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Article

Moderate Intervention with Carotenoid-Rich Vegetable Products Reduces Lipid Peroxidation in Men¹

Institute of Nutritional Physiology, Federal Research Centre for Nutrition, D-76131 Karlsruhe, Germany and ^* Academic Medical Centre, University of Amsterdam, Department of Surgery, 110 Amsterdam, The Netherlands

²To whom correspondence should be addressed.

	ABSTRACT

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Because of their antioxidant properties, carotenoids may have beneficial effects in preventing cancer and cardiovascular disease. However, in humans consuming carotenoid-rich vegetables, data concerning the antioxidant effects of carotenoids are rather scarce. A human intervention trial was conducted, therefore, to determine whether a moderately increased consumption of carotenoid-rich vegetables would influence the antioxidant status in 23 healthy men. This short-term feeding study lasted 8 wk during which the men consumed a low carotenoid diet. A 2-wk low carotenoid period was followed by daily consumption of 330 mL tomato juice, then by 330 mL carrot juice and then by 10 g of spinach powder, each for 2 wk. Antioxidant status [water-soluble antioxidants in serum, ferric reducing ability of plasma (FRAP) and antioxidant enzyme activities] and lipid peroxidation (plasma malondialdehyde and ex vivo oxidation of LDL) were determined. In a subgroup of 10 men, lipoprotein carotenoids were measured. The consumption of carotenoid-rich vegetables significantly increased selected carotenoids in lipoproteins but had only minor effects on their relative distribution pattern. Tomato juice consumption reduced plasma thiobarbituric acid reactive substances (TBARS) by 12% (P < 0.05) and lipoprotein oxidizability in terms of an increased lag time (18%, P < 0.05). Carrot juice and spinach powder had no effect on lipid peroxidation. Water-soluble antioxidants, FRAP, glutathione peroxidase and reductase activities did not change during any study period. In evaluating the low carotenoid diet, we conclude that the additional consumption of carotenoid-rich vegetable products enhanced lipoprotein carotenoid concentrations, but only tomato juice reduced LDL oxidation in healthy men.

KEY WORDS: • vegetable • humans • antioxidant • carotenoid • lipoprotein

	INTRODUCTION

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

 
The oxidative modification of LDL is considered to play an important role in the pathogenesis of atherosclerosis (Diaz et al. 1997 ). In addition, lipid oxidation products are involved in the formation of mutagenic DNA adducts, which may contribute to carcinogenesis (Chung et al. 1996 ). It has been hypothesized that dietary antioxidants protect LDL from oxidation and should therefore reduce the risk of atherosclerosis and cancer. In fact, dietary antioxidants such as ascorbic acid, vitamin E and ß-carotene have been demonstrated to prevent LDL oxidation in vitro (Frei et al. 1996 , Jialal and Devaraj 1996 ). In vivo studies, however, have yielded contradictory results. Some studies reported that ß-carotene supplementation inhibited LDL oxidation (Levy et al. 1996 , Nyysönen et al. 1994 ), whereas others did not find an inhibition of LDL oxidation (Gaziano et al. 1995 , Reaven et al. 1993 ).

Although information exists concerning the absorption, metabolism and action of ß-carotene in humans, little is known about these processes for other carotenoids. Nutrition research has focused recently on other carotenoids including -carotene, lycopene and lutein (Stahl and Sies 1996 , Yeum et al. 1996 ). These include carotenoids to be found in high concentrations in vegetables, such as lycopene in tomato and lutein in spinach, cabbage or kale. One of the major characteristics of these carotenoids is their high antioxidative potential measured as Trolox equivalent antioxidant capacity (TEAC)⁴ . Among carotenoids, TEAC values were highest for lycopene, ß-carotene and lutein (Miller et al. 1996 ). Because antioxidant mechanisms are likely involved in the pathogenesis of cardiovascular diseases and cancer (Gaziano and Hennekens 1993 , Ziegler 1991 ), it is tempting to assume that the health benefits associated with the consumption of carotenoid-rich vegetables are due at least in part to antioxidant properties of the carotenoids. This hypothesis is supported by recent studies that showed an increase in plasma antioxidant capacity in humans (Cao et al. 1998a and 1998b , Miller et al. 1998 ) and protection against lipid peroxidation as measured by thiobarbituric acid reactive substances (TBARS) and breath pentane (Miller et al. 1998 ) upon increased consumption of fruit and vegetables. Nevertheless, the protection of antioxidant carotenoids from food against LDL oxidation in vivo in humans requires further elucidation (Agarwal and Rao 1998 , Rao and Agarwal 1998 ).

