| | Effect of whole walnuts and walnut-skin extracts on oxidant status in miceReceived 4 February 2009; accepted 2 September 2009. published online 29 January 2010. Abstract ObjectiveTo evaluate the effect of the intake of whole walnuts and walnut fractions on the oxidant status in mice. MethodsThirty-six C57BL/6J male mice were randomized to be fed one of three diets: 1) a standard diet (control group), 2) a standard diet with 10% of whole walnuts (walnut-diet group), or 3) a standard diet with 2% of walnut skins (walnut-skin-diet group) for 8 wk. The plasma antioxidant capacity was measured by oxygen radical-absorbance capacity and plasma ferric-reducing antioxidant potential. Conjugated diene formation and reduced glutathione levels were also analyzed. ResultsWe observed no changes in plasma oxidation capability between the walnut and walnut-skin groups with the exception of conjugated dienes. Plasma total antioxidant capacity and the ratio between reduced and oxidized forms of glutathione were lower in the walnut and walnut-skin groups than in the control group. ConclusionThe decrease in the antioxidant burden observed in enzymatic and non-enzymatic antioxidant systems after sustained consumption of a whole-walnut or a walnut-skin diet in mice may be related to the plasma oxidation capability being maintained in the groups consuming the walnut diets. Introduction  Cardiovascular disease is the major cause of death in the developed world. Since Goldstein et al. [1] established the oxidative hypothesis for atherosclerosis, oxidative stress has been considered to be one of the major risk factors for atherosclerosis and cardiovascular disease. Therefore, improving the whole-body antioxidant capacity may help to prevent many chronic and age-related diseases, including cardiovascular disease. Antioxidants are derived from intrinsic and extrinsic antioxidant systems and are constitutively present in human blood and tissue. Diet is a natural source of such extrinsic antioxidants as vitamins, flavonoids, carotenoids, or proteins and, in recent years, greater interest has been paid to the involvement of nutritional compounds in the development or progression of oxidative diseases. Several epidemiologic studies have supported the dietary antioxidant hypothesis and demonstrated that high consumption of plant foods or nutritional factors derived from plants protect against cardiovascular disease because of the numerous phytochemical compounds they contain [2]. Recently, nuts have been included in this group of potentially healthy foodstuffs. In this respect, many observational studies in large cohorts have consistently shown a negative and dose-dependent association between nut intake and the risk of cardiovascular disease, which suggests that regular nut consumption reduces the risk of cardiovascular disease by 30–60% in several population groups, independently of other confounding lifestyle factors. The US Food and Drug Administration has supported these findings and claimed that walnuts have a role in reducing the risk of heart disease. The healthy benefits of nuts, especially walnuts, are mainly attributed to their high content of ω-3 fatty acids and low saturated fatty acids, and their favorable effects on lipid profiles. Moreover, their high content of vitamin E, polyphenols, flavonoids, arginine, and fiber means that they can have a considerable modulatory effect on the antioxidant system because they decrease the oxidative damage caused by lipids and lipoproteins and thus help prevent the evolution of atherosclerotic plaque. However, the unsaturated fatty acids in nuts are highly susceptible to oxidation. Thus, the effect of nut consumption on the balance between the pro-oxidant and antioxidant capacities in the body could be crucial to determining the real effect of nuts on cardiovascular health. Because antioxidant bioavailability depends on the amount of food ingested and the food matrix, it should be taken into account when the antioxidant effects of nuts or nuts extracts are tested. For this reason, the aim of the present study was to evaluate the effect of the consumption of whole walnuts and walnut-skin fractions on oxidant status in mice. Materials and methods Animals and diets Thirty-six C57BL/6J male mice 5 wk of age (Charles River