Original Paper
Ophthalmic Res 2017;58:231–241 DOI: 10.1159/000479930
Epilutein for Early-Stage Age-Related
Macular Degeneration: A Randomized
and Prospective Study
Raimondo Forte
a
Lucia Panzella
b
Ida Cesarano
a
Gilda Cennamo
a
Thomas Eidenberger
c
Alessandra Napolitano
b
Departments of a Ophthalmology and b Chemical Sciences, University of Naples Federico II, Naples , Italy; c School of Engineering and Environmental Sciences, Upper Austria University of Applied Sciences, Wels , Austria
0.03 ODU at M2 ( p = 0.04). Sixteen patients (mean age 72.0 ± 6.3 years, 29 eyes) were included in group 2. Mean MPOD was 0.215 ± 0.03 at BL, which reduced to 0.202 ± 0.03 ODU at M1 ( p = 0.003) and 0.207 ± 0.02 ODU at M2 ( p < 0.001). A rise in the systemic level of total xanthophylls was observed at M1 for both groups. At M2, total xanthophylls were significantly increased only in group 1 and decreased in group 2. Conclusion: In patients with early-stage AMD, the administration of lutein in combination with epilutein was associated with an increased MPOD compared to the admin-istration of lutein alone. © 2017 S. Karger AG, Basel
Introduction
The macular pigment (MP) of the human retina is composed of xanthophylls. Located in the Henle fibers and inner plexiform layer, the highest MP density is found in the fovea [1, 2] . These fibers play an important
Keywords
Age-related macular degeneration · Lutein · Epilutein · Macular pigment optical density
Abstract
Purpose: The hypothesis that oral supplementation of the epilutein/lutein combination could augment the macular pigment optical density (MPOD) in patients with age-related macular degeneration (AMD) was tested. Methods: In a pro-spective randomized interventional study, 40 consecutive patients with early-stage AMD were recruited. After a 2-week run-in period, patients were randomly treated with a daily oral administration of 8 mg epilutein and 2 mg lutein (group 1) or 10 mg lutein (group 2) for 2 months. At baseline (BL) and 1-month (M1) and 2-month visits (M2), all patients under-went a complete ophthalmological examination, including measurement of MPOD in a 7° area (Visucam 200; Carl Zeiss Meditec, Milan, Italy). Xanthophylls were quantified in plas-ma, as well as the HDL, non-HDL, and erythrocyte fractions at each study visit. Results: Twenty-one patients (mean age 69.4 ± 6.7 years, 35 eyes) were included in group 1. Mean MPOD was 0.203 ± 0.02 optical density units (ODU) at BL, and increased to 0.214 ± 0.04 ODU at M1 ( p = 0.008) and 0.206 ±
Received: June 19, 2017
Accepted after revision: July 30, 2017 Published online: September 28, 2017
Dr. Raimondo Forte, MD, PhD
Department of Ophthalmology, University of Naples Federico II Via Pansini 15
role in protecting the retina against oxidative stress through different mechanisms [3] .
Based on a series of investigations, a number of isomers of lutein and zeaxanthin, epilutein, and 3 ′ -oxolutein were identified as the main xanthophylls occurring in dietary sources and the human retina ( Fig. 1 ) [4, 5] . The presence of these xanthophylls in the eyes is of special interest as the human body cannot synthesize them although they play important roles to maintain eye health [6] .
The eye xanthophylls, especially all- E -lutein and zea-xanthin, were shown to act as antioxidants and radical quenchers, and to protect the eye by absorbing damaging blue light. Adequate dietary supply with xanthophylls in-creases MP optical density (MPOD) and exerts beneficial effects in patients with age-related macular degeneration
(AMD) [7–14] . Main nutritional sources for
xantho-phylls are vegetables, egg yolk, fish, and dietary supple-ments delivering mainly lutein from nonedible plants. Xanthophylls occur in nature either in free or in esterified form [15] . Around 80% of the xanthophylls occurring in nutritional sources is lutein. Zeaxanthin and epilutein have been identified as minor components in nutritional sources [16, 17] . More recently, it was shown that
epilu-tein is formed during cooking of vegetables and occurs in significant amounts in fish skin; hence, epilutein may contribute larger amounts of the daily xanthophyll supply as thought previously [18] .
The pharmacokinetic profile of xanthophylls in hu-mans has been investigated [19] . The absorption of xan-thophylls after oral intake is complex and depends – amongst other things – on the form of ingestion (free or esterified), the type of co-ingested food, emulsification, incorporation in mixed micelles, and chylomicron trans-port. It has been speculated that xanthophyll absorption is saturable and that xanthophylls compete with them-selves and other carotenoids for absorption. Lutein, zea-xanthin (except for meso-zeazea-xanthin), and epilutein are found in the blood and liver [20] . Meso-zeaxanthin is ex-clusively found in retinal tissue and should be generated locally. It has been shown that lutein and zeaxanthin are absorbed by erythrocytes and appear in lipoproteins [21] . The uptake and incorporation into the retinal tissue is explained by active transport involving specific trans-porters in blood and a retinal lutein-binding protein [22] . Considering the fact that meso-zeaxanthin and epilutein contribute essentially to the xanthophyll pattern in the retinal tissue, but only epilutein is found in the blood and liver, we hypothesized that epilutein may play a role in the xanthophyll supply to the eye. This view is supported by the finding that epilutein plasma concentrations are sig-nificantly decreasing in patients progressing from weak to intermediate AMD [23] . Based on the current under-standing, epilutein can be formed from zeaxanthin di-rectly by double-bond isomerization or from lutein via 3 ′ -oxolutein by reduction [20, 24] . As all these processes are reversible, epilutein may form a metabolic pool to re-store lutein and zeaxanthin in the retinal tissue.
