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Departamento de Nutrición y Bromatología I (Nutrición)
Departamento de Farmacología
Grupo de Biotransformaciones
Departamento de Química Orgánica y Farmaceútica, Facultad de Farmacia, Universidad Complutense, Madrid, SPAIN
Address correspondence to: Professor Dr. Francisco J. Sánchez-Muniz, Departamento de Nutrición y Bromatologia I (Nutrición), Facultad de Farmacia. Universidad Complutense, E-28040-Madrid, SPAIN. E-mail: frasan{at}farm.ucm.es
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ABSTRACT |
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TOP
ABSTRACT
INTRODUCTION
SUBJECTS AND METHODS
RESULTS
CONCLUSIONS
ACKNOWLEDGMENTS
REFERENCES |
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Objective: (i) To investigate whether the erythrocyte catalase (CAT), superoxide dismutase (SOD), total glutathione, reduced glutathione (GSH), oxidized glutathione (GSSG), and lipid peroxidation (LPO), and serum uric acid and paraoxonase-1 (PON1) are modified at increased CHD-risk individuals consuming walnut-enriched meat (WM), (ii) to evaluate whether these changes were influenced by basal serum cholesterol, body mass index or smoking habit.
Design: The study was a non blinded, cross-over, placebo-controlled trial in which 22 volunteers (60% overweight and 40% obese) with increased CHD-risk were randomly assigned to receive WM or control meat (CM) during two different periods of 5 weeks.
Results: A significant interaction time*treatment (p < 0.05) was observed in all enzymes and substrates tested except HDL-C, uric acid and LPO. The treatment significantly increased CAT activity, total glutathione and GSSG (p < 0.05). Significant gender*time*treatment interaction (p = 0.043) for total glutathione was found increasing at the end of the WM period in male but not changing in female. Total glutathione and GSH/GSSG ratio (p < 0.05) were lower in smokers. Hypercholesterolemics presented higher uric acid (p < 0.05) but no enzyme activities or substrate concentrations were different from those of normocholesterolemics.
Conclusions: The WM tested appears to be a functional food as it improved the antioxidant status of increased CHD-risk volunteers. Despite its high energy content, it also appears adequate for overweight and obese people because did not exert negative effect upon body weight.
Key words: antioxidant status, functional food, lipid peroxidation, walnut-enriched meat
Abbreviations: BMI = Body mass index • CHD = Coronary heart disease • CAT = Catalase • Hb = Haemoglobin • CM = Control meat • LPO = Lipid peroxidation • MDA = Malondialdehyde • GSSG = Oxidized glutathione • ox-LDL = Oxidized-LDL • PON1 = Paraoxonase • PUFA = Polyunsaturated fatty acids • ROS = Reactive oxygen species • GSH = Reduced glutathione • SOD = Superoxide dismutase • WM = Walnut-enriched meat
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INTRODUCTION |
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ABSTRACT
INTRODUCTION
SUBJECTS AND METHODS
RESULTS
CONCLUSIONS
ACKNOWLEDGMENTS
REFERENCES |
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The nutrient and phytochemical composition of walnuts differs from that of other nuts. They are rich in 
-linoleic, -linolenic and 
-linolenic acids and in other health-related compounds such as high-biological-value proteins (e.g. arginine) fibre, vitamins, tannins, folates and polyphenols which may provide additional antiatherogenic properties [6,7].
Spanish diet has deeply changed throughout last decades with increased in total fat, saturated fatty acids (SFA) and a reduction in vegetable and fatty fish consumptions [8]. Thus, walnuts intake could improve the quality of the Spanish diet. At present, and despite all these facts, walnut consumption remains low in Spain (2.65 g/day in 2004) [9] and other Mediterranean countries [10]. Consequently, several strategies, the most important of which has been the inclusion of walnuts in functional foods, have been adopted to increase their intake [11].
