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Articles

A Vegetable/Fruit Concentrate with High Antioxidant Capacity Has No Effect on Biomarkers of Antioxidant Status in Male Smokers

Robin van den Berg^*,1, Trinette van Vliet^*, Wendy M. R. Broekmans^*, Nicole H. P. Cnubben^*, Wouter H. J. Vaes^*, Len Roza^*, Guido R.M.M. Haenen, Aalt Bast and Henk van den Berg^*

^* TNO Nutrition and Food Research, 3700 AJ Zeist, the Netherlands and Department of Pharmacology and Toxicology, Faculty of Medicine, University of Maastricht, 6200 MD, Maastricht, the Netherlands

¹To whom correspondence should be addressed at TNO Nutrition and Food Research, Department of Nutritional Physiology, Utrechtseweg 48, P.O. Box 360, 3700 AJ Zeist, the Netherlands. E-mail: R.vandenBerg{at}voeding.tno.nl

	ABSTRACT

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

 
The potential benefits of a high fruit and vegetable intake on the antioxidant status and on relevant biomarkers of oxidative damage to lipids, proteins and DNA and on (functional) markers of oxidative stress were evaluated. A randomized, free living, open placebo-controlled cross-over trial of 3 wk, with a 2-wk washout period between treatments, was performed in a group of 22 male smokers with a relatively low vegetable and fruit intake using a vegetable burger and fruit drink. The vegetable burger and fruit drink increased serum levels of vitamin C, -carotene, ß-carotene, ß-cryptoxanthin and zeaxanthin and plasma total antioxidant capacity. However, no effects were demonstrated on any marker of oxidative damage to lipids (malondialdehyde F₂-isoprostane) proteins (carbonyls) and DNA (Comet assay) and (functional) markers of oxidative stress (reduced/oxidized glutathione ratio, glutathione-S-transferase , glutathione-S-transferase and nuclear transcription factor-B). Apparently, these increased levels of antioxidants in serum were not sufficiently high to show beneficial changes with the selected biomarkers. Alternatively, oxidative stress in male smokers with a relatively low fruit and vegetable intake might have been still too low to demonstrate a beneficial effect of antioxidants.

KEY WORDS: • oxidative stress • humans • antioxidants • smokers • fruit and vegetables

	INTRODUCTION

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

 
A high consumption of fruit and vegetables has been associated with lower incidence and mortality rates of cancer, as found in several human cohort and case-control studies and for a number of common cancer sites (1) . This is in line with many animal experiments showing that vegetables that are common in human diets have antitumorigenic effects (2 3 4) .

Protection against cardiovascular disease by a high consumption of fruits and vegetables has also been suggested (5 ,6) . Additional evidence comes from preclinical studies, such as in vitro or animal studies (7) . However, such data are of limited value because of difficulties in extrapolating to the human situation, e.g., due to uncertainties with respect to bioavailability and tissue distribution or, as in animal models, differences in metabolism and metabolic rate (8) .

The potential health benefits of fruits and vegetables have been attributed to the effects of specific ingredients in fruits and vegetables, such as vitamins, dietary fiber and a wide range of bioactive compounds, such as flavonoids. Antioxidant activity is considered to play an important role in the protective effects of fruits and vegetables (9 10 11 12 13) . This is in part based on the increasing evidence that oxidative damage is involved in the pathogenesis of atherosclerosis and in carcinogenesis (14 ,15) .

The aim of the present study was to evaluate the sensitivity of biomarkers of oxidative damage and (functional) biomarkers of oxidative stress and the potential to demonstrate beneficial effects with these biomarkers in a potential risk group with a relatively low "habitual" vegetable and fruit consumption using "natural" products. In this report, we describe the results of a cross-over intervention study with antioxidants in a "natural" matrix and other bioactive compounds from fruit and vegetables in a group of male smokers with a relatively low habitual fruit and vegetable intake. We used a vegetable burger (VB)² with commercially available lyophilized vegetables, containing the equivalent of 500 g mixed "fresh" vegetables (tomatoes, carrots, onions, broccoli and red sweet pepper) per daily portion and a fruit drink (FD) prepared from fruit (orange, blueberry, apple, lemon and lime) concentrate. The vegetables and fruits selected for preparation of the test products were considered to be significant sources of antioxidants associated with the beneficial health effects, as suggested in observational epidemiological and experimental studies (12 ,16 17 18) .

The effects of the VB/FD on the antioxidant status (vitamins C and E, retinol, carotenoids), plasma trolox equivalent antioxidant capacity (TEAC) and selected biomarkers of oxidative damage [malondialdehyde (MDA), F₂-isoprostane, protein carbonyls, DNA damage (Comet assay)] and oxidative stress [reduced/oxidized glutathione ratio (GSH/GSSG ratio), glutathione-S-transferases (GST, GST)] were assessed. Activity of the nuclear transcription factor-B (NF-B) was determined in peripheral blood mononuclear cells (PBMC). This factor is involved in carcinogenic and inflammatory processes and is under redox control, i.e., activated under conditions of increased cellular oxidative stress (19) , and therefore is considered as an early functional marker of oxidative stress.

	SUBJECTS AND METHODS

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Subjects

The study was performed according to the guidelines for Good Clinical Practice of the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use at the Department of Nutritional Physiology of TNO Nutrition and Food Research, Zeist, the Netherlands. The protocol was approved by the local medical ethics committee.

Smoking, male (aged 18–50 y) subjects were recruited from the pool of volunteers of the institute. The protocol was carefully explained to the volunteers, and their written informed consent was obtained. Subjects were eligible if they fulfilled the following inclusion criteria: i) apparently healthy as indicated by a general medical questionnaire, physical examination and a "normal" clinical chemical blood profile, ii) currently smoking 10 cigarettes/d for 1 y and no intention to stop during the study period, iii) a body mass index [BMI; body weight (kg)/height (m²)] 30, iv) low use of vegetables, fruits and fruit drinks as assessed by a food frequency questionnaire (subjects with the lowest quantified vegetable/fruit intake were selected), v) moderate alcohol consumption of 28 U/wk (1 U = 10 g alcohol), vi) regular (Dutch) food pattern and vii) no use of vitamin and/or food supplements.

