Introduction

Carotenoids scavenge free radicals and thereby act as antioxidants under reduced oxygen conditions in vitro and in vivo (Krinsky, 2001). A body of scientific evidence links the antioxidant character of carotenoids with their subsequent beneficial effects on chronic diseases including cardiovascular and photosensitivity diseases, cataracts and age-related degeneration (Rousseau et al, 1992; Riso et al, 1999). The evidence for the beneficial effects of lycopene in reducing the risk of chronic diseases such as cancer and cardiovascular diseases is particularly strong (Rao & Agarwal, 2000). There is also evidence that the xanthophylls lutein and zeaxanthin may have a protective role in delaying the onset of chronic disease (Mares-Perlman et al, 2002). However, three large-scale clinical trials, namely the Alpha Tocopherol Beta- Carotene (ATBC) trial, the -Carotene and Retinol Efficacy Trial (CARET) and the Physician's Health Study, concluded that dietary supplementation with beta-carotene did not reduce the risk of chronic diseases (Pryor et al, 2000). These trials indicate that dietary supplementation with high levels of an individual carotenoid may lead to adverse effects in individuals subject to a high level of oxidative stress. It may be deduced that individual carotenoids may accumulate to unacceptable levels in selected tissues, where they may enhance oxidative deterioration due to their high concentration. Dietary supplementation with individual carotenoids may consequently provide an unacceptable risk. A wide range of carotenoid-rich extracts are used by the food industry as colouring materials in foods, and dietary supplementation with a mixture of carotenoids of varying polarity and properties is less likely to allow the development of adverse effects.

Most intervention studies investigating the effects of carotenoids on biomarkers of oxidative stress have investigated the effects of individual carotenoids. Some studies have found that carotenoids increase the oxidative stability of low-density lipoprotein (LDL) (Agarwal & Rao, 1998; Dugas et al, 1999), but other studies have found no effect on this parameter (Reaven et al, 1993; Gaziano et al, 1995). Lin et al (1998) found an increase in the oxidative stability of LDL following supplementation with a -carotene-rich carotenoid mixture. Consumption of a mixture of -carotene with vitamin E and vitamin C was found to reduce base damage in lymphocyte DNA (Duthie et al, 1996). The aim of this study was to investigate the effects of a mixture of natural carotenoids on biomarkers of oxidative stress.

In order to make the study more sensitive, it was considered desirable to use subjects whose level of oxidative stress was towards the higher end of the normal range. Since dietary supplementation with -carotene in smokers has been shown to have adverse effects, it was not considered ethical to use these subjects. Instead, the oxidative challenge of the background diet was increased by including fish oil in the background diet of both the control and test groups. Fish oil is rich in the polyunsaturated fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), and several studies have shown that fish oil increases oxidative stress when assessed by the oxidative stability of LDL (Wander et al, 1996; Sorensen et al, 1998; Foulon et al, 1999; Leigh-Firbank et al, 2002).

Methods

Subjects

A total of 32 free-living healthy nonsmoking volunteers were recruited by posters and emails in The University of Reading. The subjects comprised 17 males and 15 females aged 31.711.3 y (means.d.), of body mass index (BMI) 22.43.0 kg/m². Subjects were recruited on the basis of a health and lifestyle questionnaire and a screening blood sample. On the basis of physical examination and standard haematological tests, all volunteers were considered healthy. Subjects considered for inclusion were asked to complete a 3-day diet diary to give more accurate information on their dietary intake. One male withdrew from the study after 3 weeks.

Ethical permission for the execution of this trial was given by the Ethics and Research Committee of the University of Reading. Each individual gave their written consent.

Experimental design

The study was a randomised double-blind crossover dietary intervention of 18 weeks duration. Participants were randomly assigned to consume a daily supplement of 4 1 g capsules of fish oil or 4 1 g capsules each containing fish oil plus 24.6 mg tomato extract (Lyc-O-mato), 6.3 mg palm oil carotene extract, 2.0 mg marigold extract, 3.7 mg paprika extract and 3.7 mg bixin for 3 weeks. The total active carotenoid content of the test supplement was 7.6 mg per capsule, and the fish oil contained 65% EPA and DHA. Lyc-O-mato was purchased from Forum Products Ltd, Redhill, UK and the other carotenoid-rich extracts were supplied by Overseal Foods, Overseal, UK. The supplements were supplied in opaque brown capsules prepared by RP Scherer, Swindon, UK. After a 12-week washout period, individuals were crossed over to receive the opposite supplementation regimen. Fasting blood and urine samples were collected on days 0 and 21 of each intervention period.