A plethora of physiological disorders and degenerative diseases have been related to oxidative stress [12,13]. Oxidative stress may occur when production of reactive oxygen species (ROS) increases and/or when scavenging free radicals or repairing capacities against oxidative damage decreases or fails [14]. Measurement of malondialdehyde (MDA) and 4-hydroxyalkenals has been used as an indicator of lipid peroxidation (LPO) [15]. A variety of non-enzymatic antioxidants (e.g. glutathione, uric acid) and enzymatic antioxidants (e.g. catalase (CAT), Cu-Zn-superoxide dismutase (SOD)) have been known to play an active role against oxidative stress [13,14]. During recent years, the paraoxonase enzyme (PON-1) has become relevant, due to its implications in the protection and recovery of LDL antioxidant status [16]. Free radicals and peroxides are clearly involved in physiological phenomena but also in the pathogenesis of several diseases such as atherosclerosis and type-2 diabetes [12,13,17]. In fact, LDL peroxidation is accepted as the initial step of the atherosclerotic process [15]. Moreover, hypercholesterolemic or obese/overweight subjects have higher levels of serum and LDL peroxidation than their normocholesterolemic or slim counterparts [18].
Thus, it can be hypothesized that the intake of restructured beef steaks and sausages containing walnuts may modify oxidative stress in individuals with increased CHD risk. The present study aims (i) to compare the effects of walnut-enriched meat (WM) vs. control meat (CM) on oxidative stress by measuring CAT, SOD and PON-1 activities, as well as the concentrations of uric acid, GSH and GSSG; (ii) to ascertain if these changes depend on the body mass index (BMI), basal serum cholesterol concentrations or the smoking habit.
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SUBJECTS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
SUBJECTS AND METHODS
RESULTS
CONCLUSIONS
ACKNOWLEDGMENTS
REFERENCES |
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45 years; women (50 years and postmenopausal) and c) BMI 25-<35 kg/m2. Volunteers having BMI 30 kg/m2 were defined as obese. Moreover, at least one of the following criteria was also required: serum total cholesterol 5.69 mmol/L; smoking habit (10 cigarettes per day); and/or hypertension (systolic pressure 140 mmHg and/or diastolic pressure 90 mm Hg). Exclusion criteria included subjects with familiar hypercholesterolemia and/or type I diabetes; those taking any hypolipemiant, antihypertensive or anti-inflammatory drugs and those receiving hormonal substitutive therapy. The characteristics of volunteers are presented in Table 1.
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Study Design
Volunteers were randomly assigned to follow a non-blinded, cross-over, placebo-controlled study, consisting of two 5-week experimental periods (intervention and control). Both periods were separated by a 4 to 6-week wash-out interval during which subjects returned to their usual diet. During the intervention period, volunteers weekly consumed four 150g walnut-enriched restructured steaks and a 150g ration of walnut-enriched sausages, all containing 20% in walnut paste. The composition of both meats is presented in Table 2 and more information can be obtained in [19]. It was firmly recommended that all other meats and meat derivatives had to be excluded from the diet. During the control period, volunteers consumed identical amounts of restructured steaks and sausages that did not include walnut paste.
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Dietary Control and Compliance
Subjects received control and walnut-enriched frozen meat once a week. Special emphasis was given to compliance and management of intake with regard to frequency, dates and numbers of steaks consumed. Energy and nutrient intakes were calculated using Food Composition Tables for the foodstuff raw weights [21]. In order to avoid any possible dietary misunderstanding, volunteers recorded the amount and type of food consumed on a daily basis. Compliance was also assessed by measuring plasma -tocopherol concentrations after each experimental period [22].
Anthropometric and Blood Pressure Measurements
Trained staff measured weight, height, BMI and systolic and diastolic blood pressures of the participants at the beginning and end of the intervention and control periods.
Sample Collection
Fasting blood samples were collected between 7:30 and 10:00 a.m. at the beginning of the study and at the end of each experimental period. Blood was gently delivered into citrate tubes for SOD, CAT, GSH, and GSSG analysis and in vacutainer tubes for PON-1, serum cholesterol and uric acid determinations.
