Dietary Supplementation with a Natural Carotenoid Mixture Decreases Oxidative Stress

ORIGINAL COMMUNICATION Dietary supplementation with a natural carotenoid mixture decreases oxidative stress

S Kiokias1 and MH Gordon1*

1Hugh Sinclair Unit of Human Nutrition, School of Food Biosciences, The University of Reading, Whiteknights, Reading, UK

Objective: To determine whether dietary supplementation with a natural carotenoid mixture counteracts the enhancement of oxidative stress induced by consumption of fish oil. Design: A randomised double-blind crossover dietary intervention. Setting: Hugh Sinclair Unit of Human Nutrition, School of Food Biosciences, The University of Reading, Whiteknights PO Box 226, Reading RG6 6AP, UK. Subjects and intervention: A total of 32 free-living healthy nonsmoking volunteers were recruited by posters and e-mails in The University of Reading. One volunteer withdrew during the study. The volunteers consumed a daily supplement comprising capsules containing fish oil (4 Â 1 g) or fish oil (4 Â 1 g) containing a natural carotenoid mixture (4 Â 7.6 mg) for 3 weeks in a randomised crossover design separated by a 12 week washout phase. The carotenoid mixture provided a daily intake of b-carotene (6.0 mg), a-carotene (1.4 mg), lycopene (4.5 mg), bixin (11.7 mg), lutein (4.4 mg) and paprika carotenoids (2.2 mg). Blood and urine samples were collected on days 0 and 21 of each dietary period. Results: The carotenoid mixture reduced the fall in ex vivo oxidative stability of low-density lipoprotein (LDL) induced by the fish oil (P ¼ 0.045) and it reduced the extent of DNA damage assessed by the concentration of 8-hydroxy-20-deoxyguanosine in urine (P ¼ 0.005). There was no effect on the oxidative stability of plasma ex vivo assessed by the oxygen radical absorbance capacity test. b-Carotene, a-carotene, lycopene and lutein were increased in the plasma of subjects consuming the carotenoid mixture. Plasma triglyceride levels were reduced significantly more than the reduction for the fish oil control (P ¼ 0.035), but total cholesterol, HDL and LDL levels were not significantly changed by the consumption of the carotenoid mixture. Conclusions: Consumption of the natural carotenoid mixture lowered the increase in oxidative stress induced by the fish oil as assessed by ex vivo oxidative stability of LDL and DNA degradation product in urine. The carotenoid mixture also enhanced the plasma triglyceride-lowering effect of the fish oil. Sponsorship: The study was supported by funding from the Greek Studentship Foundation and from Unilever Bestfoods plc. Carotenoids were contributed by Overseal Foods plc. European Journal of Clinical Nutrition (2003) 57, 1135–1140. doi:10.1038/sj.ejcn.1601655