Laboratory measurements

Analyses were performed on three replicate samples for HPLC, ORAC, LDL lag phase, and plasma lipid assays, or on two replicate samples for 8-hydroxy-2'-deoxyguanosine (8-OHdG) determination. All blood samples were centrifuged at 1600 g for 10 min and the plasma stored at -80°C, until required for analysis. For HDL-chol analysis, a subsample of plasma was precipitated with dextran sulphate and magnesium chloride in order to remove apolipoprotein B-containing lipoproteins (McNamara et al, 1994), and the supernatant fraction was stored at -80°C

LDL-chol was determined using the Friedewald formula (Friedewald & Levy, 1972). Plasma samples were analysed for TG, total-chol and HDL-chol using the Monarch Automatic Analyser (Instrumentation Laboratories Ltd, Warrington, 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 g for 18 min followed by 1700 g for 10 min), and stored at -80°C for platelet phospholipid fatty acid analysis. Preparation and analysis of fatty acid methyl esters was according to Leigh-Firbank et al (2002). The platelet-poor plasma was used for the isolation of LDL as described by Leigh-Firbank et al (2002). Assessment of the oxidative stability of LDL ex vivo was performed according to Esterbauer et al (1989). Lipid peroxidation in LDL (50 g LDL protein/ml) was induced by CuSO₄ (0.005 mM), and the absorbance at 234 nm was monitored. The lag phase was used as the primary index of the susceptibility of the LDL particle to oxidation, with the end of the lag phase taken as the intercept of the tangents drawn through the lag and propagation phases of the absorbance plot against time

The oxygen radical absorbance capacity (ORAC) assay was applied to plasma as described by Cao and Prior (1999). Urine samples were analysed for creatinine by the Unit of Biochemistry at the Royal Berkshire Hospital by an enzymatic assay (Ortho Clinical Diagnostics, Amersham, UK). The 8-OHdG content of the urine was determined by a competitive ELIZA assay with a kit purchased from the Japan Institute for Control of Ageing, Fukuroi City, Japan, as described by Erhola et al (1997).

Analysis of retinol, tocopherols and carotenoids

Two different procedures were used for the less polar and more polar components. Plasma (200 l), distilled water (200 l) and ethanol (400 l for protein precipitation) containing 20 ppm of -apo-carotenal as internal standards were pipetted into a 10 ml glass tube and mixed in a vortex for 10 s. Hexane (for retinol, tocopherols and carotenes) or dichloromethane (for lutein, paprika and annatto pigments) (1.5 ml) was added and the tubes were first shaken (IKA VXR shaker, Staufen, Germany) for 10 min and then centrifuged at 3000 rpm for another 10 min. The solvent layer was separated and the solvent evaporated. The residue was dissolved in acetonitrile/tetrahydofuran/methanol (6:2:2) (for retinol, tocopherols and carotenes) or acetonitrile/2-propanol (4:6) (for lutein, paprika and annatto pigments) before application onto the HPLC column.

A Hewlett-Packard 1050 liquid chromatograph, equipped with diode array detector and Chemstation 7, was used for the identification and quantification of the carotenoids. The separation was done on a reversed-phase column (Spherisorb C-18, ODS-1, 250 4.6 mm² i.d.,5 m particle size), protected by a guard column (ODS-1), with a mixture of acetonitrile/methanol/tetrahydrofuran/ammonium acetate (1% w/v) in the ratio 68:22:7:3 for tocopherols, retinol and carotenes or acetonitirile/2-propanol/water in the ratio 39:57:4 for bixin, paprika carotenoids and lutein. The flow rate was 1m l/ min and the injection volume was 100 l.

Statistical analysis

A paired Student's t-test was used to identify significant differences between treatments or from baseline to postsupplementation. A P-value <0.05 was considered statistically significant. Prior to the t-test, data were checked for normality with the Shapiro–Wilk test. Genstat 5.0 (Lawes Agricultural Trust, Oxford, UK) was used for the data analysis.