Twenty-four men entered the study. Two subjects withdrew during the first treatment period of the study due to dislike of the test products. One subject withdrew on d 1 and was replaced. Therefore, data for 21 subjects were available on d 1 and data for 22 subjects were available on d 22 and 57.

Study design

The study was designed as a randomized, free living, open placebo-controlled cross-over trial. The study consisted of two treatment periods of 3 wk with a 2-wk washout period between periods. Each subject received test products (VB/FD) and the respective control products [control burgers and control drinks (CB/CD)] in a randomized order. Subjects were instructed to maintain their usual diet and to use one portion of either test products or control products per day during 3 wk at a self-selected moment but not replacing a meal. Half of the subjects started with the test products for 3 wk and changed over after 2 wk washout to the control products; the other half of the subjects did the opposite. Each week, volunteers were supplied with fresh products and instructed to store the products in a refrigerator.

At d 1, 22 and 57, body weight was assessed with the subject wearing indoor clothing, without shoes, wallet and keys. Systolic and diastolic blood pressure and heart rate were measured oscillometrically in a sitting position and in the right arm after a 5-min rest. Blood samples were collected for analysis of various status, damage and stress variables as described later.

Test products

A VB was prepared using commercially available lyophilized vegetables, provided by Keizer Waalwijk BV, the Netherlands (lyophilized by Kerry Ingredients BV, the Netherlands) and an FD prepared from fruit juice concentrates (SVZ International, Etten-Leur, the Netherlands).

The VB, of 100 g, contained the equivalent of 500 g mixed "fresh" vegetables, consisted of 40 g mixed freeze-dried vegetables (equivalent to 85 g broccoli, 47 g tomato, 84 g carrot, 99 g red sweet pepper and 156 g onion), mixed with 39.5 g chicken meat, 5 g of palm oil carotenoids (in corn oil; Quest International, Maarssen, the Netherlands) and a mix of spices (Table 1 ). The FD, of 330 mL, consisted of a mixture of tap water–diluted juice concentrates of orange (30%), blueberry (30%), apple (30%), lemon (5%) and lime (5%).

Blood samples

On d 1, 22 and 57, blood samples were collected from fasting subjects, between 0800 and 0930 h. For analysis of vitamins A and E, carotenoid profile, triglycerides, total cholesterol and HDL cholesterol, blood was collected in tubes containing clot activator (Vacutainer systems; Becton Dickinson, Leiden, the Netherlands) and further prepared on ice under subdued light. Within 30 min, the tubes were centrifuged (10 min at 2000 x g at 4°C). Serum samples for analysis of vitamins A and E and carotenoid concentrations were stored at <-70°C until analysis. Samples for analysis of triglycerides and total and HDL cholesterol were stored at <-18°C and analyzed within 2 mo. For analysis of 8-epi-prostaglandin F₂ (F₂-isoprostane), blood was collected in tubes containing 38 g sodium citrate/L (Vacutainer systems). Directly after collection, indomethacin (14 µmol/L) and BHT (20 µmol/L) were added, and the tubes were centrifuged (10 min at 2000 x g at 4°C). Plasma was removed for analysis of F₂-isoprostane, placed on ice and stored at <-70°C. For all other parameters, blood was collected in tubes containing lithium heparin (Vacutainer systems; Becton Dickinson). For analysis of vitamin C, directly after collection, 0.5 mL blood was added to 2.0 mL metaphosphoric acid (50 g/L; J. T. Baker, Deventer, the Netherlands) under continuous vortexing. This mixture was placed on dry ice, stored at <-70°C and analyzed within 5 d. For analysis of TEAC, MDA, protein carbonyls and GST-, within 15 min, blood was centrifuged (10 min at 2000 x g at 4°C). Plasma samples were immediately placed on dry ice and stored at <-70°C. For analysis of DNA breakage, NF-B activity and GST-, immediately after blood collection, PBMC were isolated. Heparinized blood was transferred into Leucosep tubes containing Ficoll Paque and diluted with a balanced salt solution. Samples were centrifuged (30 min at 800 x g and 4°C), and subsequently PBMC were collected, washed twice and divided over the aliquots needed. Cell concentrations were determined, and samples were directly used for analysis of GST- and for isolation of nuclear proteins (NF-B) or stored at <-70°C for analysis of DNA breakage. For analysis of GSH/GSSG ratio, blood was placed on ice, centrifuged (5 min at 5000 x g) and immediately used for determination of GSH/GSSG in erythrocytes. The resulting erythrocytes were washed, and 1 mL of 10 mmol HCl/L plus 6.5% 5-sulfosalicyl acid was added to 450 µL erythrocytes, mixed and put on ice for 10 min. Samples were centrifuged (15 min at 2000 x g at 4°C), and the supernatant was rapidly frozen on dry ice and stored at <-70°C.

Biochemical and chemical analyses

    Characterization of products. All study products (VB/FD and CB/CD) were characterized by chemical analysis of macronutrient composition and antioxidant contents. Protein, carbohydrate and total fat concentrations were analyzed using standard methodology, and energy values were calculated based on the results of macronutrient composition.

The antioxidant content was established by analyses of vitamin C, total vitamin E (only burgers) and carotenoid profile. For vitamin C analyses, VB and CB were first extracted with trichloroacetic acid and FD and CD were deproteinated with metaphosphoric acid, followed by filtration, oxidation to dehydro-L-ascorbic acid and condensation according to a method reported by Speek et al. (20) .

Total vitamin E and carotenoid concentrations were analyzed after saponification with KOH followed by extraction with diisopropylether and HPLC with fluorometric detection for vitamin E (21) and spectrophotometric detection for carotenoids (22) . The results of the macronutrient composition (protein, carbohydrate and fat), carotenoid profile (lutein, zeaxanthin, ß-cryptoxanthin, lycopene, -carotene and ß-carotene), vitamin E and vitamin C concentrations of the burgers and drinks (except for vitamin E) are summarized in Table 2 .