Therefore, we conducted a human intervention trial to determine whether a moderately increased consumption of carotenoid-rich vegetables would elevate plasma carotenoids to a concentration that was correlated negatively with the risk of cancer and cardiovascular disease in epidemiological studies. Effects of carotenoid-rich vegetable products on the antioxidant status and LDL oxidation in men were assessed. Lipoprotein carotenoids were measured to possibly relate diet-induced changes in LDL carotenoids and LDL oxidation measurements.

A study design without washout periods between the different vegetable intervention periods was chosen to mimic more closely the dietary behavior of consumers. Tomato juice, carrot juice and spinach powder were used as sources for specific carotenoids because their major carotenoids showed the highest antioxidant activity in the TEAC assay (Miller et al. 1996 ). Results of this study referring to plasma carotenoid concentrations (Müller et al. 1999 ), immunologic effects (Watzl et al. 1999 ), prevention of lymphocyte DNA damage (Pool-Zobel et al. 1997 ) and the effect of vegetable products on detoxifying enzymes (Pool-Zobel et al. 1998 ) have been published.

	SUBJECTS AND METHODS

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Subjects and study design.

The study protocol was described previously (Müller et al. 1999 ). In brief, 23 nonsmoking men aged 27–40 y in good health, as determined by a screening history and medical check, were examined. Anthropometric data are given in Table 1 . None were taking vitamin supplements or medication during the study. The study was approved by the Medical Ethical Committee of the Landesaerztekammer Baden-Wuerttemberg and all participants gave their written consent. This short-term feeding study consisted of an 8-wk experimental period, divided into four 2-wk periods. During the study the men consumed a low carotenoid diet. They adhered to their usual diet but were instructed to avoid food products with a high carotenoid content. They were given a list of products to be excluded from the diet (Müller et al. 1999 ). On the basis of food records, the average daily carotenoid intake from self-selected foods during the study was 1.32 ± 0.58 mg (Watzl et al. 1999 ). The first 2 wk served as a low carotenoid period, during which no additional carotenoid-rich food was given. For the next 14 d, the subjects ingested 330 mL/d of a commercially available tomato juice (40 mg lycopene) in addition to their meals (tomato period). During wk 5 and 6, the tomato juice was replaced by 330 mL carrot juice (15.7 mg -carotene and 22.3 mg ß-carotene) daily (carrot period). Tomato and carrot juice were provided by Schoenenberger Pflanzensaftwerke, Magstadt, Germany. Finally, during the last 2 wk, a liquid spinach powder preparation (10 g spinach powder; 11.3 mg lutein and 3.1 mg ß-carotene; Völpel, Königmoos, Germany) was given with the daily meals (spinach period). Table 2 describes the carotenoid concentrations of these products.

Blood samples were taken from fasting subjects at the beginning of the study and at the end of each week between 0700 and 0900 h. Blood was drawn from an antecubital vein into prechilled tubes containing EDTA (1.6 g/L, Monovette-Sarstedt, Nümbrecht, Germany) and immediately placed on ice in the dark. Plasma was collected after centrifugation at 1500 x g for 10 min at 4°C. For the lipid peroxidation assay, sucrose (15 g/L) was added to the plasma to prevent LDL aggregation,. For carotenoid analysis, sucrose (15 g/L) and BHT (5 mg/L) as antioxidant were added to the plasma and stored at -80°C until analysis.