Preparation of Haemolysates
Citrated blood was centrifuged at 1000 x g for 10 min at 4°C, and the plasma and buffy coat removed. Erythrocytes were washed with PBS (pH 7.00, containing 140 mM NaCl) three times and erythrocytes were haemolyzed with ice-cold distilled water. Haemoglobin (Hb) content was determined by using the cyanmethemoglobin method [23]. Haemolysates were used to determine SOD and CAT enzymatic activities and GSH and GSSG concentrations.
Total and HDL-Cholesterol
Serum total and HDL-cholesterol were measured by enzymatic colorimetric method (CHOD-PAP, Boehringer Mannheim; RA-XT auto analyzer, Technicom; and RA 2000, Technicom).
Uric Acid
Uric acid was determined by the method of Trinder [24].
SOD Activity
SOD (EC 1.15.11) was determined by the Marklund and Marklund method [25], based on pyrogallol autoxidation. One unit of enzyme activity was defined as 50% inhibition of the rate of pyrogallol autoxidation. Results were expressed as IU/g Hb.
CAT Activity
CAT (EC 1.11.1.6) activity was estimated by the Aebl method [21], monitoring the rate of disappearance of hydrogen peroxide at 240 nm. CAT activity is expressed as IU/g Hb.
Total Glutathione, GSH and GSSG Concentrations
Total glutathione (GSH plus GSSG) was measured by fluorometry, according to the Hissin and Hilf method [27], using o-phthaldialdehyde. GSH and GSSG results were expressed as µmol/g Hb.
PON1 Activity
PON1 (EC 3.1.8.1) activity was determined by measuring the rate of hydrolysis of paraoxon in p-nitrophenol catalyzed by the enzyme at 37°C and 405nm [28]. Frozen aliquots of pool sera were used as internal control. One unit of PON-1 activity was defined as 1 µmol of p-nitrophenol formed per L per minute.
LPO
The LPO measurement was performed using the Bioxytech LPO-586TM kit (Oxis Research, Portland, USA) and was based on the reaction of a chromogenic reagent, N-methyl-2-phenylindole with MDA and 4-hydroxyalkenals at 45 °C.
Statistics
Data are presented as means ± SD. Meat and diet compositions were analysed by one-way ANOVA. Data were analysed by a two-factor (time and treatment) repeated-measures analysis of variance. When there was a significant interaction time*treatment, the change over time within each group was assessed by a one-factor ANOVA. The inter-subjects effects of gender, serum cholesterol, BMI and smoking habit were also tested. Data were significant at p<0.05. The SPSS 13.0 statistical package was employed.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
SUBJECTS AND METHODS
RESULTS
CONCLUSIONS
ACKNOWLEDGMENTS
REFERENCES |
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Enzyme Activities
CAT.
A significant time*treatment interaction on CAT activity was found (p = 0.006). During the WM period CAT activity increased significantly (p = 0.008) (Table 4). No significant differences due to basal serum cholesterol, BMI and smoking habit were found (data not shown).
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PON1.
A significant time*treatment interaction was found (p = 0.005). It significantly increased during the walnut meat-period but decreased during the control-meat one (p < 0.05) (Table 4). No significant differences on PON1 activity due to serum basal cholesterol, BMI and smoking habit were found (data not shown).
Total Glutathione.
A significant time*treatment interaction was found (p = 0.018), increasing more during the intervention period than during the control one (Table 4). A triple gender*time*treatment interaction (p = 0.043) was found (Fig. 1). Men increased total glutathione following the WM but women were not significantly affected. Smoking affected negatively (p = 0.043) the total glutathione concentration (data not shown).
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Uric Acid.
No significant time or treatment effects were found (Table 4). A significant interaction treatment*BMI was found (p = 0.002) as uric acid concentrations of obese volunteers increased during the control-meat period more than during the intervention period. A triple interaction serum cholesterol levels*time*treatment was found (p = 0.037). In normocholesterolemics but not in hypercholesterolemics, uric acid significantly increased during the walnut-meat period and decreased during the control-meat period (p < 0.05) (data not shown).