The in vitro antioxidant capacity was measured as the Trolox equivalent antioxidant capacity (TEAC) as described by van den Berg et al. (24) . Briefly, VB and CB were extracted with 70% methanol. Samples of VB/CB or FD/CD were added to an 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonate) radical solution, and the decrease in radical concentration was monitored spectrophotometrically.

    Blood lipid and antioxidant measurements. Serum lipids and serum triglycerides were analyzed by enzymatic hydrolysis with subsequent enzymatic determination of the liberated glycerol by colorimetry (Boehringer, Mannheim, Germany). Total cholesterol was analyzed by enzymatic conversion to a stable chromogen, which can easily be detected with colorimetry (Boehringer). HDL cholesterol was analyzed by precipitation with polyethylene glycol, centrifugation and enzymatic detection by colorimetry (Boehringer), and LDL cholesterol was calculated using the Friedewald formula (25) .

Total antioxidant capacity as measured by the TEAC was determined according to the method described (24) . Briefly, plasma samples were deproteinized by adding an equal volume of 10% trichloroacetic acid. After centrifugation, the supernatant was added to an 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonate) radical solution. The decrease in radical concentration was monitored spectrophotometrically and related to the decrease obtained with Trolox.

Serum vitamin A and E and carotenoid concentrations were quantified by reversed phase HPLC using a modified version of a method described previously (22) . Tocopherols were quantified by fluorometric detection, and vitamin A and carotenoid profiles were quantified with diode array detection.

Vitamin C in deproteinized blood was analyzed by an HPLC method with fluorometric detection (20) . Vitamin C was oxidized to dehydroascorbic acid and subsequently condensed with 1,2-diaminobenzene to quinoxaline, which was detected.

    Lipid, protein and DNA oxidative damage. MDA was determined in plasma by derivatization of MDA with thiobarbituric acid and quantified by HPLC separation with a fluorometric detector according to a method described by Fukunaga et al. (26) . Plasma 8-epi-prostaglandin F₂ was measured according to a method described by Nourooz-Zadeh et al. (27) with some modifications. This method involves solid-phase extraction and conversion to pentafluorobenzyl ester and trimethylsilyl ether derivatives. 8-Epi-prostaglandin F₂ was quantified using negative-ion chemical ionization high resolution mass spectrometry. Plasma protein carbonyl content was quantified by the reaction with 2,4-dinitrophenylhydrazine, as previously described by Buss et al. (28) , using an enzyme-linked immunosorbent assay.

DNA oxidative damage, measured as the Comet assay, was conducted as described by Collins et al. (29) . The DNA strand breaks were analyzed in untreated, H₂O₂-treated (to monitor resistance of the cells to oxidative stress) and endonuclease III–treated (to detect levels of oxidized pyrimidine bases) isolated human PBMC. Comets were analyzed quantitatively (100 cells per samples) using image analysis software (Comet Assay II; Perceptive Instruments, Suffolk, U.K.). The tail moments were converted to visual scoring system (Collins score) from class 0 (no damage) to class 4 (almost all DNA in tail).

    Markers of oxidative stress. The GSH/GSSG ratio was determined in erythrocytes according to the method described by Baker et al. (30) using the enzymatic DTNB-reductase recycling method. Reduced glutathione (GSH) is oxidized by DTNB leading to the formation of oxidized glutathione (GSSG) and TNB (5-thio-2-nitrobenzoic acid). The formation of TNB was measured at 405 nm. GSSG was determined after derivatization of GSH with 2-vinylpyridine.

GST- enzyme activity in plasma was analyzed using the Biotrin HEPKIT-Alpha kit (Biotrin International, Dublin, Ireland) for quantitative determination of GST-, and GST- in PBMC was analyzed using the Biotrin HEPKIT-Pi kit (Biotrin International) for quantitative determination of GST-.

NF-B activity in PBMC was analyzed according to method described by van den Berg et al. (31) as a "functional" marker of oxidative stress. The electromobility shift assay (EMSA) with phosphor imaging was used to detect NF-B. For the quantification of NF-B activity, a commercially obtained HeLa nuclear (protein) extract (Promega Benelux, Leiden, The Netherlands) was used as a standard on each gel. NF-B activity was expressed as HeLa equivalents/10 µg protein.

Statistical analysis

Data were analyzed using the SAS statistical software package (SAS/STAT Version 6; SAS Institute, Cary, NC). All data are expressed as means ± SD. Effects of consumption of the vegetable and fruit concentrates on variables measured were tested by analysis of variance using the General Linear Model procedure. Only the 22 subjects with results for both treatments were included in the analysis. Results are based on those 22, unless indicted otherwise in the tables. Differences were considered significant at 0.05.

	RESULTS

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Volunteers

The 22 men which entered the study were 33 ± 11 y old (range, 19–49 y), had a BMI of 23.6 ± 3.0 kg/m² (range, 19.1–28.5 kg/m²) and smoked 18 ± 7 cigarettes/d (range, 10–40 cigarettes/d). The vegetable and fruit intake of the men, as assessed by a food frequency questionnaire, was estimated to be 167 g/d, which is around the 50th percentile of the Dutch male population (174 g/d) (32) . The test products were well tolerated by the subjects, and compliance to the study protocol was good (>98% of all study products were used), as judged from the reported product consumption and the return of the remaining products. Subjects’ smoking habits were the same for both treatment periods, as judged by the returned question forms. None of the subjects had to be excluded due to illness.

Body weight, blood pressure, heart rate and serum lipid concentrations were not affected by the treatments (Table 3 ).

    Antioxidant status. Serum vitamin C level was significantly increased (P < 0.0001) after the VB/FD treatment compared with the CB/CD treatment (Table 4 ). Serum levels of -carotene, ß-carotene, ß-cryptoxanthin and zeaxanthin were also significantly higher (P < 0.0001) after the VB/FD treatment, whereas serum lycopene and lutein levels did not differ between treatment periods (Table 4) . Plasma TEAC was significantly increased after consumption of the VB and FD compared with the control products (Table 4) . Serum retinol and tocopherol concentrations were not affected by the VB/FD treatment (Table 4) .