Blood antioxidant measurements.

Albumin was determined by using a bromcresol green reagent (Boehringer Mannheim, Germany). Bilirubin (DCA method) and uric acid (enzymatic Trinder method) were measured with test kits (RANDOX, Ardmore, N. Ireland). Serum levels of vitamin C were determined spectrophotometrically after derivatization with dinitrophenylhydrazin (Omaye et al. 1979 ). Intra- and interassay variations were <2 and <6%, respectively.

Glutathione determination.

Total glutathione (GSHt) and oxidized glutathione (GSSG) were determined in whole blood, plasma and erythrocytes using a microtiter plate assay based on the method described by Baker et al. (1990) and Richie et al. (1996) . Samples were deproteinized using 5-sulfosalicyclic acid (Sigma, Deisenhofen, Germany). The reaction was followed at 405 nm wavelength and 30°C for 30 min at a rate of 1 measurement/min using a temperature-controlled microtiter plate reader (Molecular Devices, München, Germany). GSHt and GSSG concentrations were determined in triplicate and calculated from standard curves of GSH and GSSG (both from Sigma).

FRAP-assay.

To measure "antioxidant power," the ferric reducing ability of plasma (FRAP)-assay as described by Benzie and Strain (1996) was used with minor modifications. In brief, in a 96-well microtiter plate, plasma samples (10 µL) were added to 30 µL of H₂O and the reaction was started by further adding 300 µL prewarmed (30°C) FRAP reagent. The reaction mixture was incubated for 8 min at 30°C and absorbance was determined in a microtiter plate reader (MWG Biotech, Ebersberg, Germany) at a wavelength of 585 nm. Intra- and interassay variations were <5%.

Enzyme activity measurements.

Superoxide dismutase (SOD) and glutathione peroxidase (GPX) activities in erythrocytes were assayed using commercial test kits (Randox Laboratories, Crumlin, UK), which were adapted to a microplate reader (Molecular Devices). Catalase activity (CAT) in erythrocytes was assayed by the method of Aebi (1983) . The activity of glutathione reductase (GOR) in plasma was assayed using a slightly modified procedure of Goldberg and Spooner (1983) adapted to a microplate reader. All enzyme assays were done in duplicate or triplicate for individual samples. Intra- and interassay variations were between 5 and 7% and 7 and 9%, respectively. Hemoglobin concentrations in whole blood and erythrocyte hemolysates were analyzed using a Sysmex F-300 analyzer (Sysmex, Hamburg, Germany).

Fatty acid determination.

Serum fatty acid components were determined by the method of Müller et al. (1990) on a Fisons 8000 gas chromatograph (Thermoquest, Egelsbach, Germany) using split/splitless injection and flame ionization as detection. The fused silica column (length, 25 m; i.d., 0.25 mm; df, 0.25 µm) coated with chemically bound polyethylene glycol was purchased from Supelco (Sigma). The temperature program started at 50°C (3 min), followed by a rise of 10°C/min to 190°C and a rise of 3°C/min to 230°C (20 min).

Malondialdehyde.

Plasma malondialdehyde was determined as thiobarbituric acid reactive substances (TBARS) using a fluorometric method (Yagi 1984 ). Emission was measured at 548 nm emission wavelength in a fluorescence spectrophotometer (PTI Systems, Wedel, Germany) with an excitation wavelength of 533 nm. Intra- and interassay variations were <4 and <6%, respectively.

Preparation of LDL for oxidation.

LDL was isolated by a short-run ultracentrifugation method based on nonequilibrium density-gradient ultracentrifugation (Kleinveld et al. 1992 ). Centrifugation was carried out in polycarbonate centrifuge tubes by using a Beckman SW-55 Ti rotor at 236,000 x g for 2 h at 15°C (Beckman L7–80 ultracentrifuge, Beckman Instruments, Palo Alto, CA). After centrifugation the LDL-containing fraction was located in the upper half of the tube and collected by aspiration. Purity of the LDL fraction was confirmed by agarose gel electrophoresis (Hydragel, Sebia, Fulda, Germany). EDTA and salts were removed from LDL by gel filtration on Pharmacia PD 10 disposable columns (Amersham Pharmacia Biotech, Freiburg, Germany). The LDL oxidation was assayed on the day of preparation.