Keywords: carotenoids; bioavailability; antioxidant; oxidative stress

Introduction in vivo (Krinsky, 2001). A body of scientific evidence links the Carotenoids scavenge free radicals and thereby act as antioxidant character of carotenoids with their subsequent antioxidants under reduced oxygen conditions in vitro and beneficial effects on chronic diseases including cardiovascular and photosensitivity diseases, cataracts and age-related degeneration (Rousseau et al, 1992; Riso et al, 1999). The *Correspondence: MH Gordon, Hugh Sinclair Unit of Human Nutrition, evidence for the beneficial effects of lycopene in reducing School of Food Biosciences, The University of Reading, Whiteknights, the risk of chronic diseases such as cancer and cardiovascular P.O.Box 226, Reading, RG6 6AP, UK. diseases is particularly strong (Rao & Agarwal, 2000). There is E-mail: [email protected] Guarantor: MH Gordon. also evidence that the xanthophylls lutein and zeaxanthin Contributors: SK performed the practical work and analysed the data may have a protective role in delaying the onset of chronic from the study, and contributed to the preparation of the manuscript. disease (Mares-Perlman et al, 2002). However, three large- MHG contributed to the experimental design, data analysis and scale clinical trials, namely the Alpha Tocopherol Beta- preparation of the manuscript. Received 12 July 2002; revised 6 September 2002; Carotene (ATBC) trial, the b-Carotene and Retinol Efficacy accepted 12 September 2002 Trial (CARET) and the Physician’s Health Study, concluded Dietary supplementation with natural carotenoid mixture S Kiokias and MH Gordon 1136 that dietary supplementation with beta-carotene did not day diet diary to give more accurate information on their reduce the risk of chronic diseases (Pryor et al, 2000). These dietary intake. One male withdrew from the study after 3 trials indicate that dietary supplementation with high levels weeks. of an individual carotenoid may lead to adverse effects in Ethical permission for the execution of this trial was given individuals subject to a high level of oxidative stress. It may by the Ethics and Research Committee of the University of be deduced that individual carotenoids may accumulate to Reading. Each individual gave their written consent. unacceptable levels in selected tissues, where they may enhance oxidative deterioration due to their high concentration. Dietary supplementation with individual carote- Experimental design noids may consequently provide an unacceptable risk. A The study was a randomised double-blind crossover dietary wide range of carotenoid-rich extracts are used by the food intervention of 18 weeks duration. Participants were ran- industry as colouring materials in foods, and dietary domly assigned to consume a daily supplement of 4 Â 1g supplementation with a mixture of carotenoids of varying capsules of fish oil or 4 Â 1 g capsules each containing fish oil polarity and properties is less likely to allow the develop- plus 24.6 mg tomato extract (Lyc-O-mato), 6.3 mg palm oil ment of adverse effects. carotene extract, 2.0 mg marigold extract, 3.7 mg paprika Most intervention studies investigating the effects of extract and 3.7 mg bixin for 3 weeks. The total active carotenoids on biomarkers of oxidative stress have investi- carotenoid content of the test supplement was 7.6 mg per gated the effects of individual carotenoids. Some studies capsule, and the fish oil contained 65% EPA and DHA. Lyc- have found that carotenoids increase the oxidative stability O-mato was purchased from Forum Products Ltd, Redhill, UK of low-density lipoprotein (LDL) (Agarwal & Rao, 1998; and the other carotenoid-rich extracts were supplied by Dugas et al, 1999), but other studies have found no effect on Overseal Foods, Overseal, UK. The supplements were sup- this parameter (Reaven et al, 1993; Gaziano et al, 1995). Lin plied in opaque brown capsules prepared by RP Scherer, et al (1998) found an increase in the oxidative stability of Swindon, UK. After a 12-week washout period, individuals LDL following supplementation with a b-carotene-rich were crossed over to receive the opposite supplementation carotenoid mixture. Consumption of a mixture of b-carotene regimen. Fasting blood and urine samples were collected on with vitamin E and vitamin C was found to reduce base days 0 and 21 of each intervention period. damage in lymphocyte DNA (Duthie et al, 1996). The aim of this study was to investigate the effects of a mixture of Laboratory measurements. Analyses were performed on natural carotenoids on biomarkers of oxidative stress. three replicate samples for HPLC, ORAC, LDL lag phase, and In order to make the study more sensitive, it was plasma lipid assays, or on two replicate samples for 8- considered desirable to use subjects whose level of oxidative hydroxy-20-deoxyguanosine (8-OHdG) determination. All stress was towards the higher end of the normal range. Since blood samples were centrifuged at 1600 Â g for 10 min and dietary supplementation with b-carotene in smokers has the plasma stored at À801C, until required for analysis. For been shown to have adverse effects, it was not considered HDL-chol analysis, a subsample of plasma was precipitated ethical to use these subjects. Instead, the oxidative challenge with dextran sulphate and magnesium chloride in order to of the background diet was increased by including fish oil in remove apolipoprotein B-containing lipoproteins (McNa- the background diet of both the control and test groups. Fish mara et al, 1994), and the supernatant fraction was stored oil is rich in the polyunsaturated fatty acids eicosapentae- at À801C. noic acid (EPA) and docosahexaenoic acid (DHA), and several LDL-chol was determined using the Friedewald formula studies have shown that fish oil increases oxidative stress (Friedewald & Levy, 1972). Plasma samples were analysed for when assessed by the oxidative stability of LDL (Wander et al, TG, total-chol and HDL-chol using the Monarch Automatic 1996; Sorensen et al, 1998; Foulon et al, 1999; Leigh-Firbank Analyser (Instrumentation Laboratories Ltd, Warrington, et al, 2002). Ches., UK) as described by Minihane et al (2000). Platelets were extracted from the whole blood according to the method of Indu and Ghafoorunissa (1992) (300 Â g for Methods 18 min followed by 1700 Â g for 10 min), and stored at À801C Subjects for platelet phospholipid fatty acid analysis. Preparation and A total of 32 free-living healthy nonsmoking volunteers were analysis of fatty acid methyl esters was according to Leigh- recruited by posters and emails in The University of Reading. Firbank et al (2002). The platelet-poor plasma was used for The subjects comprised 17 males and 15 females aged the isolation of LDL as described by Leigh-Firbank et al 31.7711.3 y (mean7s.d.), of body mass index (BMI) (2002). Assessment of the oxidative stability of LDL ex vivo 22.473.0 kg/m2. Subjects were recruited on the basis of a was performed according to Esterbauer et al (1989). Lipid health and lifestyle questionnaire and a screening blood peroxidation in LDL (50 mg LDL protein/ml) was induced by