	DISCUSSION

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

The aim of this study was to evaluate the sensitivity of a number of biomarkers of oxidative damage and (functional) biomarkers of oxidative stress and to demonstrate beneficial effects with these biomarkers, in a potential risk group of smoking males with a relatively low habitual fruit and vegetable consumption, by increasing their intake of natural antioxidants and of other bioactive compounds from fruit and vegetables.

From the results as summarized in Tables 5 and 6 , it is, however, clear that no effect on markers of oxidative lipid, protein, DNA damage and functional markers of oxidative stress could be demonstrated after a treatment period of 3 wk with these test products.

To account for these findings and apparent discrepancies with reported studies, the following items are discussed: i) the additional antioxidant dose achieved by the test products in relation to the anticipated response and ii) the study design, i.e., "suitability" of the study population and "power" of the study.

Antioxidant dose achieved by the test products in relation to the anticipated response (antioxidant status).

Analysis of the VB/FD, summarized in Table 2 , showed that these products indeed contained substantial amounts of antioxidants and had a high potential (TEAC). The high TEAC of the VB and the FD is largely explained by the high vitamin C content (118 mg in the VB and 53 mg in the FD) and the contribution from other natural antioxidants, such as flavonoids. Consumption of these products did indeed result in an increase in the serum levels of vitamin C, -carotene, ß-carotene, ß-cryptoxanthin and zeaxanthin, respectively, as well as in an increased total plasma antioxidant capacity, measured as the TEAC (Table 4) . The main contribution to the product TEAC comes from vitamin C and the flavonoids, whereas carotenoids do not, or only minimally, contribute (24) . The total flavonoid content measured in the product was 28 mg for the VB and 57 mg in the FD, and 20–30% of the TEAC might be explained by the flavonoids and other bioactive compounds with antioxidant capacity present. An uncertain factor is the bioavailability and postprandial metabolism of flavonoids contained in the products. As far as data are available, it seems that bioavailability of flavonoids is generally low (36) . Plasma flavonoid levels were not determined, but the increase in plasma TEAC might be in part explained by these compounds (or metabolites).

The additional amount of vitamin E provided by the VB was 8.2 mg -tocopherol equivalents (-TE) and 3.0 mg -TE for the CB. According to Traber and Sies (37) , a daily dietary intake of 15–30 mg -tocopherol would be needed to reach an "optimal" plasma -tocopherol level above 30 µmol/L, associated with lower cardiovascular and cancer risk. We have no data on the actual total dietary vitamin E intake in our subjects, but serum -tocopherol levels were slightly below 30 µmol/L. Observational epidemiological and intervention studies indicate that the major effect (if any) of vitamin E supply are found only with a daily intake higher than 74 mg -TE (34) . Therefore, the amount used in the present study apparently was not sufficient to increase the serum tocopherol level and/or to affect parameters of oxidative damage and stress.

The carotenoid content of the VB was at least 50–70% lower as expected from the contents in the equivalent "fresh" vegetables. Lycopene content was 1.3 mg, whereas 3 mg was expected on the basis of 47 g "fresh" tomatoes. For lutein, the expected content was 3 mg, but only 0.85 mg was detected. Apparently, losses have occurred in production of the VB, which included blanching and freeze-drying (35) .

The lowest lycopene intake reported to increase plasma lycopene levels is 3.3 mg in heat processed vegetable juice given for 15 d (38) . In other studies, using tomato products, higher amounts were achieved, such as 40 mg/d (39) , 16.5 mg/d (40) or 10 mg/d (41) to increase plasma lycopene levels. The bioavailability of lycopene, which depends on the matrix, is also an important factor. Bohm and Bitsch (42) reported that lycopene from capsules and tomato juice (processed tomatoes) was better absorbed from the intestine than lycopene from raw tomatoes. They also showed that administration of 5 mg lycopene/d did not affect plasma antioxidant capacity. If the protective effect of tomatoes on prostate cancer risk is indeed due to lycopene then the protective lycopene intake should be 6.5 mg/d (17) . In the present study, the additional intake of lutein and lycopene from the burger was apparently too low to achieve an increase of serum levels.

The VB/FD provided per day an additional 4.0 mg -carotene and 9.0 mg ß-carotene. This resulted in an increase in plasma - and ß-carotene. ß-Cryptoxanthin and zeaxanthin levels were also increased, but the obtained higher levels were not associated with changes in oxidative stress and damage-related markers. Using data reported in the various epidemiological trials on cardiovascular risk and mainly observational studies, a threshold level for serum ß-carotene of 0.4 µmol/L has been derived (43) . This level is reached at an intake of 1.5–2 mg ß-carotene/d. We have no data on the actual total ß-carotene dietary intake in our subjects, but ß-carotene levels were 0.3 µmol/L after CB/CD and 0.67 µmol/L after VB/FD treatment. "High dose" supplementation with synthetic ß-carotene in smokers has been associated with an increased risk for lung cancer (44 ,45) .

Previously, reduced genetic damage by natural products has been reported in humans, using the Comet assay (39) . After a 2-wk carotenoid depletion period, followed by a daily intake for 2 wk of 330 mL tomato juice (containing 40 mg lycopene) or 330 mL carrot juice (containing 22.3 mg -carotene and 15.7 mg ß-carotene) or 10 g dried spinach powder (containing 11.3 mg lutein), a significant decrease in endogenous levels of strand breaks in lymphocyte DNA has been found.

Vitamin C is an important contributor to the oxidant defense system and has been shown to protect against in vivo oxidation of lipids and DNA in humans, particularly in persons exposed to enhanced oxidative stress, such as smokers (33 ,46) . Numerous epidemiological studies strongly suggest that 90–100 mg vitamin C/d (33) , and plasma concentrations of >50 µmol/L (47) have been associated with a lower incidence and mortality from cardiovascular disease and cancer.

