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3 Departamento de Nutrición y Bromatología I (Nutrición), 4 Biotransformations Group, Departamento de Química Orgánica y Farmacéutica, Facultad de Farmacia, Universidad Complutense de Madrid, Madrid E-28040, Spain; and 5 Departamento de Medicina Preventiva y CIBER Fisiopatología de la Obesidad y Nutrición, Facultad de Medicina, Universidad de Valencia, Valencia E-46010, Spain
* To whom correspondence should be addressed. E-mail: frasan{at}farm.ucm.es.
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ABSTRACT |
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 TOP ABSTRACT IntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
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Introduction |
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TOPABSTRACTIntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
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Major PON 1 polymorphisms include the replacement of glutamine (Q or A) by arginine (R or B) at position 192 (5), and that of leucine (L) by methionine (M) at position 55 (6). PON1-192R and PON1-L55 carriers hydrolyze paraoxon faster than noncarriers (7,8). However, it has been suggested that the arylesterase activity of both allozymes toward phenylacetate (PA) is similar (9). The ability of HDL to protect LDL against peroxidation in vitro is significantly lower in HDL particles containing PON1-192R (10) than in those with PON1-Q192.
Studies evaluating the interaction of diet and PON1 polymorphisms on PON1 activity and lipoprotein metabolism are scarce (11–13). Most of the studies focus on measuring paraoxonase activity (11–13) but not that of arylesterase (14). Intake of different vegetable-based foods has been found to reduce lipid peroxidation in PON1-192R carriers (14) and to diminish paraoxonase activity in PON1-L55 and PON1-192R subjects (13). Walnuts, which are rich in PUFA and antioxidant substances, protect against cardiovascular disease (CVD) (15–17). Thus, we hypothesized that frequent walnut intake, in the form of walnut-enriched meat (WM), might decrease arylesterase activity and improve lipoprotein profile in individuals at increased risk of CVD by raising their PUFA and antioxidant intake. Furthermore, we hypothesized that those changes might be related to the effects and interaction of the PON1-Q192R and L55M polymorphisms.
The objectives of the present study were to assess differences between subjects at increased risk for CVD, taking into account PON1-Q192R and PON1-L55M polymorphisms, with regard to: 1) basal arylesterase activity and lipid peroxidation (LPO), LDL cholesterol (LDL-C), HDL-C, total cholesterol (TC) and oxidized LDL (ox-LDL) concentrations; 2) the relations between arylesterase activity and lipid variables; and 3) the effect of WM consumption on arylesterase activity and lipid variables.
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Materials and Methods |
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TOPABSTRACTIntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
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45 y and postmenopausal women 50 y, and 3) BMI 25 and <35 kg/m2. In addition, at least one of the following criteria was required: serum TC 5.69 mmol/L, smoking habit of 10 cigarettes/d, and/or hypertension (systolic blood pressure 140 mm Hg and/or diastolic blood pressure 90 mm Hg). Exclusion criteria included: volunteers with familiar hypercholesterolemia and/or type I or type II diabetes; those taking any hypolipemiant, antihypertensive, or antiinflammatory drugs, and those receiving substitutive hormonal therapy. The basal characteristics of the subjects before dietary intervention are presented in Table 1. Procedures followed were in accordance with the Ethics Committee standards of the Puerta de Hierro University Hospital (Madrid, Spain) and the Helsinki Declaration. Participants provided informed consent before the start of the study.
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Study design. Subjects were randomly assigned to diet-order groups in a nonblinded, crossover, placebo-controlled study, consisting of two 5-wk (18) experimental periods: WM and control meat (CM). This time of treatment has been effective in identifying lipid changes in response to dietary nut intervention in humans (18). Both periods were separated by a 4- to 6-wk washout interval during which subjects consumed normal diets. During the intervention period, volunteers consumed four 150 g/wk WM steaks and a 150 g/wk ration of WM sausages, all containing 20% walnut paste (Table 2). During the control meat (CM) period, volunteers consumed identical amounts of restructured steaks and sausages that did not include walnut paste (Table 2). Volunteers were firmly advised to avoid consuming all other meats and meat derivatives during both periods.
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-tocopherol concentrations after each experimental period. As previously published (20), at the end of the WM period, plasma -tocopherol was 
20% greater than the basal concentration, whereas no change occurred during the CM period. Energy and nutrient intakes were calculated with DIAL 2005 hardware, using Food Composition Tables for the foodstuff raw weights (21) (Table 3).
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Measure of arylesterase activity. Arylesterase activity was measured with simulated body fluid (SBF) as buffer and PA as substrate at pH 7.34–7.4 and 37°C (23). Reaction rates of arylesterase were followed at 270 nm in thermostatically controlled 10-mm lightpath quartz cuvettes using a Shimadzu UV-2401PC spectrophotometer. The final reaction volume in the cuvettes was 2.0 mL and the total time was 3 min. One unit of arylesterase activity is equal to 1 mol of PA hydrolyzed/(L · min).
