Lutein, zeaxanthin and meso-zeaxanthin are the only carotenoids present in the macular pigment of the retina(Reference Davies and Morland1) and it is suggested that they could protect the retina by blue light filtering(Reference Davies and Morland1), thereby decreasing chances for photochemical light damage(Reference Landrum, Bone and Kilburn2, Reference Wu, Seregard and Algvere3). In addition, they are capable of scavenging free radicals(Reference Wu, Seregard and Algvere3). An initial study by Seddon et al. (Reference Seddon, Ajani and Sperduto4) observed an inverse association between a diet with a high content of lutein and the prevalence of exudative age-related macular degeneration, the most common cause of irreversible, severe loss of vision among the elderly in the Western countries. A definite proof for a causal relationship however is still lacking(Reference Trumbo and Ellwood5). Several studies addressed the possible role of macular pigment more explicitly by measuring the macular pigment optical density (MPOD) in patients with, or at risk of age-related macular degeneration, also with ambiguous results(Reference Beatty, Murray and Henson6–Reference Kanis, Berendschot and van Norren10). Macular pigment is entirely of dietary origin and it has been show that MPOD can be increased by a dietary modification(Reference Hammond, Johnson and Russell11) or by supplements(Reference Berendschot, Goldbohm and Klöpping12–Reference Koh, Murray and Nolan15). Functional foods containing plant sterol or stanol esters may lower serum concentrations of fat-soluble carotenoids(Reference Plat and Mensink16–Reference Colgan, Floyd and Noone18). The relevance of these reductions on health is unknown, and only a few human intervention studies addressed the long-term effects of decreased carotenoid concentrations(Reference Neuhouser, Rock and Kristal17, Reference Broekmans, Klopping-Ketelaars and Weststrate19). Therefore, we carried out a human intervention trial to evaluate the effects of plant stanol and sterol esters on serum lutein/zeaxanthin reduction and the possible change in MPOD as a consequence of this reduction.
Methods
Subjects
Subjects were recruited via local newspaper advertisements and posters in the university and hospital buildings. Inclusion criteria were: current treatment with a 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor (statin), age 18–65 years, BMI ≤ 32 kg/m2, no proteinuria or glucosuria, diastolic blood pressure ≤ 95 mm Hg and systolic blood pressure ≤ 200 mm Hg. Using statins was an inclusion criteria since another purpose of the study was to analyse the long-term effects of plant sterol or stanol esters on serum lipoprotein metabolism and markers of endothelial dysfunction and vascular stiffness in patients on stable statin treatment(Reference de Jong, Plat and Lütjohann20). Exclusion criteria were clinical manifestations of liver disorders, having diabetes mellitus type 2 or having had cardiovascular or cerebral events within a period of 6 months prior to the study. The Ethics Committee of the Maastricht University had approved the protocol and all subjects signed an informed consent.
Diets and design
Subjects were asked to replace their own margarine or butter with the experimental ‘light’ margarines (40 % fat) we supplied. They were instructed to consume 30 g ‘light’ margarine per day, and to divide the margarine over at least two meals during the day. During a 5-week run-in period, all subjects used a control margarine without added plant sterols or stanols. At the end of the run-in period subjects were randomly allocated to one of the three experimental groups, stratified for sex and age. For the following 85 weeks, the first group continued with the control margarine, the second group with a plant sterol-enriched margarine (2·5 g plant sterols/d) and the third group with a plant stanol-enriched margarine (2·5 g plant stanols/d). Plant sterols and stanols were provided as fatty acid esters obtained by transesterification of free plant sterols and stanols with sunflower oil-based fatty acids (Unilever, Vlaardingen, The Netherlands) or rapeseed oil-based fatty acids (Raisio Group, Raisio, Finland), respectively. Neither the plant stanol ester nor sterol ester margarines contained lutein or zeaxanthin.
Measurements
Blood sampling
Fasting blood samples were taken by venepuncture in weeks 4, 5, 89 and 90. Blood was sampled in 10 ml serum separator and EDTA tubes. Serum was obtained by centrifugation at 2000 g for 30 min at 4°C, minimally 1 h after blood sampling, and was used for analysis of cholesterol concentrations. EDTA plasma was obtained by centrifugation at 2000 g for 30 min at 4°C, and was used for analysing lutein/zeaxanthin concentrations. All samples were snap-frozen and stored in small aliquots directly after sampling at − 80°C until further analysis.
Cholesterol and lutein/zeaxanthin
Cholesterol and lutein/zeaxanthin concentrations were determined as described(Reference Plat and Mensink16). Samples from one subject of weeks 4 and 5 as well as of weeks 89 and 90 were pooled before analysis and measured in the same analytical run to minimize the potential influence of factors other than the dietary intervention and as well as the intra-assay variation.
Macular pigment optical density
MPOD was determined at weeks 5 and 90 by a full spectral analysis(Reference Berendschot and van Norren21) of light reflected at the fovea, measured with the Utrecht Foveal Reflection Analyser(Reference Zagers, van de and Berendschot22). The subjects' pupils were dilated with 0·5 % tropicamide and 1 % phenilefrine.
Statistical analyses
Since serum lutein/zeaxanthin depends on the amount of lipoprotein carriers in plasma(Reference Plat and Mensink16) we present total cholesterol standardized (μmol/mmol cholesterol) lutein/zeaxanthin concentrations, to correct for changes in serum cholesterol concentrations. Overall differences in changes (week 90 − week 5) between groups were tested by ANOVA. In addition, to correct for possible influence of the baseline concentrations on the serum changes, we applied an ANCOVA with the end value as dependent, diet as fixed factor and baseline as covariate. To look for a possible association between the serum lutein/zeaxanthin and the MPOD we calculated the Pearson correlation coefficient. All data analysis was performed with the SPSS statistical software package version 14.0.0 (SPSS Inc., Chicago, IL, USA).