Laboratories, Barcelona, Spain) were housed and maintained in an environmentally controlled room (20–22°C, 12-h alternating light/dark cycle, and a relative humidity of 60%). Twelve mice were fed a standard diet (control group), another group of 12 was fed a standard diet with 10% of whole walnuts (walnut-diet group), and a third group of 12 was fed a standard diet with 2% of walnut skins (walnut-skin-diet group). A total of 10% of nuts in the diet is the caloric equivalent of the daily nutritional recommendations of nuts in humans (30 g/d). Nuts were mechanically crushed and added to the standard feed. Walnut skins were removed from the walnut by hot water blanching while the walnuts were being prepared and they are generally treated as a waste product. The percentage of 2% was calculated using the normal ratio between whole nuts and the cover fraction so that the amount of antioxidant consumption was the same. The diets were administered ad libitum for 8 wk (Panlab, Barcelona, Spain). Borges S.A. (Reus, Spain) donated the walnuts and walnut-skin extracts used in the study. Food consumption was measured twice a week and animal weight once a week. Nutritional differences between diets are presented in Table 1. At the end of this period, and after an overnight fast, the mice were anesthetized with an intraperitoneal injection of ketamine-xylazine (100–10 mg/kg, respectively) dissolved in 0.9% saline. Blood was obtained by heart puncture and was collected in tubes containing ethylenediaminetetra-acetic acid (EDTA) as an anticoagulant and antioxidant. Plasma was obtained and stored at −80°C until use. All procedures applied in the study were approved by the ethics committee of animal research of the Rovira i Virgili University (Tarragona, Spain) and they were carried out according to the Spanish and the European Community Guides for animal care. From the plasma samples, the following parameters were determined: 1) conjugated dienes, 2) reduced (GSH) and oxidized (GSSG) glutathione, 3) plasma antioxidant capacity (ORAC), and d) plasma ferric-reducing antioxidant potential (FRAP). Also, the antioxidant capacity of the solid sample preparations (walnuts, walnut skins, standard diet, walnut diet, and walnut-skin diet) was determined. Conjugated dienes PD-10 desalting columns (GE Healthcare, Uppsala, Sweden) were used to remove EDTA from the plasma. Cu+2 was used to oxidize plasma lipids and conjugated diene formation was measured at 234 nm, at 37°C for 5 h, as described by Spranger et al. [3]. A total of 50 μL of plasma was eluted through the column with phosphate buffered saline up to a final dilution of 1:75. The oxidation was then started in a spectrophotometric cuvette with 1.5 mL of the eluted sample, 1.47 mL of phosphate buffered saline, and 30 μL of 5 mM CuCl2 solution. From the kinetic profile of each sample measured, several indexes describing the plasma oxidation capability were determined. The lag phase, defined as the interval (minutes) between the intercept of the linear least-square slope of the curve and the initial-absorbance axis, was measured. The maximal rate of oxidation was calculated from the slope of the absorbance curve during the propagation phase (expressed as nanomoles per minute per milliliter of plasma) using the molar absorptivity for conjugated dienes (E240 = 29500 L · mol−1 · cm−1). The same absorptivity value was used to determine the maximal amount of dienes formed. Conjugated dienes were measured as a marker of the amount of lipid peroxidation products. GSH and GSSG The GSH and GSSG were determined by Hissin and Hilf's [4] fluorimetric method using a Perkin Elmer LS 50B (Waltham, MA, USA) spectrofluorometerat an excitation of 350 nm and emission wavelengths of 420 nm. For GSH assay plasma samples were preserved in acid and frozen at −80°C. The samples were diluted 1:10 in phosphate-EDTA buffer, pH 8.0. The final assay mixture contained 100 μL of the diluted plasma, 1.8 mL of phosphate-EDTA buffer, and 100 μL of o-phthalaldehyde solution (Sigma Chemicals Co.). After thorough mixing and incubation at room temperature for 15 min, the solution was transferred to a cuvette. The GSSG assay was performed in 50 μL of plasma samples incubated with 20 μL of 0.04 M N-ethylmaleimide (Merck) for 25 min at room temperature To this mixture, 430 μL