Analytical measurements. Serum TC and HDL-C concentrations were measured by an enzymatic colorimetric method (CHOD-PAP, Boehringer Mannheim). LDL-C was calculated using the formula by Friedewald et al. (24).
Measurement of LPO in erythrocytes, based on the determination of malonyldialdehyde (MDA) and 4-hydroxyalkenals, was performed using the Bioxytech LPO-586 kit (Oxis Research).
Serum ox-LDL was measured by ELISA (Mercodia AB). The ox-LDL:LDL-C ratio was calculated to obtain an estimate of the percentage of ox-LDL particles (25).
Statistics. Values are presented as means ± SD. The Pearson product moment correlation coefficient was used to evaluate the association between 2 continuous variables. Chi-square analysis was used to determine whether the genotype distribution was in Hardy-Weinberg equilibrium and to compare categorical variables. Student's t test was used to assess differences in macronutrient intakes during the WM and CM periods. This study was designed to have a power of 80% to detect a 50% relative difference between basal arylesterase activity in subjects carrying different PON1-L55M and PON1-Q192R polymorphisms, assuming a pooled SD of 35% for arylesterase activity. Two-way ANOVA was used to assess the effect of the different polymorphisms on basal arylesterase activity, lipid variables, and LPO concentrations, considering BMI, smoking habit, sex, and age as covariables. When an interaction of both polymorphisms was observed, [LL,(QR+RR)] and [(LM+MM),QQ] groups were compared using Student's t tests. Groups [LL,QQ] and [(LM+MM),(QR+RR)] were not included in the analysis to avoid a Type II error. Placebo-controlled dietary study data were analyzed by 4-factor repeated-measures ANOVA, with time and treatment as intrasubject factors and PON1 polymorphisms as intersubject factors. Significant interactions between intra- and/or intersubject effects were analyzed with 2-way ANOVA. Statistics were performed with the SPSS 13.0 statistical package and SIMFIT 5.4 (26). Significance was determined at P < 0.05.
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Results |
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TOPABSTRACTIntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
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Influence of PON1-L55M and PON1-Q192R polymorphisms. PON1-192Q homozygous subjects had higher basal ox-LDL:LDL ratio (P = 0.042) and lower basal serum TC (P = 0.042), HDL-C (P = 0.047), and LDL-C (P = 0.050) concentrations and arylesterase activity (P = 0.036) than PON1-192R carriers (Table 4). PON1-L55 homozygous individuals had higher basal arylesterase activity than PON1-55M carriers (Table 4). We found significant PON1-L55M x PON1-Q192R interactions for basal serum TC, HDL-C, and LDL-C concentrations (P < 0.05) and a marginal interaction for arylesterase activity (P < 0.1) were found (Table 4).
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Associations with arylesterase activity before the dietary intervention. Basal arylesterase activities were positively correlated with basel serum HDL-C and TC concentrations, and negatively correlated with basal LPO concentration and the ox-LDL:LDL-C ratio (Table 5). When stratified by PON1-L55M, basal arylesterase activity was significantly correlated with basal HDL-C concentration and significantly negatively correlated with the basal ox-LDL:LDL ratio in PON1-55M carriers. When stratified by PON1-Q192R, basal arylesterase activity was significantly correlated with basal HDL-C concentrations and negatively correlated with the ox-LDL:LDL-C ratio in PON1-Q192 individuals (Table 5) but not in PON1-192R carriers (data not shown). Basal arylesterase activity and basal LPO levels were negatively correlated in all genotypes (Table 5).
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PON1 polymorphisms and the effect of WM. PON1-L55 subjects had higher arylesterase activity throughout the study than their PON1-55M counterparts (P = 0.034) (Fig. 1A). Furthermore, arylesterase activity was lower at wk 5 of the WM period than at wk 0 in PON1-55M carriers (P = 0.012) (Fig. 1A). The magnitude of this decrease (4 U/L) was the same in these subjects after CM consumption, but it was not significant (P = 0.77). The time x treatment x PON1-Q192R polymorphism interaction affected the LPO concentration (P = 0.040) whereby PON1-192R carriers had a lower concentration at wk 5 of the WM period compared with wk 0 (P = 0.031) (Fig. 1B). All other lipid variables did not differ between the PON1-L55M and PON1-Q192R polymorphism carriers during consumption of either WM or CM.
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Discussion |
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TOPABSTRACTIntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
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Because the present study was performed using subjects at increased risk of CVD, the genotypic distribution of the sample studied for PON1-Q192R and PON1-L55M (Table 1) was the same as that reported by Tomás et al. (27) in controls and hypercholesterolemic individuals.