Results
Recruitment and follow-up
As described elsewhere, fifty-four subjects completed the study(Reference de Jong, Plat and Lütjohann20). However, during the study six subjects changed their statin medication. Therefore, their data were not included in the analysis. One other subject showed a decrease in total cholesterol concentration of almost five standard deviations above the mean of all others. This subject was considered an outlier and also excluded from the analysis. Thus, data of forty-seven subjects was available for analysis (Table 1). MPOD was obtained at a different study site than all other measures. This caused some logistical problems, and MPOD was measure in only thirty-four subjects at both study visits (see also Table 1). Compliance was evaluated by evaluating changes in serum plant sterol and stanol concentrations throughout the study period as reported elsewhere(Reference de Jong, Plat and Lütjohann20).
Table 1 Characteristics of the forty-seven volunteers that were included in the study and of the thirty-four volunteers that had their macular pigment optical density determined twice*
(Mean values and standard deviations)
* For details of subjects and procedures, see Methods.
Serum lipids and lutein/zeaxanthin
The average total cholesterol concentration over the entire study period was lowered by 6·8 % or 0·38 mmol/l (P = 0·005) and 8·8 % or 0·48 mmol/l (P = 0·001) in the plant sterol ester and plant stanol ester groups as compared to the control group(Reference de Jong, Plat and Lütjohann20). These reductions could almost entirely be ascribed to reductions in LDL-cholesterol, and were not significantly different between the plant sterol and plant stanol ester groups (P = 0·18).
The reduction in cholesterol-standardized serum lutein/zeaxanthin concentrations was significantly larger in the sterol ester group as compared to the stanol ester group (P < 0·001), whereas compared to the control the reduction in the plant sterol ester group nearly reached significance (P = 0·07; Table 2). The rather unexpected mean increase in cholesterol-standardized serum lutein/zeaxanthin concentration in the stanol ester group was significantly different (P = 0·032) from the change in lipid-adjusted serum lutein/zeaxanthin concentrations in the control group. In an ANCOVA analysis, the cholesterol-standardized serum lutein/zeaxanthin concentrations at study end were not only explained by differences in baseline concentrations (P < 0·001), but there was also a significant contribution of the different diets (P = 0·017).
Table 2 Mean lutein serum concentrations corrected for total cholesterol, and macular pigment optical density (MPOD) during the study*
(Mean values and standard deviations)
* For details of subjects and procedures, see Methods.
† P values represent whether the differences between the three groups are significant.
Macular pigment optical density
There were no differences between the MPOD at baseline and despite the differences in serum lutein/zeaxanthin concentrations in the three groups, there were no differences in MPOD changes between the groups (P = 0·76).
At baseline, the MPOD did not correlate with the cholesterol-standardized serum lutein/zeaxanthin concentration (r 0·20, P = 0·22). If we stratified for gender we found a nearly significant positive correlation for men (r 0·37, P = 0·080) and a nearly significant negative correlation for women (r − 0·47, P = 0·07). Also, at baseline the MPOD for men of 0·50 (sd 0·14) was significantly higher compared with the MPOD for women of 0·41 (sd 0·05) (P = 0·008), despite the almost equal cholesterol-standardized serum lutein/zeaxanthin concentrations of 0·066 (sd 0·18) for men and 0·063 (sd 0·022) for women (P = 0·58). We found no correlation between the change in the MPOD and the change in lutein/zeaxanthin concentration.
Discussion
It is well recognized that plant sterol and stanol esters consistently lower serum LDL-cholesterol concentrations. As a result, also concentrations of fat-soluble antioxidants are lowered. We have now shown in this 85-week intervention study that again lutein/zeaxanthin plasma concentrations were significantly lowered. Interestingly, the reduction in lutein/zeaxanthin was only observed in the plant sterol ester group and not in the plant stanol ester group, for which we do not have an explanation.
Macular pigment consists of lutein, zeaxanthin and meso-zeaxanthin and could be directly affected by a change in serum lutein/zeaxanthin(Reference Hammond, Johnson and Russell11–Reference Koh, Murray and Nolan15). In comparison with the significant increases in MPOD in supplementation studies, the present study showed that significantly different changes in serum cholesterol-adjusted lutein/zeaxanthin between the three groups did not result in a significant difference between the groups in MPOD. This may be due to the small number of subjects that underwent MPOD measurements. On the other hand, the present results are in accordance with the results of Cooper et al. (Reference Cooper, Curran-Celentano and Ciulla23) and Broekmans et al. (Reference Broekmans, Klopping-Ketelaars and Weststrate19) who reported that consumption of a dietary fat replacer was not associated with reduced MPOD.
Despite the small numbers, at baseline the MPOD was significantly higher for men than for women, in line with results from other studies(Reference Broekmans, Berendschot and Klöpping24–Reference Ciulla, Curran-Celentano and Cooper27). Also, the differences in the correlation between the MPOD and the cholesterol-standardized serum lutein/zeaxanthin concentration for men and women have been observed before(Reference Broekmans, Berendschot and Klöpping24, Reference Johnson, Hammond and Yeum28, Reference Burke, Curran-Celentano and Wenzel29).
In conclusion, we found a significantly lowered cholesterol-standardized lutein/zeaxanthin concentration after 85 weeks of consumption of margarines enriched with plant sterol or stanol esters. However, this did not translate into a change in MPOD.
Acknowledgements
J. P. and R. P. M. planned the study. A. de J. and T. T. J. M. B. collected and analysed the data. T. T. J. M. B. drafted the article. J. P., R. P. M. and A. de J. reviewed and commented on the manuscript. There are no conflicts of interest.