Results

The compliance of the participants was assessed by the change in EPA and DHA in the platelet phospholipids (Table 1). The increase in EPA and DHA was highly significant (P=0.001 for both EPA and DHA for both groups). The increase in both fatty acids was not significantly different for the control and test groups (P=0.90 and 0.65 for EPA and DHA, respectively). Analysis of the diet diaries indicated that there was no significant change in the dietary intake of PUFA, SFA, selenium, vitamin A or vitamin E during the trial, but the level of vitamin C showed a tendency to increase from period 1 to period 2, although the change from 146.672.2 to 159.671.2 mg/day did not reach statistical significance (P=0.07). This change corresponded to the change in season during the study, and can be ascribed to the increase in fruit consumption during the summer months.

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 weeks.

Full table

The group supplemented with fish oil containing the carotenoid mixture showed a clear increase in the plasma levels of - and -carotenes, lycopene and lutein (P<0.05). After 3 weeks supplementation, plasma concentration of - and -carotenes had been increased by 0.320.21 and 0.010.02 mg/l, respectively, whereas lycopene and lutein had increased by 0.150.16 and 0.040.06 mg l^-1 respectively (Figure 1). Bixin and the paprika carotenoids, capsanthin and capsorubin, were not detected in the plasma. No significant change in plasma carotenoid levels was observed in the control group. There were no significant changes in plasma levels of retinol and -tocopherol in either group during the supplementation periods.

Figure 1.

Effect of dietary supplementation with the carotenoid mixture on plasma carotenoid levels (mg/l).

Full figure and legend (29K)

The baseline plasma lipid levels and changes following the intervention are shown in Table 1.

Statistical analysis showed that after 3 weeks of dietary supplementation, total cholesterol, LDL- and HDL-cholesterol levels did not change significantly following supplementation, and these parameters were not significantly different between the control and test groups.

However, plasma triglycerides (TG) were significantly decreased after 3 weeks of each dietary treatment when compared with baseline levels. Carotenoid supplementation reduced the final TG levels by 31.5% from baseline, whereas the control group showed a reduction of 15.3%. This change was significantly greater for the test group (P=0.035).

Dietary supplementation with fish oil containing carotenoids caused a 20.4% reduction in LDL lag phase before initiation of conjugated diene formation ex vivo compared with 26.6% for the control group who consumed a supplement containing only fish oil (Table 2). The change in lag phase was significantly less for the test group than for the control (P=0.045). There was no significant change in plasma -tocopherol levels because of the supplementation for either group (P=0.66 for the control and 0.40 for the test group). The levels were 29.207.34 and 29.469.17 M before supplementation for the control and test groups, respectively, with a change of +0.435.59 and +1.369.12 M for the two groups following supplementation.

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

Full table

The plasma oxidative stability assessed by the ORAC method did not change significantly from baseline for the two groups following the supplementation (Table 2), and the changes were not significantly different from each other (P=0.12).

DNA damage assessed by the ratio of 8-OHdG:creatinine (Table 2) increased significantly from baseline following supplementation in the control group, but there was a nonsignificant decrease in the ratio for the test group (P=0.15). The change in the ratio because of supplementation was significantly different for the two groups (P=0.005).

Discussion

Dietary compliance of the participants was confirmed by the significant increase in EPA and DHA in the platelet phospholipids of both groups. The increase was similar for both groups because of the fish oil consumed by both the control and test groups. The carotenoids consumed by the test group had no effect on the change in platelet fatty acid composition. The fish oil consumed provided 0.82 g/day of EPA and 0.46 g/day of DHA, but the change in platelet EPA was only 1.77 times greater for EPA than for DHA, when the changes are expressed in mol/100 mol fatty acids. When calculated as percentage increase in the baseline level of the individual fatty acids, the increase was 144% for EPA and 24% for DHA. The increase in the plasma levels of - and -carotenes, lycopene and lutein confirm previous data that these carotenoids are well absorbed. In the case of lutein, the carotenoid was present in the supplement as a mixture of free lutein and lutein esters.

The absence of bixin, capsanthin and capsorubin is because of the rapid clearance of the carotenoids from plasma, rather than lack of absorption (Levy et al, 1997; Etoh et al, 2000). The rapid clearance of polar carotenoids is in contrast to the plasma response to the less polar carotenoids, which are located in LDL particles.

