%) reported enjoying this type of meat. Forty percent of the participants, how ever, commented unfavorably on the low palatability of LM.

Anthropometric and blood pressure measurements

The weight, height, BMI and systolic and diastolic blood pressures of participants were measured by trained staff at the beginning of the study and at wk 3 and 5 of each dietary period.

Sample collection

Overnight fasting blood samples were collected from all participants at baseline and at 3 and 5 wk of each period. Volunteers gave blood samples, had their blood pressure and body weight recorded and submitted their dietary records on each hospital visit.

Blood samples were mixed with 3.8% trisodium citrate (9:1 (V/V), blood/citrate). The anticoagulated blood was centrifuged at 200 x g for 10-min to prepare platelet-rich plasma (PRP). A hemocytometer was used to perform platelet counts in PRP samples diluted with saline solution. The number of platelets in PRP samples was adjusted with saline solution to 300,000/mm3. Platelet aggregation was determined in PRP using ADP (Chromopag ADP. IZASA, Spain) as the aggregating agent with an electronic aggregometer (model 500, Chronolog Corporation, IZASA-Coulter, Havertown, PA), as reported by Cardinal and Flower.28 Aggregation data were expressed as the maximum aggregation rate obtained at 5 min (cm/5 min) and the time required to reach the maximum aggregation rate (min).

TXB2, a stable metabolite of TXA2, and 6-ketoprostaglandin (PG) F1a, a stable metabolite of PGI2, were extracted from citrated plasma samples using silica microcolumns (Chromabond® C18) coupled to a vacuum system (Manifold Vacuum Gauge Controller; J.T. Baker, Phillipsburg; N.J. USA).29 After extraction at pH 3, an aliquot of the dry residue was reconstituted with the assay buffer for TXB2 determination (TXB2/2,3-Dinor-TXB2(125I) RIA kit Izotop, Budapest, Hungary) and another for the PGI2 analysis (6-keto-PGFI /2,3-dinor-6-keto-PGFI (125I) RIA kit Izotop). A Packard Mod Cobra auto-gamma counting system (Packard Instrument Company, Inc., Packard-Becker B.V.; BK Groningen, The Netherlands) was used to measure radioactivity in all samples.

DNA isolation and APOA4 genotyping

DNA was extracted from peripheral blood cells using the Ultraclean Bloodspin kit (MoBio LaboratoriesInc, Carlsbad, California, USA). Amplification of a region of the APOA4 was done using the polymerase chain reaction (PCR) with genomic DNA, 1.5 mM MgCl2, 200 mM of dNTPs, and 0·6 mM of each oligonucleotide primer (P1: 5'-TCACTGGCAGAGCTGGGTGG- 3' and P2: 5'-CATCTGCACCTGCTCCTGCTGCTGCTCCAG- 3') in a final volume of 25 ml, according to the method previously described by Hixson and Powers.30 The 309-bp amplification product was digested with the restriction enzyme PvuII to distinguish between APOA4 360Gln and 360His alleles. Volunteers being homozygous for the His allele were classified as APOA4-1 while those carrying the Gln allele as APOA4-2. Reagents were purchased from Promega (Madison, USA) and PCR was performed using a DNA thermocycler (Mastercycler-ep380®, Eppendorf, Hamburg, Germany).

Statistic study

Changes in TXB2 and PGI2 concentrations over the 5-wk trial period were considered, a priori, to be the primary outcome variables. This study was designed to have a power of 80% to detect a 20% difference in TXB2 and PGI2 production between both meat-diet periods. A pooled SD of 30% for the change from baseline TXB2 and PGI2, based on previous studies [4,8,9,31] was assumed for this calculation. Data are presented as means and standard error (SE). Net percentage difference between responses of subjects who consumed walnut enriched meat and low fat meat were considered taking into account basal values for the low fat meat period. The significance of differences in entry parameters between the APOA4-1 and APOA4-2 groups was determined by two-tailed unpaired Student's t-test. Differences in aggregation rates and eicosanoid levels between both genotype groups in response to diet were analyzed by repeated-measures analysis of variance. The SPSS 15.0 statistical package was used. Data were considered as statistically significant at P<0.05.



Table II shows the characteristics of subjects carrying the APOA4 variants. All volunteers, regardless of their gene variant maintained their body weight, BMI and blood pressure during the entire length of the study (data not shown).


WM had higher PUFA content and a lower n-6/n-3 ratio than LM (Table I). Food intake did not differ significantly throughout both dietary periods between volunteers of each of the two APOA4 variants. The contribution of SFA to dietary energy was lower with the WM diet than with the LM one, while energy contributions of PUFA n-6 and PUFA n-3 and the n-6/n-3 ratio were higher (Table III). Data on platelet aggregation, eicosanoid production and the thrombogenesis ratio at basal conditions and at wk 3 and 5 of each period for both APOA4 variants are presented in Table IV shows maximum aggregation and TXB2 values at wk 5 that were lower (P<0.05) for APOA4-2 individuals in the WM period than during the LM period. TXB2 production and the TXB2/6-keto-PGFI1α ratio decreased after 5 wk significantly more (P<0.05) in APOA4-2 than in APOA4-1 carriers on the WM diet than on the LM counterpart. However, levels of 6-keto-PGFI1α were higher (P<0.05) in APOA4-1 carriers than in APOA4-2 individuals after the 5-wk WM diet than after the 5-wk LM period (Table IV).



Up to date, this is the first study investigating differences on response to diet on platelet aggregation and eicosanoid production in APOA4 360 polymorphism variant volunteers. Moreover, no studies have previously been done on the effects of walnut consumption in individuals with APOA4 allelic variants.