sample. On the basis of physical examination and standard CuSO4 (0.005 mM), and the absorbance at 234 nm was haematological tests, all volunteers were considered healthy. monitored. The lag phase was used as the primary index of Subjects considered for inclusion were asked to complete a 3- the susceptibility of the LDL particle to oxidation, with the

European Journal of Clinical Nutrition Dietary supplementation with natural carotenoid mixture S Kiokias and MH Gordon 1137 end of the lag phase taken as the intercept of the tangents 39:57:4 for bixin, paprika carotenoids and lutein. The flow drawn through the lag and propagation phases of the rate was 1m l/ min and the injection volume was 100 ml. absorbance plot against time. The oxygen radical absorbance capacity (ORAC) assay was applied to plasma as described by Cao and Prior (1999). Statistical analysis Urine samples were analysed for creatinine by the Unit of A paired Student’s t-test was used to identify significant Biochemistry at the Royal Berkshire Hospital by an enzy- differences between treatments or from baseline to post- matic assay (Ortho Clinical Diagnostics, Amersham, UK). supplementation. A P-value o0.05 was considered statisti- The 8-OHdG content of the urine was determined by a cally significant. Prior to the t-test, data were checked for competitive ELIZA assay with a kit purchased from the Japan normality with the Shapiro–Wilk test. Genstat 5.0 (Lawes Institute for Control of Ageing, Fukuroi City, Japan, as Agricultural Trust, Oxford, UK) was used for the data described by Erhola et al (1997). analysis.