Fasting plasma TG levels were significantly decreased after 3 weeks of each dietary treatment when compared with baseline levels. This effect is mainly associated with the well-recognised effect of fish oil (n-3 fatty acids) on lowering plasma TG (Leigh-Firbank et al, 2002). It was found that the carotenoid-supplemented group reduced the final TG levels by 31.5%, whereas the control only gave a reduction of 15.3%. This suggests a possible interaction between the effects of the carotenoid mixture and fish oil in lowering plasma TG. The observation that a combination of carotenoids and fish oil is more effective than fish oil alone in lowering plasma TG has not been reported in any previous study and further studies are required to confirm this effect.

It was found that, in both the groups, there was no significant change of the mean ORAC values (P>0.05) after 3 weeks of supplementation, in comparison with the baseline levels. This probably reflects the fact that the test is most sensitive to antioxidants in the aqueous phase, since the radical initiator and the test protein -phycoerythrin are both in the aqueous phase. The lack of a significant change in the control group between the ORAC value at baseline and after 3 weeks supplementation with fish oil confirms the fact that this test is not sensitive to changes in the lipid phase. However, the high variability of the analysis, and the low statistical power of the study contributed to the lack of significance for the small change observed on supplementation with the carotenoid-rich test sample.

Copper-catalysed oxidation of LDL ex vivo is sensitive to changes in the lipid phase, and the lag time was significantly shorter for both groups after 3 weeks of dietary supplementation as compared with baseline values (control: 26.6% reduction; carotenoid test group: 20.4% reduction from baseline levels). This is expected due to the fish oil consumed, which was rich in the highly unsaturated fatty acids EPA and DHA, which were incorporated into LDL phospholipids. However, the average decrease in the lag phase from baseline in the carotenoid test group was significantly lower (P=0.045) than in the control group, reflecting thereby a protective effect of the carotenoid mixture on oxidative modification of LDL. In the literature, there are several reports of the antioxidant activity of carotenoids (in particular -carotene) against LDL oxidation, but the results are mixed. Lin et al (1998) reported decreased oxidation of LDL ex vivo in healthy women non-smokers, who consumed a supplement (11 mg/day) that was rich in -carotene, but which also contained -carotene, lycopene, zeaxanthin and lutein, Agarwal and Rao (1998) found increased resistance to oxidation of LDL following supplementation with lycopene at levels above 39 mg/day. However, Gaziano et al (1995); and Reaven et al (1993) found no beneficial effects following supplementation with relatively high levels of -carotene. Since bixin and the polar paprika carotenoids, capsanthin and capsorubin were not detected in the plasma, it can be concluded that these carotenoids were not present in the LDL particles. The antioxidant effect appears to be because of the mixture of - and -carotenes, lycopene and lutein that are known to be transported in LDL (Esterbauer et al, 1992).

The measurement of urinary 8-OHdG was selected as an appropriate method to evaluate DNA degradation. The values were quoted as a ratio of 8-OHdG:creatinine to allow for the fact that a single urine sample was collected rather than a 24 h collection. It was observed that in the control group, after 3 weeks of fish oil supplementation, the average level of urinary 8-OHdG was significantly increased in comparison with the baseline. The 19% increase in urinary 8-OHdG in the control group following fish oil supplementation is a measure of the increase in oxidative stress, and can be compared with the difference between smokers and nonsmokers reported by Loft et al (1992), who found that urinary 8-OHdG was 50% higher in smokers than in nonsmokers.

However, in the carotenoid supplemented group, the average 8-OHdG level was not changed significantly from the baseline value. The average increase from baseline was +4.2 ng 8-OHdG/mg creatinine for the control group, whereas a decrease of -1.6 ng 8-OHdG/mg creatinine was observed for the carotenoid group. This group responded significantly differently from the control group (P=0.005), suggesting that the carotenoid mixture dispersed in fish oil was effective at off-setting the increased DNA damage caused by fish oil consumption. Beneficial effects of carotenoids on decreasing DNA degradation have also been observed for lycopene and carotenes in other trials (Duthie et al, 1996; Pool-Zobel et al, 1997; Smith et al, 1999), whereas some trials (Agarwal & Rao, 1998; Collins et al, 1998) have found no effect. Most of the trials where effects have been observed have involved supplementation with higher levels of lycopene, but the current study has clearly shown that mixtures of carotenoids containing only 4.5 mg lycopene per day can be effective in decreasing DNA damage.