LDL oxidation.

The in vitro oxidation of LDL was performed by using a modification of the procedure described by Esterbauer et al. (1989) . The LDL concentration in the PBS solution was determined by measuring total cholesterol with the CHOD-PAP enzymatic test kit (Boehringer) and adjusted for the oxidation assay to 0.1 µmol/L LDL (0.204 mmol/L cholesterol), assuming an LDL molecular weight of 2.5 MDa and a cholesterol concentration of 31.6 g/100 g (Ramos et al. 1995 ). The LDL oxidation process was followed by recording the conjugated diene absorption at 234 nm in a Perkin Elmer spectrophotometer (Lambda 15, Perkin Elmer, Überlingen, Germany). The instrument was equipped with a water-heated autocell holder for simultaneous measurement of six samples. Oxidation was started by adding CuCl₂ to a final concentration of 20 µmol/L. The recording of the 234 nm absorption was started immediately after the addition of CuCl₂ and continued at intervals of 3 min for 4 h. The recorded absorption data were finally processed on a computer. Intra- and interassay variations were <5 and <8%, respectively.

Isolation and carotenoid analysis of plasma lipoproteins.

In a subset of 10 men, carotenoids were analyzed in the major human plasma lipoprotein fractions, i.e., VLDL, LDL and HDL. EDTA plasma with sucrose (15 g/L) and BHT (5 mg/L) added was used for plasma lipoprotein separation by sequential floatation ultracentrifugation adapted to the method of Clevidence and Bieri (1993) . Purity of each lipoprotein fraction was determined by agarose gel electrophoresis (Hydragel, Sebia). Ether/ethanol extraction and analysis of carotenoids by HPLC have been described previously (Müller et al. 1999 ).

Statistics.

Results are given as means ± SD, unless otherwise stated. ANOVA and the Friedman test for nonparametric testing were used to compare the depletion period with the different intervention periods. Comparisons of means were performed using the appropriate ANOVA post-test (Tukey-Kramer or Dunn’s multiple comparison test). Differences were considered to be significant at P < 0.05. Linear regression analysis was performed and the coefficient of correlation (r) was calculated. Statistical calculations were done by using the InStat 2.02 statistical program (Graph Pad Software, San Diego, CA) and StatView 5 (SAS Institute, Cary, NC).

	RESULTS

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Vegetable juice consumption was well tolerated by all men and none had to be excluded from the study due to illness or noncompliance. Plasma carotenoid concentrations have already been published (Müller et al. 1999 ).

Carotenoid concentrations in lipoproteins.

Lycopene increased significantly in VLDL, LDL and HDL after tomato juice consumption. Both - and ß-carotene were elevated in the lipoprotein fractions after carrot juice consumption and after spinach powder consumption, and lutein was increased in lipoproteins after consumption of a spinach preparation (Table 3 ). After carrot juice consumption, -tocopherol (4.3 ± 1.3 vs. 6.6 ± 2.2 µmol/L, P < 0.01) and ubichinone-10 (39 ± 15 vs.72 ± 33 nmol/L, P < 0.05) were significantly reduced in HDL, but not in VLDL and LDL compared with the low carotenoid period.

Consumption of carotenoid-rich vegetable products had only minor effects on the relative distribution of carotenoids among the lipoprotein fractions (VLDL, LDL and HDL; Table 4 ) despite the significant changes in carotenoid concentrations in lipoproteins. In VLDL and LDL, no significant changes in the carotenoid distribution were found except for phytoene, which showed a significant relative reduction in VLDL and an increase in LDL after the spinach period. In HDL, relative reductions of ß-carotene, all-trans- and cis-lycopene, phytofluene and phytoene were observed during various intervention periods (Table 4) .