The VB/FD provided an additional vitamin C intake of 170.5 mg/d. This resulted in an increase in plasma vitamin C level of 20 µmol/L. This increase likely explains the increase in plasma TEAC (+38 µmol/L). Calculated using the TEAC of 1 for vitamin C (24) , the contribution of vitamin C to the increase in TEAC is 83%. However, for analysis of plasma TEAC, no precautions could be taken to prevent loss of vitamin C, as done for analysis of vitamin C, and therefore the contribution of vitamin C to the increase in TEAC might be overestimated.

In our subjects, vitamin C levels were 40 µmol/L before and 58 µmol/L after VB/FD treatment. This is in line with kinetic data showing an increase in fasting plasma levels of vitamin C to 60 µmol/L, after a 200-mg dose (48) .

High dose supplementation with vitamin C (500 mg/d) has been shown to elevate red blood cell glutathione in healthy adults (49) . Erythrocyte glutathione oxidation is considered to be a consistent marker of oxidative stress (50) .

In our study, the GSH/GSSG ratio was not altered, but GSH levels appeared to be lower than baseline after both treatments. Hininger et al. (41) reported increased GSH levels in smokers, which also decreased to the "normal" nonsmoking levels after a fruit and vegetable–rich diet. This increase was unrelated to an increase in GSSG. The observed higher GSH in erythrocytes of smokers might reflect an adaptation to enhanced production of reactive oxygen species from cigarettes.

Duthie et al. (51) reported that supplementation of the diet for 20 wk with vitamin C (100 mg/d), in combination with vitamin E (280 mg/d) and ß-carotene (25 mg/d), resulted in a highly significant decrease in endogenous oxidative base damage in lymphocyte DNA of both smokers and nonsmokers. In addition, lymphocytes of antioxidant supplemented subjects showed an increased resistance to oxidative damage when challenged in vitro with H₂O₂.

Effect on (functional) markers of oxidative stress.

Antioxidant status and markers of oxidative damage (DNA adducts, lipid and protein oxidation products) are frequently used to assess antioxidant and pro-oxidant effects. However, gene expression, being an important modulator of cell functions, has been shown, in some cases, to be under redox control. Changes in gene expression can provide a sensitive marker of oxidative stress (or changes in oxidative status). A well-defined transcription factor, NF-B, has been identified to be regulated by the intracellular redox state. Activation of the transcription factor NF-B was measured in this study to assess the potential of these markers as a ‘functional’ marker of oxidative stress. This redox-controlled mechanism is quite distinct from the response to antioxidant that involves up-regulation of certain specific genes (e.g., glutathione-S-transferase) as a consequence, for example, of the antioxidant responsive element present in the promotor. Such genes respond to a diverse selection of antioxidants and might reflect changes in oxidative status. Induction of both glutathione-S-transferases (GST- and GST-), a family of phase II enzymes, was measured in this study to assess the potential of these markers as markers of oxidative stress.

Induction of phase II enzymes has been postulated as a key mechanism for the anticarcinogenic effects of fruits, vegetables and antioxidants (52 53 54) . GST are able to conjugate electrophiles with glutathione or by a glutathione-dependent peroxidase activity. They may also participate in the repair of cellular macromolecules damaged by oxidative stress. In addition, GST- can react directly with reactive oxygen species via a sensitive SH-group or be inactivated by disulfide formation that can be reversed by glutathione. Therefore, it has a specific response to oxidative stress (55) . Several genetic elements in the promoter regions of the genes encoding phase II enzymes have been identified. GST- is regulated by the redox status of the cell (55) . In the regulatory element of the GST- gene, an antioxidant responsive element, as well as a NF-B–binding site, has been identified.

In our study, GST- was lower after treatment with the VB/FD compared with the CB/CD. This is in contrast with a previous study with nonsmokers, where an induction of GST- was observed in 6 of the 23 subjects after treatment with tomato and carrot juices (39) . Smokers may have higher levels of GST- compared with nonsmokers to protect them against oxidative stress caused by smoking. A higher intake of antioxidants prevents formation of reactive oxygen species, and therefore GST- is down-regulated.

NF-B is activated by oxidative stress, and this enhanced activity can be modulated by antioxidants (56) . To assess in vivo NF-B activity, the activation was measured in freshly isolated PBMC. In a previous study, we found a higher NF-B activity in male "heavy" smokers compared with nonsmokers (57) . Because of the high TEAC of the VB/FD, the relatively high concentrations of vitamin C and quercetin, a decrease in NF-B activation was anticipated. In vitro reduction of NF-B activation by antioxidants has been reported, such as by vitamin C, vitamin E and resveratrol (19 ,58) . However, we did not find a counteraction in the present study.

Study design: "suitability" of the study population and test products, "power" of the study.

The study was designed as a randomized, free living, open, placebo-controlled, cross-over study to simulate the normal home situation and intervention with processed vegetables, rather than with food supplements. Compliance was checked and considered adequate. Subjects supplemented the product to their usual diet. However, no further data on the actual fruit and vegetable intake during the study were collected. Replacement of other food products and changes in dietary pattern as a result of the intervention per se therefore cannot be excluded but, if any, should be balanced by the cross-over design with isoenergetic placebo products. Also, the selection of male smokers with a relatively low fruit and vegetable intake on the basis of food questionnaires might have been insufficiently discriminative to select subjects with increased oxidative stress. In some studies, e.g., Bub et al. (59) , a depletion stage was included in which subjects are fed a "low carotenoid diet" before the actual intervention, which might increase the sensitivity and power of the design to demonstrate effects.

Smokers are considered to be exposed to an increased oxidative stress, because of their apparent lower antioxidant intake (60) in combination with a relatively high free radical exposure. Several studies have been reported showing lower antioxidant vitamin and/or carotenoid plasma levels in smokers (41 ,61 ,62) . This also is true with respect to markers of oxidative damage, such as increased plasma conjugated dienes (63) , increased oxidative DNA damage (51) or increased plasma levels of F₂-isoprostanes (64) .