The significance of urinary 8-OHdG has been discussed by Cooke et al (2000). Excretion of 8-OHdG is increased in smokers or individuals with malignant diseases, but it was noted that increased excretion does not necessarily indicate increased DNA damage but may indicate a lowering of tissue levels of 8-OHdG. There have been no studies aimed at correlating reduction in urinary 8-OHdG excretion with reduced incidence of disease.

In conclusion, our study has shown that daily supplementation with a natural carotenoid mixture (30 mg) in subjects consuming fish oil can significantly increase the lag time before oxidative deterioration ex vivo, and can decrease the extent of DNA damage assessed by urinary 8-OHdG. The beneficial effects were probably because of the less polar carotenoids (- and -carotenes, lycopene and lutein), since the more polar carotenoids (bixin and the polar paprika carotenoids, capsanthin and capsorubin) were not detected in the plasma of subjects consuming the supplement.

References

1. Agarwal S & Rao V (1998): Tomato lycopene and LDL oxidation. A human dietary intervention study. Lipids 33, 981–985. | Article | PubMed | ChemPort |
2. Cao GH & Prior RL (1999): Measurement of oxygen radical absorbance capacity in biological samples. Methods Enzymol. 299, 50–62. | PubMed | ChemPort |
3. Collins RA, Olmedella B, South C, Granado F & Duthie S (1998): Serum carotenoids and oxidative DNA damage in human lymphocytes. Carcinogenesis 19, 2159–2162. | Article | PubMed | ISI | ChemPort |
4. Cooke MS, Evans MD, Herbert KE & Lunec J (2000): Urinary 8-oxo-2'-deoxyguanosine—source, significance and supplements. Free Rad. Res. 32, 381–397. | ISI | ChemPort |
5. Dugas TR, Morel DW & Harrison EH (1999): Dietary supplementation with beta-carotene but not with lycopene, inhibits endothelial cell mediated oxidation of LDL lipoprotein. Free Rad. Med. Biol. 26, 1238–1244.
6. Duthie JS, Ma AG, Ross MA & Collins AR (1996): Antioxidant supplementation decreases DNA damage in human lymphocytes. Cancer. Res. 56, 1291–1293. | PubMed | ISI | ChemPort |
7. Erhola M, Toyokuni S, Okada K, Tanaka T, Hiai H, Ochi H, Uchida K, Osawa T, Nieminen MM, Alho H & Lehtinen PK (1997): Biomarker evidence of DNA oxidation in lung cancer patients: association of urinary 8-hydroxy-2'deoxyguanosine excretion with radiotherapy, chemotherapy and response to treatment. FEBS Lett. 409, 287–291. | Article | PubMed | ISI | ChemPort |
8. Esterbauer H, Striegl G, Puhl H & Rotheneder M (1989). Continuous monitoring of in vitro oxidation of human low density lipoproteins and resistance to oxidation. Free Rad. Res. Commun. 6, 67–75. | ISI | ChemPort |
9. Esterbauer H, Gebicki J, Puhl H & Jurgens G (1992): The role of lipid peroxidation and antioxidants in oxidative modification of LDL. Free Rad. Med. Biol. 13, 341–390.
10. Etoh H, Utsunomiya Y, Komori A, Murakami Y, Oshima S & Inakuma T (2000): Carotenoids in human blood plasma after ingesting paprika juice. Biosci. Biotechnol. Biochem. 64, 1096–1098.
11. Foulon T, Richard J, Rayen N, Bournain JL, Beani C, Laborte F & Hadjian A (1999): Effects of fish oil fatty acids on plasma lipids and lipoproteins and oxidant antioxidant imbalance in healthy subjects. Scand. J. Clin. Lab. Invest 59, 239–248. | Article | PubMed | ChemPort |
12. Friedewald WT & Levy RI (1972): Estimation of the concentration of low-density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge. Clin. Chem. 18, 499–502. | PubMed | ISI | ChemPort |
13. Gaziano MJ, Hatta A, Flynn M & Krinsky NI (1995): Supplementation with -carotene in vivo and in vitro does not inhibit LDL oxidation. Atherosclerosis 112, 187–195.