Analysis of retinol, tocopherols and carotenoids Results Two different procedures were used for the less polar and The compliance of the participants was assessed by the more polar components. Plasma (200 ml), distilled water change in EPA and DHA in the platelet phospholipids (200 ml) and ethanol (400 ml for protein precipitation) con- (Table 1). The increase in EPA and DHA was highly taining 20 ppm of b-apo-carotenal as internal standards were significant (P ¼ 0.001 for both EPA and DHA for both pipetted into a 10 ml glass tube and mixed in a vortex for groups). The increase in both fatty acids was not significantly 10 s. Hexane (for retinol, tocopherols and carotenes) or different for the control and test groups (P ¼ 0.90 and 0.65 dichloromethane (for lutein, paprika and annatto pigments) for EPA and DHA, respectively). Analysis of the diet diaries (1.5 ml) was added and the tubes were first shaken (IKA VXR indicated that there was no significant change in the dietary shaker, Staufen, Germany) for 10 min and then centrifuged intake of PUFA, SFA, selenium, vitamin A or vitamin E during at 3000 rpm for another 10 min. The solvent layer was the trial, but the level of vitamin C showed a tendency to separated and the solvent evaporated. The residue was increase from period 1 to period 2, although the change from dissolved in acetonitrile/tetrahydofuran/methanol (6:2:2) 146.6772.2 to 159.6771.2 mg/day did not reach statistical (for retinol, tocopherols and carotenes) or acetonitrile/2- significance (P ¼ 0.07). This change corresponded to the propanol (4:6) (for lutein, paprika and annatto pigments) change in season during the study, and can be ascribed to the before application onto the HPLC column. increase in fruit consumption during the summer months. A Hewlett-Packard 1050 liquid chromatograph, equipped The group supplemented with fish oil containing the with diode array detector and Chemstation 7, was used for carotenoid mixture showed a clear increase in the plasma the identification and quantification of the carotenoids. The levels of b- and a-carotenes, lycopene and lutein (Po0.05). separation was done on a reversed-phase column (Spherisorb After 3 weeks supplementation, plasma concentration of b- C-18, ODS-1, 250 Â 4.6 mm2 i.d.,5 mm particle size), pro- and a-carotenes had been increased by 0.3270.21 and tected by a guard column (ODS-1), with a mixture of 0.0170.02 mg/l, respectively, whereas lycopene and lutein acetonitrile/methanol/tetrahydrofuran/ammonium acetate had increased by 0.1570.16 and 0.0470.06 mg lÀ1respec- (1% w/v) in the ratio 68:22:7:3 for tocopherols, retinol and respectively (Figure 1). Bixin and the paprika carotenoids, carotenes or acetonitirile/2-propanol/water in the ratio capsanthin and capsorubin, were not detected in the plasma.

Table 1 Baseline characteristics of the study group and the responsiveness to fish oil (control) or fish oil plus carotenoids (test group) intervention for 3 weeksa

Control group Test group

Baseline Change P Baseline Change P

Fasting Total-chol (mmol/l) 4.8670.08 À0.0970.26 0.09 4.7870.07 À0.1070.28 0.07 TG (mmol/l) 0.8570.22 À0.1370.22 0.002 0.8970.29 À0.2870.24 0.001 HDL-chol (mmol/l) 1.4570.34 À0.0470.12 0.09 1.4470.33 +0.0170.11 0.29 LDL-chol (mmol/l) 3.0570.67 0.0070.24 0.96 2.9770.71 À0.0470.41 0.59 Fatty acids (mol/100 mol) 20:5 1.7970.70 +2.5671.57 0.001 1.8870.76 +2.6171.07 0.001 22:6 6.0972.27 +1.4871.32 0.001 6.1572.36 +1.4572.05 0.001 aMean values with standard deviation for 31 subjects who completed the study.