We recently reported that (baseline) NF-B activity is higher in male smokers compared with nonsmokers (57) . However, we did not compare the present baseline data with those for a comparable group of nonsmokers. Comparison with values reported by other groups is complicated because of methodological differences and, for some markers, relatively large interassay variabilities.

Test products.

To maximize the intake of natural antioxidants, we used a VB and an FD, prepared from commercially obtained ingredients (freeze-dried vegetables and fruit extracts), selected because of their potential antioxidant contents and reported health effects. These VB/FD had indeed relatively "high" TEAC, but for some compounds, such as the carotenoids, the actual additional intake achieved was lower than anticipated, most probably because of losses during processing of the extracts/lyophilized products. As discussed above, carotenoid and vitamin E levels were apparently too low to achieve an effect, if any, on the markers evaluated in this study.

Power of the study.

The relatively low antioxidant content of the test products and/or the low level of oxidative stress in the study population may explain the lack of effects of fruits and vegetables supplementation on the selected biomarkers in our study. To assess the sensitivity of these markers, we used our data set to calculate the number of subjects needed to demonstrate a 10% difference between treatments ( = 0.05, two-sided and a power of 80%). Based on the observed within-subject variability during treatment with the control products, we calculated that for some of the biomarkers, the number of subjects was indeed insufficient. Table 7 shows that in our study, using 22 subjects, only an effect (10% difference) on protein carbonyls and plasma TEAC could have been demonstrated.

	FOOTNOTES

 
² Abbreviations used: BMI, body mass index; CB, control burger; CD, control drink; FD, fruit drink; GSH, reduced glutathione; GSSG, oxidized glutathione; GST, glutathione-S-transferase; MDA, malondialdehyde; NF-B, nuclear transcription factor-B; PBMC, peripheral blood mononuclear cells; TEAC, trolox equivalent antioxidant capacity; VB, vegetable burger.

Manuscript received January 2, 2001. Initial review completed January 25, 2001. Revision accepted March 12, 2001.

	REFERENCES

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

 

1. Block G., Patterson B., Subar A. Fruit, vegetables and cancer prevention: a review of the epidemiological evidence. Nutr. Cancer 1992;18:1-29[Medline]

2. Bresnick E., Birt D. F., Wolterman K., Wheeler M., Markin R. S. Reduction in mammary tumorigenesis in the rat by cabbage and cabbage residue. Carcinogenesis 1990;11:1159-1163[Abstract/Free Full Text]

3. Wattenberg L. W., Coccia J. B. Inhibition of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone carcinogenesis in mice by D-limonene and citrus fruit oils. Carcinogenesis 1991;12:115-117[Abstract/Free Full Text]

4. Mahmoud N. N., Carothers A. M., Grunberger D., Bilinski R. T., Churchill M. R., Martucci C., Newmark H. L., Bertagnolli M. M. Plant phenolics decrease intestinal tumors in an animal model of familial adenomatous polyposis. Carcinogenesis 2000;21:921-927[Abstract/Free Full Text]

5. Rimm E. B., Katan M. B., Ascherio A., Stampfer M. J., Willett W. C. Relation between intake of flavonoids and risk for coronary heart disease in male health professionals. Ann. Intern. Med. 1996;125:384-389[Abstract/Free Full Text]

6. Gillman M. W., Cupples L. A., Gagnon D., Posner B. M., Ellison R. C., Castelli W. P., Wolf P. A. Protective effect of fruits and vegetables on development of stroke in men. J. Am. Med. Assoc. 1995;273:1113-1117[Abstract]

7. Benzie I. F. Lipid peroxidation: a review of causes, consequences, measurement and dietary influences. Int. J. Food Sci. Nutr. 1996;47:233-261[Medline]

8. Halliwell B. Oxidative stress, nutrition and health: experimental strategies for optimization of nutritional antioxidant intake in humans. Free Radic. Res. 1996;25:57-74[Medline]

9. Pool-Zobel B. L., Bub A., Liegibel U. M., Treptow-van Lishaut S., Rechkemmer G. Mechanisms by which vegetable consumption reduces genetic damage in humans. Cancer Epidemiol. Biomarkers Prev. 1997;7:891-899[Abstract]

10. Porrini M., Riso P. Lymphocyte lycopene concentration and DNA protection from oxidative damage is increased in women after a short period of tomato consumption. J. Nutr. 2000;130:189-192[Abstract/Free Full Text]

11. Young J. F., Nielsen S. E., Haraldsdottir J., Daneshvar B., Lauridsen S. T., Knuthsen P., Crozier A., Sandstrom B., Dragsted L. O. Effect of fruit juice intake on urinary quercetin excretion and biomarkers of antioxidative status. Am. J. Clin. Nutr. 1999;69:87-94[Abstract/Free Full Text]

12. Verhagen H., Poulsen H. E., Loft S., van Poppel G., Willems M. I., van Bladeren P. J. Reduction of oxidative DNA-damage in humans by Brussels sprouts. Carcinogenesis 1995;16:969-970[Abstract/Free Full Text]

13. Van Poppel G., van den Berg H. Vitamins and cancer. Cancer Lett 1997;114:195-202[Medline]

14. Loft S., Poulsen H. E. Cancer risk and oxidative DNA damage in man. J. Mol. Med. 1996;74:297-312[Medline]

15. Baynes J. W., Thorpe S. R. Role of oxidative stress in diabetic complications: a new perspective on an old paradigm. Diabetes 1999;48:1-9[Abstract]

16. Hertog M. G., Bueno de Mesquita H. B., Fehily A. M., Sweetnam P. M., Elwood P. C., Kromhout D. Fruit and vegetable consumption and cancer mortality in the Caerphilly Study. Cancer Epidemiol. Biomarkers Prev. 1996;5:673-677[Abstract/Free Full Text]

17. Giovanucci E. Tomatoes, tomato-based products, lycopene and cancer: review of the epidemiological literature. J. Natl. Cancer Inst. 1999;91:317-331[Abstract/Free Full Text]