14. Indu M & Ghafoorunissa(1992): N-3 fatty acids in Indian diets comparison of the effects of precursor alpha-linolenic acid vs product long chain n-3 polyunsaturated fatty acids. Nutr. Res. 12, 569–582.
15. Krinsky NI (2001): Carotenoids as antioxidants. Nutrition 17, 815–817.
16. Leigh-Firbank EC, Minihane AM, Leake DS, Wright JW, Murphy MC, Griffin BA & Williams CM (2002): Eicosapentaenoic acid and docosahexaenoic acid from fish oils: differential associations with lipid responses. Br. J. Nutr. 87, 435–445. | PubMed |
17. Levy LW, Regalado E, Navarette S & Watkins RH (1997): Bixin and norbixin in human plasma: determination and study of the absorption of a single dose of annatto food color. Analyst 122, 977–980.
18. Lin Y, Burri BJ, Neidlinger TR, Muller H-G, Dueker SR & Clifford AJ (1998): Estimating the concentration of -carotene required for maximal protection of low-density lipoproteins in women. Am. J. Clin. Nutr. 67, 837–845.
19. Loft S, Vistisen K, Ewertz M, Tjonneland A, Overvad K & Poulse HE (1992): Oxidative DNA damage estimated by 8-hydroxydeoxyguanosine excretion in humans—influence of smoking, gender and body mass index. Carcinogenesis 13, 2241–2247. | PubMed |
20. Mares-Perlman JA, Millen AE, Ficek TL & Hankinson SE (2002): The body of evidence to support a protective role for lutein and zeaxanthin in delaying chronic disease. Overview. J. Nutr. 132, 518S–524S. | PubMed | ISI |
21. McNamara JR, Huang C, Massov T, Leary ET, Warnick GR, Rubins HB, Robins SJ & Schaefer EJ (1994): Modification of the dextran-Mg high-density lipoprotein cholesterol precipitation method for use with previously frozen plasma. Clin. Chem. 40, 233–239. | PubMed |
22. Minihane AM, Khan S, Leigh-Firbank EC, Talmud PJ, Williams DL, Wright JW, Murphy MC, Griffin BA & Williams CM (2000): ApoE polymorphism and fish oil supplementation in subjects with an atherogenic lipoprotein phenotype. Arter. Throm. Vasc. Biol. 20, 1990–1997.
23. Pool-Zobel BL, Bub A, Muller H & Reckemmer G (1997): Consumption of vegetables reduces genetic damage in humans: first results of a human intervention trial with carotenoid rich foods. Carcinogenesis 18, 1847–1850. | Article | PubMed | ChemPort |
24. Pryor WA, Stahl W & Rock CL (2000): Beta-carotene from biochemistry to clinical trials. Nutr. Rev. 58, 39–53. | PubMed |
25. Rao AVR & Agarwal S (2000): Role of antioxidant lycopene in cancer and heart disease. J. Am. Coll. Nutr. 19, 563–569. | PubMed | ChemPort |
26. Reaven PD, Beltz W, Parthasarthy S & Witzum JL (1993): Effect of dietary antioxidant combinations in humans: protection of LDL by vitamin E but not by -carotene. Arter. Throm. 13, 590–600.
27. Riso P, Pinder A, Santangelo A & Porrini M (1999): Does tomato consumption effectively increase the resistance of lymphocyte DNA to oxidative damage? Am. J. Clin. Nutr. 69, 712–715. | PubMed | ChemPort |
28. Rousseau EJ, Davison AJ & Dunn B (1992): Protection by -carotene and related compound against oxygen-mediated cytotoxicity and genotoxicity: implications for carcinogenesis and anticarcinogenesis. Free Rad. Med. Biol. 13, 407–433.
29. Smith JM, Inserra PF, Watson R & O'Neil KL (1999): Supplementation with fruit and vegetable extracts may decrease DNA damage in the peripheral lymphocytes of the elderly population. Nutr. Res. 19, 1507–1518.
30. Sorensen NS, Marckmann P, Hoy CE, van Duyvenvoorde W & Princen HMG (1998): Effects of fish oil enriched margarine on plasma lipids, low-density lipoprotein particle composition, size and susceptibility to oxidation. Am. J. Clin. Nutr. 68, 235–241. | PubMed |
31. Wander RC, Du SH, Ketchum SO & Rowe KE (1996): Effects of interaction of RRR-alpha-tocopheryl acetate and fish oil on LDL oxidation in postmenopausal women with and without hormone replacement therapy. Am. J. Clin. Nutr. 63, 184–193. | PubMed | ChemPort |