LPO.
LPO concentrations did not change throughout the study.
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DISCUSSION |
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Dietary Assessment, Body Weight and BMI
The addition of nuts, very rich in PUFA [6], to the restructured meat explains the higher PUFA consumption during the intervention period with respect to the control period. As in other studies, where fat contribution to total energy was lower, equal or higher than 30% [1, 17, 30–32], body weight was not affected by WM consumption. These results are interesting, taking into account both the high fat and energy contents of walnuts [6], and therefore their potential effect on body weight. According to García-Lorda et al. [33] there are three possible reasons that body weight is not affected by nut consumption (i) incomplete absorption of energy from nuts, related to the structure of lipid-storing granules in nuts or to various nut fiber components, (ii) satiating effect of the dietary fiber in nuts, and (iii) effect on energy metabolism, probably related to polyphenols, that compensates for the increase in energy availability.
Antioxidant Enzymes
Present data clearly show that the intake of WM 5 times per week for 5 weeks increased concentrations/activities of several antioxidant defense biomarkers, such as CAT, SOD, PON1, total glutathione, GSH and GSSG in study volunteers. Natural defense against ROS involves a number of enzymatic and non enzymatic antioxidant mechanisms [14]. The particular composition of walnuts, which are rich in antioxidant compounds such as retinol, ß-carotenes, vitamin E, -tocopherol, -tocopherol, 
-tocopherol, folic acid and vitamin C [6, 34], seems to be responsible for improving the antioxidant status of study participants. In the present paper, the intake of the WM supplies approximately 29 mg of -tocopherol/week [22] and increased -tocopherol levels by a 8.9% (IC95%=10–16.8%) [35]. The same authors [22] observed an increase of vitamin E ( and -tocopherols) in serum and triglyceride-rich lipoproteins, suggesting a good bioavailability of vitamin E from WM in humans. ROS can stimulate oxidation of LDL and oxidized-LDL (ox-LDL), which is not recognized by apolipoprotein B100-LDL receptors. Thus, ox-LDL can be taken up by the scavenger receptors in macrophages, leading to foam cell and atherosclerotic plaque formation [15, 36]. Recently, a walnut extract containing ellagic acid, gallic acid and flavonoids was reported to inhibit oxidation of human plasma and LDL [37]. Fukuda et al. [38] analyzed the antioxidant effects of polyphenols from walnuts comparing the action of these antioxidants with that of others, such as ascorbic acid. After analyzing SOD activity and other parameters, these authors concluded that ellagitannin polyphenols from walnuts may act as potent antioxidants. The potential ability of vitamin E to help prevent CHD deserves special attention. Sabate [4] reported that a regular consumption of nuts lowers the risk of myocardial infarction and death from ischemic heart disease. In animal studies, -tocopherol supplementation increased SOD activity more than -tocopherol supplementation [39]. Nonetheless, walnuts contain PUFA that are susceptible to oxidation, and the resulting products could be potentially cytotoxic [4]. To date, several studies have been conducted in order to test the effect of lipids on oxidative damage [40].