of 0.1 N NaOH was added to obtain a pH of 12.0. A total of 100 μL of this mixture was taken to measure GSSG, using the procedure outlined above for the GSH assay, except that 0.1 N NaOH was used as diluent instead of phosphate-EDTA buffer (o-phthalaldehyde reacted with GSSG, yielding readily measurable fluorescent intensity at pH 12.0). The GSSG/ GSH couple in plasma can respond to cellular and extracellular oxidative processes. Plasma antioxidant capacity (ORAC assay) Plasma samples were diluted 500-fold in 75 mM potassium phosphate buffer (pH 7.4) before analysis. The reaction mode used 20 μL of the sample and 370 μL of fluorescein 48 nM in a multiwell plate. The reagents were mixed and incubated for 10 s before the initial fluorescence was recorded. Peroxyl radicals were generated by 10 μL of 2,2′-azobis(2-amidinopropane) dihydrochloride reagent. Fluorescence readings were taken every minute for 120 min at 485-nm (λ excitation) and 538-nm (λ emission) wavelengths on a Fluoroskan Ascent fluorescence plate reader (Labsystems, Helsinki, Finland). Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) was used as standard. The final ORAC values were calculated by using a regression equation between the Trolox concentration and the net area under the fluorescein decay curve and were expressed as Trolox equivalents per liter of plasma (millimoles of Trolox equivalents per liter of plasma) [5], [6]. Solid sample antioxidant capacity (ORAC Assay) Total antioxidant capacity was measured in solid sample preparations (walnuts, walnut skins, control feed, walnut feed, and walnut-skin feed) as has been described previously [7]. For the lipophilic ORAC assay, 1 g of each triturated sample was extracted with hexane/dichloromethane (1/1). Extracts were dried under nitrogen flow in a 30°C water bath and residues were reconstituted in 1 mL of acetone. For the hydrophilic ORAC assay, samples were extracted with acetone/water/acetic acid (70/29.5/0.5). For lipophilic and hydrophilic ORACs, a 100-fold dilution was performed before the analysis. ORACs were expressed as Trolox equivalents per gram of sample (micromoles of Trolox equivalents per gram of sample). Plasma FRAP assay The FRAP assay was performed as described by Benzie and Strain [8]. Plasma antioxidants were evaluated as reductants of Fe3+ to Fe2+, which is chelated by 2,4,6-tripyridyl-s-triazine to form a Fe2+–2,4,6-tripyridyl-s-triazine complex absorbing at 593 nm. Absorbance was monitored by a UV–VIS spectrophotometer (Lambda 25, Perkin Elmer, Beaconsfield, UK), equipped with an eight-cell holder and a thermostatically controlled bath. In short, 1 mL of working prewarmed 37°C FRAP reagent (10 vol of 300 mM acetate buffer, pH 3.6, + 1 vol of 10 mM 2,4,6-tripyridyl-s-triazine in 40 mM HCl + 1 vol of 20 mM FeCl3 + 1.2 vol of distilled H2O) was mixed with 30 μL of test sample and standards. All measurements were made in duplicate after 4 min of reaction. Results were compared with a standard curve prepared with different concentrations of ferrous sulfate, and FRAP values were expressed as millimoles of Fe2+ per liter of plasma. Statistical analysis The variable normal distribution was tested by the Kolmogorov-Smirnov test. Analysis of variance was used to compare mean groups. Results were expressed as mean ± standard error of the mean. The level of statistical significance for all tests was P < 0.05. Statistical analyses were performed with SPSS (SPSS, Inc., Chicago, IL, USA). Results Figure 1A shows the ORAC values of the walnut-skin extracts and the whole walnuts used to enrich the mice's diets. Walnut-skin extracts showed greater antioxidant capacity than whole-walnut extracts in their hydrophilic fraction, which indicates the presence of numerous potential antioxidant compounds in walnut skin. The ORAC values observed in the walnut and walnut-skin diets used in this study were higher than in the control diet, largely because of the hydrophilic fraction (Fig. 1B). No significant differences were observed in mouse growth and in the amount of food consumption among the three groups during the study (Table 2). | ∗ Mean ± SE. †Analysis of variance. |