18. Broekmans W.M.R., Klopping-Ketelaars I.A.A., Schuurman C.R.W.C., Verhagen H., van den Berg H., Kok F. J., van Poppel G. Fruits and vegetables increase plasma carotenoids and vitamins and decrease homocysteine in humans. J. Nutr. 2000;130:1578-1583[Abstract/Free Full Text]

19. Sen C. K., Packer L. Antioxidant and redox regulation of gene transcription. FASEB J 1996;10:709-720[Abstract]

20. Speek A. J., Schrijver J., Schreurs W.H.P. Fluorimetric determination of total vitamin C and total vitamin iso-C in foodstuff and beverages by high performance liquid chromatography with pre-column derivatization. J. Agr. Food Chem. 1984;32:352-355

21. Speek A. J., Schrijver J., Schreurs W.H.P. The vitamin E composition of some seed oils as determined by high performance liquid chromatography with fluorometric detection. J. Food Sci. 1985;50:121-124

22. Van Vliet T., van Schaik F., van Schoonhoven J., Schrijver J. Determination of several retinoids, carotenoids and E vitamers by high-performance liquid chromatography: application to plasma and tissues of rats fed a diet rich in either beta-carotene or canthaxanthin. J. Chromatogr. 1991;553:179-186[Medline]

23. Hertog M. G., Hollman P. C., Venema D. P. Optimization of a quantitative HPLC determination of potentially anticarcinogenic flavonoids in vegetable and fruits. J. Agric. Food. Chem. 1992;40:1591-1598

24. Van den Berg R., Haenen G.R.M.M., van den Berg H., Bast A. Applicability of an improved Trolox equivalent antioxidant capacity (TEAC) assay for evaluation of antioxidant capacity measurements of mixtures. Food Chem 1999;66:511-517

25. Friedewald W. T., Levy R. I., Fredrickson D. S. Estimation of the concentration of low-density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge. Clin. Chem. 1972;18:499-502[Abstract]

26. Fukunaga K., Yoshida M., Nakazono N. A simple, rapid, highly sensitive and reproducible quantification method for plasma malondialdehyde by high-performance liquid chromatography. Biomed. Chromatogr 1998;12:300-303[Medline]

27. Nourooz-Zadeh J., Gopaul N. K., Barrow S., Mallet A. I., Anggard E. E. Analysis of F₂-isoprostanes as indicators of non-enzymatic lipid peroxidation in vivo by gas chromatography-mass spectrometry: development of a solid-phase extraction procedure. J. Chromatogr. B. Biomed. Appl. 1995;667:199-208[Medline]

28. Buss H., Chan T. P., Sluis K. B., Domigan N. M., Winterbourn C. C. Protein carbonyl measurement by a sensitive ELISA method. Free Radic. Biol. Med. 1997;23:361-366[Medline]

29. Collins A. R., Duthie S. J., Dobson V. L. Direct enzymic detection of endogenous oxidative base damage in human lymphocyte DNA. Carcinogenesis 1993;14:1733-1735[Abstract/Free Full Text]

30. Baker M. A., Cernigilia G. J., Aman A. Microtiterplate assay for the measurement of glutathione and glutathione disulphide in large number of biological samples. Anal. Biochem. 1990;190:360-365[Medline]

31. Van den Berg, R., Haenen, G.R.M.M., van den Berg, H. & Bast, A. (2001) Nuclear factor-B as a potential biomarker of oxidative stress. Br. J. Nutr. (in press).

32. Hulshof K.F.A.M., Kistenmaker C., Bouwmans M., Löwik M.R.H. Energy and nutrient intake of the Dutch population—Dutch Nutrition Surveillance System 1997–1998 1998:1-13TNO report V98.805 (in Dutch)

33. Carr A. C., Frei B. Towards a new recommended dietary allowance for vitamin C based on antioxidant and health effect in humans. Am. J. Clin. Nutr. 1999;69:1086-1107[Abstract/Free Full Text]

34. Bramley P. M., Elmadfa I., Kafatos A., Kelly F. J., Manios Y., Roxborough H. E., Schuch W., Sheehy P.J.A., Wagner K-H. Vitamin E. J. Sci. Food Agric. 2000;80:913-938

35. Van den Berg H., Faulks R., Fernando-Granado H., Hirschberg J., Olmedilla B., Sandman G., Southon S., Stahl W. The potential for the improvement of carotenoid levels in food and the likely systemic effects. J. Sci. Food Agric. 2000;80:880-912

36. Hollman P. C. Bioavailability of flavonoids. Eur. J. Clin. Nutr. 1997;51:S66-S69

37. Traber, M. G. & Sies, H. (1996) Vitamin E in humans: demand and delivery. In: Annual Reviews in Nutrition (McCormick, D. B., ed.), pp. 321–347, Vol 16, Annual Reviews, Palo Alto, CA.

38. Yeum K. J., Booth S. L., Sadowski J. A., Liu C., Tang G., Krinsky N. I., Russell R. M. Human plasma carotenoid response to the ingestion of controlled diets high in fruits and vegetables. Am. J. Clin. Nutr. 1996;64:594-602[Abstract/Free Full Text]

39. Pool-Zobel B. L., Bub A., Müller H., Wollowski I., Rechkemmer G. Consumption of vegetables reduces genetic damage in humans: first results of an intervention trial with carotenoid-rich foods. Carcinogenesis 1997;18:1847-1850[Abstract/Free Full Text]

40. Riso P., Pinder A., Santangelo A., Porrini M. Does tomato consumption effectively increase the resistance of lymphocyte DNA to oxidative damage?. Am. J. Clin. Nutr 1999;69:712-718[Abstract/Free Full Text]

41. Hininger I., Chopra M., Thurnham D. I., Laporte F., Richard M. J., Favier A., Roussel A. M. Effect of increased fruit and vegetable intake on the susceptibility of lipoprotein to oxidation in smokers. Eur. J. Clin. Nutr. 1997;51:601-606[Medline]