Tomato juice consumption for 2 wk reduced lipid peroxidation in healthy men (Table 5 ). The initial concentration of conjugated dienes in LDL did not change during the study. In vitro lipoprotein oxidizability was reduced as seen by an increased lag time (18%) at the end of the tomato juice period (P < 0.001). At that point, all-trans- and cis-lycopene and lycopene oxidation products in plasma and LDL had increased significantly. LDL lycopenes were negatively correlated with lag time (all-trans-lycopene, r = -0.421, P = 0.22; cis-lycopene, r = -0.571, P = 0.08; lycopene oxidation products, r = -0.816, P = 0.004). There were no significant correlations between lag time and other carotenoids at the end of the tomato juice period. During the carrot and spinach periods, lag time was not elevated compared with baseline values. At the end of the carrot juice period, strong correlations were found between lag time and - and ß-carotene (r = 0.695, P = 0.026; r = 0.785, P = 0.007, respectively). No correlation between lag time and lutein was observed after the spinach period. During the tomato juice intervention, plasma TBARS were significantly reduced (P < 0.05) by 12% and increased to baseline values at the end of the carrot juice period.

Water-soluble serum antioxidants.

Water-soluble serum antioxidants, uric acid, bilirubin, albumin, glutathione and vitamin C, and the reducing capacity of plasma measured as the FRAP did not change during the vegetable juice intervention (data not shown). Serum uric acid, bilirubin and albumin were within the normal range. Serum vitamin C concentrations ranged from 66.3 ± 12.8 to 73.5 ± 12 µmol/L. There was a strong correlation between uric acid and FRAP (r = 0.898, P < 0.001) but none for bilirubin, albumin, glutathione or vitamin C.

Compared with the end of the depletion period, tomato juice consumption reduced whole blood GSHt (0.72 ± 0.02 vs. 0.87 ± 0.04 mmol/L, P < 0.05) and increased whole blood GSSG (0.09 ± 0.01 vs. 0.07 ± 0.01 mmol/L, P < 0.05). Tomato juice had no effect on plasma or erythrocyte GSHt and GSSG. Carrot juice consumption reduced whole blood GSHt (0.60 ± 0.02 vs. 0.87 ± 0.04 mmol/L, P < 0.05) and whole blood GSSG (0.05 ± 0.002 vs. 0.07 ± 0.01 mmol/L, P < 0.05) as well as erythrocyte GSHt (0.17 ± 0.01 vs. 0.26 ± 0.01 nmol/10⁶ cells, P < 0.05), erythocyte GSSG (0.02 ± 0.001 vs. 0.05 ± 0.002 nmol/10⁶ cells, P < 0.05) and plasma GSSG (1.5 ± 0.1 vs. 2.4 ± 0.3 µmol/L, P < 0.05). Plasma GSHt did not change during the carrot juice period. Spinach consumption reduced whole blood GSHt (0.59 ± 0.03 vs. 0.87 ± 0.04 mmol/L, P < 0.05) and erythrocyte GSHt (0.13 ± 0.004 vs. 0.26 ± 0.01 nmol/10⁶ cells, P < 0.05), whereas other glutathione measurements were unaltered. However, these changes did not correlate with any of the carotenoids and derivatives measured in this study.

No significant changes in erythrocyte GPX and plasma GOR activity were observed. Erythrocyte SOD activity [U/g hemoglobin (Hb)] decreased during the low carotenoid period (from 936 ± 206 on d -14 to 816 ± 16 on d 0 (P < 0.05) and increased significantly after the first week of tomato consumption (d 7, 961 ± 216 vs. d 0, P < 0.05). In contrast, erythrocyte CAT activity (U/g Hb) increased during the low carotenoid period (from 165 ± 21 on d -14 to 184 ± 18 on d 0, P < 0.05), was reduced after the tomato period (d 14, 165 ± 24 vs. d 0, P < 0.05), but was elevated again after spinach consumption (d 42, 221 ± 34 vs. d 0, P < 0.05). All enzyme activities were within the normal ranges (Aebi 1983 , Barnett and King 1995 , Goldberg and Spooner 1983 ).