European Journal of Clinical Nutrition Dietary supplementation with natural carotenoid mixture S Kiokias and MH Gordon 1138 0.8 baseline before supplementation for the control and test groups, respectively, with a change of +0.4375.59 and 0.6 3-weeks +1.3679.12 mM for the two groups following supplementation. 0.4 The plasma oxidative stability assessed by the ORAC method did not change significantly from baseline for the 0.2 two groups following the supplementation (Table 2), and the changes were not significantly different from each other (P ¼ 0.12). plasma concentration (mg/l) 0.0 beta-carotene lycopene lutein alpha-carotene DNA damage assessed by the ratio of 8-OHdG:creatinine (Table 2) increased significantly from baseline following Figure 1 Effect of dietary supplementation with the carotenoid supplementation in the control group, but there was a mixture on plasma carotenoid levels (mg/l). nonsignificant decrease in the ratio for the test group (P ¼ 0.15). The change in the ratio because of supplementation was significantly different for the two groups (P ¼ 0.005). No significant change in plasma carotenoid levels was observed in the control group. There were no significant changes in plasma levels of retinol and a-tocopherol in either group during the supplementation periods. Discussion The baseline plasma lipid levels and changes following the Dietary compliance of the participants was confirmed by the intervention are shown in Table 1. significant increase in EPA and DHA in the platelet Statistical analysis showed that after 3 weeks of dietary phospholipids of both groups. The increase was similar for supplementation, total cholesterol, LDL- and HDL-choles- both groups because of the fish oil consumed by both the terol levels did not change significantly following supple- control and test groups. The carotenoids consumed by the mentation, and these parameters were not significantly test group had no effect on the change in platelet fatty acid different between the control and test groups. composition. The fish oil consumed provided 0.82 g/day of However, plasma triglycerides (TG) were significantly EPA and 0.46 g/day of DHA, but the change in platelet EPA decreased after 3 weeks of each dietary treatment when was only 1.77 times greater for EPA than for DHA, when the compared with baseline levels. Carotenoid supplementation changes are expressed in mol/100 mol fatty acids. When reduced the final TG levels by 31.5% from baseline, whereas calculated as percentage increase in the baseline level of the the control group showed a reduction of 15.3%. This change individual fatty acids, the increase was 144% for EPA and was significantly greater for the test group (P ¼ 0.035). 24% for DHA. The increase in the plasma levels of b- and Dietary supplementation with fish oil containing carote- a-carotenes, lycopene and lutein confirm previous data that noids caused a 20.4% reduction in LDL lag phase before these carotenoids are well absorbed. In the case of lutein, the initiation of conjugated diene formation ex vivo compared carotenoid was present in the supplement as a mixture of with 26.6% for the control group who consumed a supple- free lutein and lutein esters. ment containing only fish oil (Table 2). The change in lag The absence of bixin, capsanthin and capsorubin is phase was significantly less for the test group than for the because of the rapid clearance of the carotenoids from control (P ¼ 0.045). There was no significant change in plasma, rather than lack of absorption (Levy et al, 1997; Etoh plasma a-tocopherol levels because of the supplementation et al, 2000). The rapid clearance of polar carotenoids is in for either group (P ¼ 0.66 for the control and 0.40 for the test contrast to the plasma response to the less polar carotenoids, group). The levels were 29.2077.34 and 29.4679.17 mM which are located in LDL particles.

Table 2 Changes in biomarkers of oxidative stress during the study (mean values with standard deviation)

Control group Test group

Baseline Changea P Baseline Changea P

LDL lag phase (min) 72.4715.3 À19.3714.4 0.001 69.0717.3 À14.1713.1b 0.001 ORAC value (mM) 5957142 +1.9789 0.9 5977128 +277111 0.19 8-OHdG/creatinine (ng/mg) 21.6712.6 +4.277.6 0.004 24.0713.3 À1.674.9b 0.15

aAfter 3 weeks of supplementation. bSignificantly different from control group (Po0.05).