Considering the high fat and energy contents of walnuts,32 and thus their potential effect on increasing body weight, it is interesting to note the absence of any effect on body weight or BMI in carriers of either variant studied as a result of WM intake. Although no data is available regarding the effect of APOA4 polymorphism on thrombogenesis, the documented benefits of the APOA4-2 variant on lipid metabolism33 may indicate that this variant could also prove to have beneficial effects on parameters related to thrombogenesis such as platelet aggregation, eicosanoid production and the thrombogenic ratio. This hypothesis is based on the presence of lipoprotein receptors on platelets.34.

According to the present data, platelet aggregation decreased in all volunteers, regardless of their APOA4 variant. However, TXB2 production in of the APOA4-2 carriers consuming WM-diet vs. LM-diet decreased to a greater degree than that of their APOA4-1 counterparts (81.3 vs. 24.6%, respectively). In contrast, PGI2 production increased (36.5%) in APOA4-1 individuals while PGI2 levels decreased slightly (2.2%) in APOA4-2 carriers. No clear explanation can be drown to explain present data; however, as both APOA4 polymorphism carriers consumed similar amounts of n-6 PUFA and n-3 PUFA intakes in each separate period, it can be suggested that APOA4-2 carriers appear to be more efficient in enriching platelets in n-3 fatty acids. This will be tested in future studies.

An optimal balance in the TXA2/PGI2 ratio may help prevent thrombotic conditions, but this ratio appears to be affected by dietary fatty acid content.7 Chan et al7 found that TXB2 production in men significantly decreased when an oil mixture with a high linoleic/linolenic ratio was substituted by an intermediate or low linoleic/linolenic acid mixture. However, thromboxane synthase activity decreased and PGI2 synthesis increased when the linoleic/linolenic ratio was approximately 10.7 Present data suggest that WM diet vs. LM diet, due to differences in their linoleic/linolenic acid ratios, affected TXA2, TXA2/PGI2 and PGI2 levels in APOA4-1 individuals as expected.7 However, dietary exchange did not appear to affect PGI2 levels in APOA4-2 subjects. We have no clear explanation for the PGI2 results, but differences between the biophysical properties of APOA4-1 and APOA4-2 isoproteins may be implicated. APOA4-2 isoprotein has higher surface activity than APOA4-1.20 Moreover, polyunsaturated phospholipids and n-3 polyunsaturated fatty acids (PUFA) more than n-6 PUFA have an expanded interfacial conformation. While there is abundant scientific evidence regarding the benefits of decreasing TXB2 by replacing it with TXB3,35 there is no consensus on the benefits of substituting PGI2 for PGI3, as the latter has been found to be equally active,36 less active37 or more active38 than the former.

Various walnut compounds may be responsible for the modifications in parameters related to thrombogenesis observed in the present study. Tocopherol, for example, may decrease thrombogenicity. 39,40 Phenolic antioxidants are known to inhibit prostaglandin synthesis as a result of reduced lipid peroxide levels.41 Glutathione peroxidase also suppresses PG biosynthesis by removing lipid hydroperoxides.41 Thus, platelet aggregation and levels of the enzymes that play a role in eicosanoid production may drop due to the action of the polyphenols found in walnuts and certain vegetables in the form of condensed tannins].39,41-43 Proanthocyanidins, naturally occuring plant metabolites commonly found in fruits, vegetables, nuts, seeds, flowers and bark,44 form part of a specific group of polyphenolic compounds called flavonoids.45 These compounds are reported to have anti-inflammatory and vasodilatory properties,44,45 to inhibit lipid peroxidation, platelet aggregation and capillary permeability and to affect, among others, the phospholipase A2, cyclooxigenase and lipoxygenase enzyme systems40,43-45 No data is currently available on the possible role of APOA4 polymorphism on the absorption and/or bioavailability of these compounds.

In conclusion, the effect of WM consumption on TXB2 production and the thrombogenic ratio suggest that substitution of LM meat products with WM helps decrease thrombogenic risk in habitual meat consumers. However, results suggest that dietary treatment affected more the TXB2 but less the PGI2 productions in APOA4-2 carriers. Nonetheless, due to the relatively low number of APOA4-2 volunteers more future studies are needed to better understand the physiological effects of changes induced by consumption of meat enriched in walnut paste, and to assess the importance of the gene mutation in response to a diet enriched in linolenic acids and some minor compounds from walnut.



We are grateful for the valuable assistance in thromboxane and prostacyclin determination given by the Department of Physiology of the Medical School of the Universidad Complutense of Madrid. Thanks are due to the Universidad Complutense of Madrid for the predoctoral fellowship of Meritxell Nus.

All authors have significantly contributed to the paper and agree with the present version of the manuscript. FJ S-M is the corresponding author and Guarantor of the paper and has contributed to the study design, volunteers' selection, data discussion and writing of the paper. JB and SB and have contributed to the data acquisition and analysis and writing of the paper, AC, MN and JL have contributed to the volunteers' selection, data analysis and writing of the paper. DC and MG have contributed to the sample genotyping and writing of the paper. The study was performed according to the ethical bases of the Helsinki Declaration. Volunteers provided informed consent previous to the start of the study.

The authors declare no conflicts of interest



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Francisco J. Sánchez-Muniz.
Plaza Ramón y Cajal s/n.

Recibido: 6-IX-2009.
Revisado: 7-IX-2009.
Aceptado: 11-XI-2009.

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