Figure 2 shows the results of the different plasma oxidation markers in the three groups of mice at the end of the interventions. The plasma antioxidant capacity in the groups fed with walnut-skin diets was significantly lower than in the control group. Mice fed with the walnut-skin diet showed significantly lower ORAC and FRAP values than the mice in the control group. In the mice on the walnut diet, only the plasma FRAP values significantly differed from the control group. Plasma GSH concentrations were lower in the groups that received walnut or walnut-skin diets in comparison with the control group, although these differences were only statistically significant in the case of the mice fed with the walnut-skin diet (P = 0.020). No differences were observed in plasma concentrations of GSSG between groups. Therefore, the GSSG/GSH ratio was significantly higher in the walnut-skin group. The diene oxidation rate and diene lag phase were not significantly different among groups. However, the mice fed with the walnut diet showed a plasma concentration of conjugated dienes that was significantly higher than that of the mice in the control and walnut-skin groups (P < 0.03). Discussion This is the first chronic study to evaluate the effect of whole-walnut and walnut-skin consumption on oxidative stress markers and the plasma antioxidant capacity in mice. The aim was to investigate whether the oxidation of polyunsaturated fatty acids (PUFAs) from nuts could be compensated for or not by the antioxidant properties attributed to their antioxidant compounds. Our results show that the antioxidant capacity at the plasma level was decreased in the mice on the walnut and walnut-skin diets in comparison with the control group. Despite this, the plasma oxidizing capability seemed to be preserved. The putative health benefits of walnuts have been mainly attributed to their antioxidant capacity, which is associated with their content of flavonoids—mainly distributed in the skin—and fiber. However, most of the supportive evidence is based on in vitro experiments or in vivo feeding studies that measured the effect of a single potential antioxidant compound. Because the susceptibility of fatty acids to oxidation is thought to be directly dependent on their degree of unsaturation [9] and nuts are rich in monounsaturated fatty acids and PUFAs [10], nuts could per se be prone to easy oxidation independently of their content of antioxidant compounds. Therefore, the debate about whether whole nuts can or cannot improve oxidative status in vivo is still ongoing. Only two studies have evaluated the acute effect of nut consumption on oxidative status in animals. In relation to control groups, an increase was observed in the postprandial plasma antioxidant capacity in walnut-fed rats [11] and the lag time of low-density lipoprotein (LDL) oxidation 180 min after administering 40 μM of gallic acid equivalents from almonds by stomach gavage in hamsters [12]. In contrast, long-term clinical trials in humans evaluating the effect of nut consumption on oxidative status have produced controversial results. Although some studies have reported a significant decrease in such oxidative markers as plasma malondialdehyde and isoprostanes after nut consumption [13], [14], others have failed to find any positive effect on conjugated diene synthesis in LDL or even in the antioxidant enzymatic activity [15], [16], [17]. Although the walnut and walnut-skin diets have shown a higher antioxidant capacity in vitro, in the present study we found that the total plasma antioxidant capacity, measured by ORAC and FRAP, was significantly lower in the animals that were chronically fed with a walnut diet or a walnut-skin diet in comparison with the control group. Although walnuts are very rich in antioxidant compounds, the total amount of fat to be oxidized is greater. Walnuts are also richer in ω-6 PUFAs than in monounsaturated fatty acids, which are present in larger amounts in other nuts. The ω-6 PUFAs are more easily oxidized than monounsaturated fatty acids [18], [19], [20], [21] and ω-3 PUFAs [10], [22]. These differences in oxidizing capacity between types of fats could partly explain our results in those mice fed with a walnut diet. We also observed that, in comparison with the control group, the formation of total plasma-conjugated dienes was significantly higher, whereas no changes in the lag phase were observed between groups. Moreover, although the mice on the