European Journal of Clinical Nutrition Dietary supplementation with natural carotenoid mixture S Kiokias and MH Gordon 1139 Fasting plasma TG levels were significantly decreased after appears to be because of the mixture of a- and b-carotenes, 3 weeks of each dietary treatment when compared with lycopene and lutein that are known to be transported in LDL baseline levels. This effect is mainly associated with the well- (Esterbauer et al, 1992). recognised effect of fish oil (n-3 fatty acids) on lowering The measurement of urinary 8-OHdG was selected as an plasma TG (Leigh-Firbank et al, 2002). It was found that the appropriate method to evaluate DNA degradation. The carotenoid-supplemented group reduced the final TG levels values were quoted as a ratio of 8-OHdG:creatinine to allow by 31.5%, whereas the control only gave a reduction of for the fact that a single urine sample was collected rather 15.3%. This suggests a possible interaction between the than a 24 h collection. It was observed that in the control effects of the carotenoid mixture and fish oil in lowering group, after 3 weeks of fish oil supplementation, the average plasma TG. The observation that a combination of carote- level of urinary 8-OHdG was significantly increased in noids and fish oil is more effective than fish oil alone in comparison with the baseline. The 19% increase in urinary lowering plasma TG has not been reported in any previous 8-OHdG in the control group following fish oil supplemen- study and further studies are required to confirm this effect. tation is a measure of the increase in oxidative stress, and can It was found that, in both the groups, there was no be compared with the difference between smokers and significant change of the mean ORAC values (P40.05) nonsmokers reported by Loft et al (1992), who found that after 3 weeks of supplementation, in comparison with the urinary 8-OHdG was 50% higher in smokers than in baseline levels. This probably reflects the fact that the nonsmokers. test is most sensitive to antioxidants in the aqueous However, in the carotenoid supplemented group, the phase, since the radical initiator and the test protein average 8-OHdG level was not changed significantly b-phycoerythrin are both in the aqueous phase. The lack of from the baseline value. The average increase from baseline a significant change in the control group between the ORAC was +4.2 ng 8-OHdG/mg creatinine for the control value at baseline and after 3 weeks supplementation with group, whereas a decrease of À1.6 ng 8-OHdG/mg creatinine fish oil confirms the fact that this test is not sensitive to was observed for the carotenoid group. This group responded changes in the lipid phase. However, the high variability of significantly differently from the control group (P ¼ 0.005), the analysis, and the low statistical power of the study suggesting that the carotenoid mixture dispersed in contributed to the lack of significance for the small change fish oil was effective at off-setting the increased DNA observed on supplementation with the carotenoid-rich test damage caused by fish oil consumption. Beneficial effects sample. of carotenoids on decreasing DNA degradation have Copper-catalysed oxidation of LDL ex vivo is sensitive to also been observed for lycopene and carotenes in other changes in the lipid phase, and the lag time was significantly trials (Duthie et al, 1996; Pool-Zobel et al, 1997; Smith et al, shorter for both groups after 3 weeks of dietary supplemen- 1999), whereas some trials (Agarwal & Rao, 1998; Collins tation as compared with baseline values (control: 26.6% et al, 1998) have found no effect. Most of the trials where reduction; carotenoid test group: 20.4% reduction from effects have been observed have involved supplementation baseline levels). This is expected due to the fish oil with higher levels of lycopene, but the current study has consumed, which was rich in the highly unsaturated fatty clearly shown that mixtures of carotenoids containing only acids EPA and DHA, which were incorporated into LDL 4.5 mg lycopene per day can be effective in decreasing DNA phospholipids. However, the average decrease in the lag damage. phase from baseline in the carotenoid test group was The significance of urinary 8-OHdG has been discussed by significantly lower (P ¼ 0.045) than in the control group, Cooke et al (2000). Excretion of 8-OHdG is increased in reflecting thereby a protective effect of the carotenoid smokers or individuals with malignant diseases, but it was mixture on oxidative modification of LDL. In the literature, noted that increased excretion does not necessarily indicate there are several reports of the antioxidant activity of increased DNA damage but may indicate a lowering of tissue carotenoids (in particular b-carotene) against LDL oxidation, levels of 8-OHdG. There have been no studies aimed at but the results are mixed. Lin et al (1998) reported decreased correlating reduction in urinary 8-OHdG excretion with oxidation of LDL ex vivo in healthy women non-smokers, reduced incidence of disease. who consumed a supplement (B11 mg/day) that was rich in In conclusion, our study has shown that daily supplemen- b-carotene, but which also contained a-carotene, lycopene, tation with a natural carotenoid mixture (30 mg) in subjects zeaxanthin and lutein, Agarwal and Rao (1998) found consuming fish oil can significantly increase the lag increased resistance to oxidation of LDL following supple- time before oxidative deterioration ex vivo, and can mentation with lycopene at levels above 39 mg/day. How- decrease the extent of DNA damage assessed by urinary 8- ever, Gaziano et al (1995); and Reaven et al (1993) found no OHdG. The beneficial effects were probably because of the beneficial effects following supplementation with relatively less polar carotenoids (a- and b-carotenes, lycopene and high levels of b-carotene. Since bixin and the polar paprika lutein), since the more polar carotenoids (bixin and the polar carotenoids, capsanthin and capsorubin were not detected in paprika carotenoids, capsanthin and capsorubin) were not the plasma, it can be concluded that these carotenoids were detected in the plasma of subjects consuming the not present in the LDL particles. The antioxidant effect supplement.

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