	DISCUSSION

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It was our aim to evaluate whether a moderate short-term dietary intervention with carotenoid-rich vegetable products affects antioxidant status and LDL oxidation in humans. We further wanted to investigate whether diet-related increases in lipoprotein carotenoids might be responsible for changes in lipoprotein oxidation.

A study design without washout periods between the different vegetable intervention periods was chosen to mimic more closely the dietary behavior of consumers. Although we measured carotenoid concentrations in plasma and lipoproteins, we cannot exclude relevant functional carryover effects of individual carotenoids at the end of each study period. In a comparable study without washout periods between the interventions, the effect of tomato consumption on lymphocyte DNA damage was investigated (Riso et al. 1999 ). Although a significant carryover effect was observed with lycopene plasma concentration, no carryover effect was present when lymphocyte resistance to oxidative stress was measured. This suggests that functional carryover effects may not be the major limitation with this study design. During the intervention, the men had a different carotenoid uptake with the vegetable products. They ingested 330 mL tomato juice (40 mg lycopene), 330 mL carrot juice (15.7 mg -carotene and 22.3 mg ß-carotene) and 10 g spinach powder (11.3 mg lutein and 3.1 mg ß-carotene) daily. It was our objective to study the health effects of reasonable serving sizes of vegetable products and not to compare different carotenoids on an equimolar basis. As a result of this approach, we cannot exclude the possibility that the different doses of carotenoids may have influenced the antioxidant measurements.

In this study, the consumption of carotenoid-rich food increased lipoprotein carotenoids, showing that the major carotenoid in the food is also the major carotenoid appearing in lipoproteins. The intake of carotenoid-rich vegetable products had no effect on relative distribution of carotenoids in VLDL and LDL, except for phytoene. This might be caused by the ingestion of the phytoene-free spinach preparation after 4 wk consumption of phytoene-rich products, which can be seen by the overall decrease of the phytoene concentration in all lipoprotein fractions. Similarly, the relative changes in HDL carotenoid distributions may be explained by the corresponding changes in HDL carotenoid concentrations. The relative distribution pattern of carotenoids among lipoprotein fractions in this study is consistent with previous reports (Clevidence and Bieri 1993 , Paetau et al. 1998 ). Taken together, the data show that the appearance of carotenoids in lipoproteins reflects the uptake from carotenoid-rich food and does not substantially influence the relative distribution pattern, although plasma carotenoid concentrations increased several fold.