The intake of WM increased PON1 activity independently of HDL-C levels. The HDL-C concentrations were not affected by the treatment corroborating results of other studies [32], although Tapsell et al. [17] found that HDL-C increases in type 2 Diabetes patients consuming walnuts in the framework of a low-fat modified-fat diet. The lack of correlation between PON1 and HDL-C between volunteers could be due to the presence of different PON1 polymorphism. Thus, PON1-192R hydrolyzes paraoxon faster and diazoxon, soman and serin slower than PON1-192Q [41]. In addition, the ability of HDL to protect LDL against peroxidation in vitro is reduced significantly in HDL particles containing PON1-192R with respect to PON1-192Q [42]. The increase in PON1 concentrations would be a consequence of the intake of a relatively high amount of PUFA, counterbalanced by the consumption of nut antioxidants. Many other authors have investigated the influence of diet on PON1 activity but available results are controversial. Sutherland et al. [43] reported that PON1 activity decreases when the diet contains oil which has been used repeatedly for frying. The same result was observed in rabbits consuming atherogenic diets [44]. On the other hand, Kudchodkar et al. [45] have observed that PON1 activity is higher in rats consuming olive oil than in those with an intake of saturated oil or fish oils. Although intake of antioxidants may be thought to favor PON1 status, present data remain inconclusive, as supplementation with vitamin C and E seems to increase PON1 activity [46], while intake of vegetables, rich in antioxidants, reduces it [47]. This suggests that PON1 would be more active in the absence of antioxidants. For this reason, this enzyme would not be expressed in vegetarians, who consume large amounts of antioxidants and therefore do not need high activity of this enzyme.
Due to a large percentage of volunteers were mildly hypercholesterolemics, it can be assumed that the group as a whole displayed an impaired GSH profile. Simon et al. [48] and Yalcin et al. [49] suggested that the resistance of erythrocytes to oxidative stress decreases in hypercholesterolemic individuals. The GSH/GSSG ratio is considered as a marker of oxidative stress. As a consequence of higher PUFA intake during the WM period the GSH/GSSG ratio was higher than during all the CM period. This data seem controversial having into account the general improvement of the antioxidant status found in the present paper, but it has been proposed that some substrates promote the so called "hormesis" property to induce a potent antioxidant defense increase after producing relatively small peroxidation [50] Moreover, the elevation of microsomal and cytosolic glutathione S-transferase activities proved the utilization of GSH in the formation of glutathione S-conjugates. Due to this, it can be hypothesized that a suitable amount of GSH would be metabolically used by the glutathione S-transferases, and the increase in the GSH level could be lower than expected [51].
In agreement with Dünken et al. [52], basal concentrations of GSH (Fig. 2) and total glutathione were significantly lower in smokers than in non smokers. Although only 18% of the participants of the present study were smokers, in general we observed a more positive response in smokers than in non-smokers, because GSH basal differences due to smoking habit disappear, suggesting that nut consumption counterbalance the oxidative stress induced by numerous free radical compounds present in the gas phase [53].
Although information suggests that beef consumption increases uric acid in hypercholesterolemics [54] present results suggest that uric acid was not affected throughout the whole study and that the walnut inclusion in meat prevents the obese volunteers from the increase of uric acid after consuming high amount of meat [55].
The consumption of WM induced a decrease of 
34% LPO concentration in erythrocytes while that of the CM approximately 8%. These results suggest that the lipid peroxidation is not increased by WM consumption despite its higher PUFA content. Haque et al [56] found that the aqueous extract of walnut reduced in mice the LPO content in liver and kidney induced by chemotherapy toxicity.
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CONCLUSIONS |
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TOP
ABSTRACT
INTRODUCTION
SUBJECTS AND METHODS
RESULTS
CONCLUSIONS
ACKNOWLEDGMENTS
REFERENCES |
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ACKNOWLEDGMENTS |
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TOP
ABSTRACT
INTRODUCTION
SUBJECTS AND METHODS
RESULTS
CONCLUSIONS
ACKNOWLEDGMENTS
REFERENCES |
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Received April 20, 2006. Accepted September 16, 2006.
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TOP
ABSTRACT
INTRODUCTION
SUBJECTS AND METHODS
RESULTS
CONCLUSIONS
ACKNOWLEDGMENTS
REFERENCES |
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dms/qhcnuts3.html (accessed 28 October 2004).This article has been cited by other articles:
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  D. K Banel and F. B Hu Effects of walnut consumption on blood lipids and other cardiovascular risk factors: a meta-analysis and systematic review Am. J. Clinical Nutrition, July 1, 2009; 90(1): 56 - 63. [Abstract] [Full Text] [PDF]
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