walnut-skin diet had higher levels of conjugated dienes than the control group, they had significantly lower levels than the mice on the walnut diet. Once again, those animals consuming larger amounts of ω-6 PUFAs derived from walnuts showed a higher synthesis of oxidized products. Similar negative results on conjugated diene formation in LDL were observed in four clinical trials performed in humans to evaluate the chronic effect of walnut consumption, although only one of them reported a significant increase in the maximal rate of oxidation of the treated group in comparison with the control group [15], [16], [23], [24]. Lower concentrations of GSH and higher GSSG/GSH ratios were also observed in both walnut-diet groups in our study. The differences were significant in the mice fed with the walnut-skin diet. These results suggest that the antioxidant enzymatic capacity is also modulated after the chronic consumption of walnuts. Because the total antioxidant capacity of blood was worse in mice on a walnut-skin diet than in mice on a walnut diet, it could be hypothesized that long-term treatments with larger amounts of phenolic compounds have a pro-oxidant capacity. Our results are in agreement with previous studies that evaluated the effect of phenolic compounds in rabbits [25], golden Syrian hamsters [26], and apolipoprotein E–deficient mice [27], which reported that larger amounts of potentially antioxidant compounds could have a harmful effect on oxidative status and the progression of atherosclerosis. The lack of protection provided by the antioxidant compounds consumed in walnuts may also be partly attributed to the fact that these compounds are less bioavailable when they are consumed as the whole nut than as alternative forms. Although the bioavailability of selected single flavonoid compounds has been widely reported [28], [29], little information is available about the concurrent absorption of antioxidant compounds from whole nuts. The results of our study suggest that the consumption of walnuts and walnut skins has no deleterious effect on LDL oxidizing capability, despite their higher content of ω-6 PUFAs and the depletion of the antioxidant burden in enzymatic and non-enzymatic antioxidant systems in mice. Conclusion The decrease in the antioxidant burden observed in enzymatic and non-enzymatic antioxidant systems after the sustained consumption of a whole-walnut or a walnut-skin diet in mice could be related to the plasma oxidizing capability being maintained in the groups consuming the walnut diets. Acknowledgments The authors thank M. Covas for her critical revision of the manuscript. Borges S.A. (Reus, Spain) donated the walnuts and walnut-skin extracts used in the study. References [1]. [1]Goldstein JL, Ho YK, Basu SK, Brown MS. Binding site on macrophages that mediates uptake and degradation of acetylated density lipoprotein, producing massive cholesterol deposition. Proc Nat Acad Sci U S A. 1979;76:333–337. [2]. [2]Willcox BJ, Curb JD, Rodriguez BL. Antioxidants in cardiovascular health and disease: key lessons from epidemiologic studies. Am J Cardiol. 2008;101(suppl 10A):75D–86D. [3]. [3]Spranger T, Finckh B, Fingerhut R, Kohlschütter A, Beisiegel U, Kontush A. 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[29]. [29]Lesser S, Cermak R, Wolffram S. Bioavailability of quercetin in pigs is influenced by the dietary fat content. J Nutr. 2004;134:1508–1511. MEDLINE a Human Nutrition Unit, Department of Biochemistry and Biotechnology, Facultat de Medicinia i Ciències de la Salut, ISPV, Universitat Rovira i Virgili, Reus, Spain b CIBER Fisiopatología de la Obesidad y Nutrición, Instituto de Salud Carlos III, Santiago de Compostela, Spain c Unit of Pharmacology, Facultat de Medicinia i Ciències de la Salut, Universitat Rovira i Virgili, Reus, Spain d Nutrition and Dietetics Unit, Internal Medicine Department, Hospital Universitari de Sant Joan, Reus, Spain  Corresponding author. Tel.: +34-977-75-93-12; fax: +34-977-75-93-22.
Patricia López-Uriarte is the recipient of a predoctoral fellowship from the Catalan government's Department of Universities, Research, and the Information Society. PII: S0899-9007(09)00371-2 doi:10.1016/j.nut.2009.09.002 © 2010 Published by Elsevier Inc. | |
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