Tomato juice consumption for 2 wk significantly increased plasma and lipoprotein lycopene concentrations. Compared with the other two vegetable products, tomato juice was most effective in this study as observed by the reduced lipid peroxidation product TBARS in plasma and the ex vivo oxidation of LDL, with an increased lag time and reduced diene production rates. This suggests that lycopene accounts for the LDL-protecting effect during the tomato juice intervention period because plasma and LDL lycopene increased significantly after tomato juice consumption. However, other compounds from tomato juice may also protect LDL from oxidation, contributing to the prolonged lag time. Nevertheless, in vitro studies have shown that lycopene is a potent antioxidant (DiMascio et al.1989 , Miller et al. 1996 , Woodall et al. 1997 ) that protects LDL (Oshima et al. 1996 , Romanchik et al.1997 ) and other lipid structures from oxidation (Klebanov et al.1998 , Stahl et al. 1998 ). To date, there is little information on the effects of tomato consumption on markers of oxidative stress in vivo. Recently, Sutherland et al. (1999) found that tomato juice consumption (400 mL/d, 4 wk) increased plasma lycopene concentrations but had no effect on lipid peroxidation (lag time, TBARS, LDL lipid peroxides) in 15 cyclosporine-treated patients with stable kidney transplants. In that study, baseline plasma lycopene concentrations in the patients were approximately five times higher than in healthy men. Cyclosporine treatment, which may increase susceptibility of LDL to oxidation (Apanay et al. 1994 ), and the high baseline plasma lycopene concentrations may account for the lack of observed effect in the kidney transplant patients. On the other hand, Agarwal and Rao (1998) and Rao and Agarwal (1998) showed that consumption of tomato products reduced lipid peroxidation and DNA damage in humans as seen in the reduction of serum and LDL TBARS and lymphocyte 8-oxo-2'-deoxyguanosine content. In a previous paper from our intervention trial, we also found reduced DNA damage, measured as single strand breaks in peripheral blood mononuclear cells (PBMC), after tomato juice consumption (Pool-Zobel et al. 1997 ). Whether these protective effects of tomato juice are mediated by lycopene alone or by other antioxidants such as polyphenols and phenolic acids present in tomatoes, could not be determined here because we did not measure other tomato-specific phytochemicals in plasma. However, we determined the major antioxidants in plasma and lipoproteins. Concentrations of vitamin C, -tocopherol and ubichinone-10 in plasma, and -tocopherol and ubichinone-10 in LDL were not affected by any of the dietary interventions in this study and may therefore not contribute to the observed antioxidant effects after tomato juice consumption. The serum fatty acid composition, which also influences the oxidizability of LDL (Thomas et al.1994 , Tsimikas and Reaven 1998 ) did not change during the study. We conclude, therefore, that the serum fatty acid composition does not contribute to the reduced LDL oxidizability after tomato juice consumption.

In contrast to tomato juice, carrot juice had less pronounced effects on antioxidant defense. No changes in plasma TBARS and lag time were found. However, the diene production rate in the ex vivo LDL oxidation assay was reduced significantly after 2 wk of carrot juice consumption. We also found a decrease in oxidative DNA damage in PBMC at this time point (Pool-Zobel et al.1997 ). To date, studies on the antioxidant properties of carotenoids in humans have been designed using ß-carotene as a supplement, which reduced lipoprotein oxidation in healthy volunteers (Levy et al. 1996 , Nyysönen et al.1994 ), children suffering from cystic fibrosis (Winklhofer-Roob et al.1995 ) and coronary artery patients (Mosca et al.1997 ). However, some studies showed that supplementation with ß-carotene in vivo did not inhibit LDL oxidation (Gaziano et al. 1995 , Reaven et al. 1993 ). In these studies, rather high doses (50–100 mg) of ß-carotene were given, and the authors discussed their results as possible prooxidant effects of increased ß-carotene levels at least during the in vitro assay of LDL oxidation. These findings and the results of the ATBC (Hennekens et al. 1996 ) and CARET studies (Omenn et al.1996 ), in which ß-carotene supplementation even increased lung cancer risk, suggest that the consumption of whole diets rich in carotenoids (and other phytochemicals) and not the supplementation with single compounds may be important to prevent LDL oxidation and/or disease development.

The findings of Hininger et al. (1997) support this conclusion. They showed an inhibition of the susceptibility of LDL to oxidation after carotenoid-rich food intake for 2 wk in which carrots, tomatoes, and cabbage + spinach provided an additional daily amount of 10 mg ß-carotene, 10 mg lycopene and 10 mg lutein. Compared with our intervention trial, which increased plasma carotenoids several fold, those authors reported only minor effects on plasma carotenoid concentrations. In the nonsmoking group, which is comparable to our study group, only - and ß-carotene and retinol increased significantly in plasma. They concluded that the protective effect of fruit and vegetables on susceptibility of LDL to oxidation may also be related to biological interactions between carotenoids and other antioxidants and possible synergistic effects. The study of Hininger et al. (1997) and our present and earlier (Pool-Zobel et al. 1997 ) results are indicative of an increased antioxidant capacity in blood lipid fractions and cells as a result of the consumption of vegetable juice. This increase in antioxidant capacity could be explained by the increase in blood carotenoid levels. However, to be able to make such a conclusion, we had to study the effects of the vegetable consumption on endogenous antioxidants and systems involved in the detoxification of reactive oxygen species.