In accordance with the study by Sentí et al. (28), PON1-Q192 individuals in our study had lower HDL-C levels than PON1-192R carriers. To our knowledge, our data are the first to show that low levels of LDL-C, TC, and arylesterase activity are also associated with the PON1-Q192 genotype. Furthermore, we conclude that differences in TC, HDL-C, and LDL-C concentrations and arylesterase activity are increased by the interaction of PON1-192 and PON1-55 polymorphisms. We also demonstrated that PON1-L55 and PON1-192R are associated with higher activity toward PA than PON1-55M and PON1-Q192. These results could be due to the use of SBF, instead of Tris/HCl (29), to measure arylesterase activity (23). SBF seems to be a more suitable reagent than Tris/HCl for the enzyme activity assessment, especially in samples with very low arylesterase activity.
Bub et al. (14) suggested that PON1 of 192Q healthy homozygous subjects is more effective against LDL peroxidation, measured as LDL oxidation lag time, than PON1 of their 192R counterparts; however, carriers of these 2 polymorphisms did not differ in MDA concentrations. We did not find significant differences in LPO and ox-LDL levels between carriers of both PON1-Q192R polymorphisms at increased risk of CVD. PON1-192Q individuals had a higher estimated percentage of ox-LDL particles than PON1-192R carriers. This was more evident in [(LM+MM), QQ] subjects, who also had the lowest arylesterase activity, explaining, at least in part, the highest estimated percentage of ox-LDL in those volunteers. Because the ox-LDL:LDL-C ratio was considered a marker of oxidative stress (25), and [(LM+MM), QQ] subjects had a 36% higher ox-LDL:LDL-C ratio than [LL, (QR+RR)], we hypothesized that [(LM+MM), QQ] individuals had lower arylesterase activity primarily because they were more susceptible to oxidative stress than [LL, (QR+RR)] carriers.
We found a higher correlation between arylesterase activity and HDL-C than that reported elsewhere (30). The negative correlation between arylesterase activity and LPO, ox-LDL concentration and the ox-LDL:LDL ratio supports the protective effect of PON1 on LDL oxidation as well as the susceptibility of PON1 to oxidative stress. These correlations were more pronounced in subjects with PON1-Q192 and PON1-55M polymorphisms. We have shown, to our knowledge for the first time, that arylesterase activity is correlated with TC.
Previous studies reported that supplementation with vitamin C and E increases paraoxonase activity (31), whereas intake of vegetables rich in antioxidants reduces it, especially in PON1-192R carriers and PON1-L55 homozygous individuals (13). In our study, the effect of WM consumption was also regulated genetically. The unique composition of walnuts rich in retinol, ß-carotenes, vitamin E, folic acid, and vitamin C (32) could explain the significant decrease in arylesterase activity in PON1-55M carriers, whose arylesterase activity was the lowest in the study.
The second most important finding of our study was that WM consumption reduced LPO levels in individuals with a risk of CVD with at least 1 allele encoding the PON1-192R genotype, thus contributing to a decrease of their CVD risk. Bub et al. (12,14) found that intake of tomato but not carrot juice reduced plasma MDA in young and old healthy, male, PON1-192R carriers.
In conclusion, TC, HDL-C, and LDL-C concentrations and arylesterase activity depend on the interaction of PON1-L55M and PON1-Q192R polymorphisms. Nevertheless, the PON1-Q192R polymorphism appears to be more related to antioxidant status than does the PON1-L55M polymorphism. WM consumption diminishes arylesterase activity in PON1-55M carriers and reduces LPO in PON1-192R carriers. More studies are needed to assess the importance of the interaction of PON1 polymorphisms on arylesterase activity in high- and low-risk CVD subjects. Moreover, additional studies are also necessary to evaluate the effects of long-term WM consumption.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Author disclosures: M. Nus, F. Frances, J. Librelotto, A. Canales, D. Corella, J. M. Sánchez-Montero, and F. J. Sánchez-Muniz, no conflicts of interest.
6 Abbreviations used: CVD, cardiovascular disease; C, cholesterol; CM, control meat; LPO, lipid peroxidation; LL,(QR+RR), subjects homozygous for leucine at position 55 and with almost one allele encoding for arginine at position 192; (LM+MM),QQ, subjects with almost one allele encoding for methionine at position 55 and homozygous for glutamine at position 192; MM, subjects homozygous for methionine at position 55; ox-LDL, oxidized-LDL; PON1, paraoxonase; PA, phenylacetate; PON1-L55, subjects homozygous for leucine at position 55; PON1-55M, subjects carrying at least one allele encoding for methionine at position 55; PON1-Q192, subjects homozygous for glutamine at position 192; PON-192R, subjects carrying arginine at least in one allele of position 192; SBF, simulated body fluid; TC, total cholesterol; WM, walnut-enriched meat.
Manuscript received 6 December 2006. Initial review completed 3 January 2007. Revision accepted 24 April 2007.
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