42. Bohm V., Bitsch R. Intestinal absorption of lycopene from different matrices and interactions to other carotenoids, the lipid status, and the antioxidant capacity of human plasma Eur. J. Nutr. 1999;38:118-125

43. Gey K. F. Ten-year retrospective on the antioxidant hypothesis of arteriosclerosis: threshold plasma levels of antioxidant micronutrients related to minimum cardiovascular disease. Nutr. Biochem. 1995;6:206-236

44. ATBC Study Group (the Alpha-Tocopherol, ß-Carotene Cancer Prevention Study Group) The effects of vitamin E and ß-carotene on the incidence of lung cancer and other cancers in male smokers. N. Engl. J. Med. 1994;334:1019-1035

45. Omenn G. S., Goodman G. E., Thornquist M. D., Balmes J., Cullen M. R., Glass A., Keogh J. P., Meyskens F. L., Jr, Valanis B., Williams J. H., Jr, Barnhart S., Cherniack M. G., Brodkin C. A., Hammar S. Risk factors for lung cancer and for intervention effects in CARET, the Beta-Carotene and Retinol Efficacy Trial. J. Natl. Cancer Inst. 1996;88:1550-1559[Abstract/Free Full Text]

46. Henning S. M., Zhang J. Z., McKee R. W., Swendseid M. E., Jacob R. A. Glutathione blood levels and other oxidant defense indices in men fed diets low in vitamin C. J. Nutr. 1991;121:1969-1975

47. Gey K. F. Vitamins E plus C and interacting conutrients required for optimal health: a critical and constructive review of epidemiology and supplementation data regarding cardiovascular disease and cancer. Biofactors 1998;7:113-174[Medline]

48. Levine M., Rumsey S. C., Wang Y., Park J., Kwon O., Amano N. In situ kinetics: an approach to recommended intake of vitamin C. Methods Enzymol 1997;281:425-437[Medline]

49. Johnston C. S., Meyer C. G., Srilakshmi J. C. Vitamin C elevates red blood cell glutathione in healthy adults. Am. J. Clin. Nutr. 1993;58:103-105[Abstract/Free Full Text]

50. Exner R., Wessner B., Manhart N., Roth E. Therapeutic potential of glutathione. Wien. Klin. Wochenschr. 2000;112:610-616[Medline]

51. Duthie S. J., Ma A., Ross M. A., Collins A. R. Antioxidant supplementation decreases oxidative DNA damage in human lymphocytes. Cancer Res 1996;56:1291-1295[Abstract/Free Full Text]

52. Bogaards J. J., Verhagen H., Willems M. I., Van Poppel G., van Bladeren P. J. Consumption of Brussels sprouts results in -class glutathione S-transferase levels in human blood plasma. Carcinogenesis 1994;15:1073-1075[Abstract/Free Full Text]

53. Hayes J. D., Pulford D. J. The glutathione S-transferase supergene family: regulation of GST and the contribution of the isoenzymes to cancer chemoprotection and drug resistance. Crit. Rev. Biochem. Mol. Biol. 1995;30:445-600[Medline]

54. Nijhoff W. A., Mulder T. P., Verhagen H., van Poppel G., Peters W. H. Effects of consumption of Brussels sprouts on plasma and urinary glutathione S-transferase class-alpha and -pi in humans. Carcinogenesis 1995;16:955-957[Abstract/Free Full Text]

55. Xia C., Hu J., Ketterer B., Taylor J. B. The organization of the human GSTP1–1 gene promoter and its response to retinoic acid and cellular redox status. Biochem. J. 1996;313:155-161

56. Blackwell T. S., Blackwell T. R., Holden E. P., Christman B. W., Christman J. W. In vivo antioxidant treatment suppresses nuclear factor-B activation and neutrophillic lung inflammation. J. Immunol. 1996;157:1630-1637[Abstract]

57. Van den Berg R., Haenen G.R.M.M., Van den Berg H., Bast A. Nuclear factor-B activation is higher in peripheral blood mononuclear cells of male smokers. Environ. Toxicol. Pharmacol. 2001;9:147-151[Medline]

58. Manna S. K., Mukhopadhyay A., Aggarwal B. B. Resveratrol suppresses TNF-induced activation of nuclear transcription factors NF-kappa B, activator protein-1, and apoptosis: potential role of reactive oxygen intermediates and lipid peroxidation. J. Immunol. 2000;164:6509-6519[Abstract/Free Full Text]

59. Bub A., Watzl B., Abrahamse L., Delincee H., Adam S., Wever J., Muller H., Rechkemmer G. Moderate intervention with carotenoid-rich vegetable products reduces lipid peroxidation in men J. Nutr 2000;130:2200-2206

60. Marangon K., Herbeth B., Lecomte E., Paul-Dauphin A., Grolier P., Chancerelle Y., Artur Y., Siest G. Diet, antioxidant status, and smoking habits in French men. Am. J. Clin. Nutr. 1998;67:231-239[Abstract]

61. Stryker W. S., Kaplan L. A., Stein E. A., Stampfer M. J., Sober A., Willett W. C. The relation of diet, cigarette smoking, and alcohol consumption to plasma beta-carotene and alpha-tocopherol levels. Am. J. Epidemiol. 1988;127:283-296[Abstract/Free Full Text]

62. Weber P., Bendich A., Schalch W. Vitamin C and human health–a review of recent data relevant to human requirements. Int. J. Vitam. Nutr. Res. 1996;66:19-30[Medline]

63. Duthie G. G., Arthur J. R., James W. P. Effects of smoking and vitamin E on blood antioxidant status. Am. J. Clin. Nutr. 1991;53:1061S-1063S[Abstract/Free Full Text]

64. Morrow J. D., Frei B., Longmire A. W., Gaziano J. M., Lynch S. M., Shyr Y., Strauss W. E., Oates J. A., Roberts L. J., II Increase in circulating products of lipid peroxidation (F₂-isoprostanes) in smokers: smoking as a cause of oxidative damage. N. Engl. J. Med. 1995;332:1198-1203[Abstract/Free Full Text]

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