GSH is one of the endogenous antioxidants that plays an important role in the cellular defense against reactive oxygen species. The GSHt and GSSG concentrations measured in this study fit very well with concentrations reported for blood, plasma and erythrocytes of healthy volunteers (Costagliola et al. 1990 , Henning et al. 1991 , Hininger et al. 1997 ). Interestingly, GSHt and GSSG levels in blood, plasma and erythrocytes were decreased significantly at different sampling periods throughout the study compared with GSHt and GSSG concentrations at the end of the carotenoid depletion phase (d 0). However, due to the diversity and the inconsistency of the decreases in GSHt and GSSG concentrations, the relationship with the vegetable intervention remains unclear. The decrease in GSHt concentrations in blood and erythrocytes during the intervention with carrot juice and spinach powder was 25%, which is on the same order of magnitude as the difference in GSHt concentration observed between smokers and nonsmokers (Costagliola et al. 1990 , Hininger et al. 1997 ). It is therefore tempting to speculate about a possible relationship between cellular GSH and the consumption of vegetables, an area of investigation requiring further research.

Other water-soluble antioxidants in serum (albumin, bilirubin, uric acid, ascorbic acid), the antioxidant power (FRAP) and the enzymes GPX and GOR did not change during the study. For the spinach period, our results are comparable to those of Castenmiller et al. (1999) , who studied the effect of carotenoid supplementation and spinach intake on blood antioxidant enzyme activities and FRAP. One group ingested 11.5 mg of lutein daily from spinach products for 3 wk. In that study, consumption of spinach had no effect on FRAP, GPX, SOD, GOR and CAT when comparing wk 0 with wk 3. This is in agreement with our findings except for CAT, for which we found an increase in activity after spinach consumption. Looking at another tomato juice intervention, Böhm and Bitsch (1999) recently showed that tomato juice consumption (5 mg/d lycopene, 2 wk) significantly increased plasma lycopene concentrations. However, total plasma antioxidant activity was not altered significantly by tomato juice intervention. These results and our findings suggest that lipid-soluble carotenoids from vegetable products do not substantially influence water-soluble antioxidants, antioxidant power and antioxidant enzyme activities in healthy humans.

In conclusion, our data show that the appearance of carotenoids in lipoproteins reflects the uptake from carotenoid-rich food and does not substantially influence the relative carotenoid distribution pattern in lipoproteins, although plasma carotenoid concentrations increased several fold. Tomato juice, but not carrot juice or spinach powder consumption reduced LDL oxidation in healthy men. Our findings also suggest that the consumption of carotenoid-rich vegetables does not influence water-soluble antioxidants and antioxidant power and may have only minor effects on antioxidant enzyme activities in healthy men.

	ACKNOWLEDGMENTS

 
The authors thank T. Gadau, U. Stadler-Prayle and M. Werner for their excellent technical assistance, S. Mittenzwei for TBARS determinations, J. Hegele for Vitamin C measurements and the volunteers from the Research Center Karlsruhe for taking part in this study.

	FOOTNOTES

 
¹ Supported by Schoenenberger Pflanzensaftwerke, Magstadt, Germany and Völpel, Königmoos, Germany who kindly supplied the vegetable products.

³ Deceased.

⁴ Abbreviations used: CAT, catalase activity; FRAP, ferric reducing ability of plasma; GOR, glutathione reductase; GPX, glutathione peroxidase; GSHt, total glutathione; GSSG, oxidized glutathione; Hb, hemoglobin; PBMC, peripheral blood mononuclear cells; SOD, superoxide dismutase; TBARS, thiobarbituric acid reactive substances; TEAC, Trolox equivalent antioxidant capacity.

Manuscript received December 28, 1999. Initial review completed March 2, 2000. Revision accepted April 12, 2000.

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