It is well established that high levels of low-density li-poproteins (LDL) and triglycerides, and low levels of high-density lipoprotein (HDL), for example, are risk fac-tors for AMD development [25] . Low lutein/zeaxanthin levels also seem associated with AMD progression al-though actual studies report either concern women younger than 75 years [26] or do not show statistical sig-nificance [27] . It was shown that xanthophylls are carried in lipoproteins and are absorbed by erythrocytes [28, 29] . To gain a deeper insight into the pharmacokinetics of lu-tein and epilulu-tein, it seems, therefore, necessary to deter-mine the xanthophyll concentrations in plasma, erythro-cytes, and various fractions of lipoproteins after oral ad-ministration. Previous studies in animals showed the possibility to increase the absorption of lutein by con-comitant administration of epilutein [30] .
HO OH All-E-lutein HO OH All-E-epilutein HO O 3-Oxolutein HO OH (3R, 3S) Meso-zeaxanthin HO OH (3R, 3R) Zeaxanthin HO OH (3S, 3S) Zeaxanthin 3 3 3 3 3 3 3 3
Fig. 1. Xanthophylls occurring in dietary sources and observed in the human eye. For lutein, possible stereoisomers and esterifica-tion posiesterifica-tions are shown. All other xanthophylls may occur with the same variations.
To test our hypothesis, a prospective, randomized, clinical study was initiated in 40 participants with early-stage AMD to investigate the effects of lutein or a combi-nation of lutein-epilutein on MPOD and the pharmaco-kinetics of lutein and epilutein after oral administration.
Materials and Methods Study Protocol
In a prospective randomized interventional study, 40 consecu-tive Caucasian patients with early-stage AMD according to the Age-Related Eye Disease Study (AREDS) research group classifi-cation were recruited at the University of Naples Federico II and at the University of Salerno. The study was conducted in accor-dance with the Declaration of Helsinki and approved by the Eth ics Committee of the Naples University Federico II on December 2014 (No. 203/14). Written informed consent was obtained from the subjects after explanation of the nature and possible conse-quences of the study. Questionnaires were used to collect lifestyle information about smoking status (smokers were defined as hav-ing smoked at least 1 cigarette per day for at least 6 months) and medical information. Body mass index (BMI) was calculated us-ing the formula of weight in kilograms divided by height in meters squared. The participants were divided into 4 groups (under-weight, healthy, over(under-weight, and obese) according to their BMI scores. According to the AREDS classification, early AMD is de-fined as a combination of small drusen and intermediate drusen (63–124 μm in diameter) [31] . Exclusion criteria were the
diagno-sis of a different stage of AMD (presence of ≥ 5 drusen >125 μm
in diameter) within the macula in the studied eye, presence of choroidal neovascularization or geographic atrophy, or signs of any other active retinal disease, such as retinal vascular (i.e., dia-betic retinopathy or retinal vein occlusion) or vitreoretinal (i.e., vitreomacular traction syndrome or epiretinal membrane) diseas-es. Eyes with lens opacities and with best-corrected visual acuity (BCVA) <20/25 were also not included in the study to ensure proper execution of MP density examinations. After a 2-week run-in period, patients were randomly assigned to daily oral treat-ment with either a combination of 8 mg all- E -epilutein and 2 mg all- E -lutein (group 1) or 10 mg all- E -lutein (group 2) for 2 months ( Fig. 1 ). At baseline (BL) and after 1 (M1) and 2 months (M2) of treatment, all patients underwent a complete ophthalmological examination, including measurement of MPOD and spectral do-main optical coherence tomography (SD-OCT). On each occa-sion, fasting blood samples were taken for pharmacokinetic in-vestigations. Analysis of blood samples included measurement of lutein and epilutein, the active part of the treatments, and 3 ′ -oxo-lutein, an oxidative reaction product of lutein and epilutein which is considered to be the first metabolite of lutein. All 3 compounds were followed in plasma, erythrocytes, and HDL and non-HDL fractions of lipoproteins to gain insight into the distribution of these compounds in blood.
Ophthalmological Examination
Monocular BCVA was determined in all subjects with the ETDRS (Early Treatment Diabetic Retinopathy Study) charts. Ret-inal status was evaluated by fundus biomicroscopy after pupil dila-tion by experienced physicians. All patients underwent
examina-tion of infrared reflectance (fundus illuminaexamina-tion, λ = 830 nm), fun-dus autofluorescence (excitation, λ = 488 nm; barrier filter, λ = 500 nm), and measurement of MPOD.
MPOD is given as the logarithmic ratio of virtual fundus reflec-tion below MP (reference area) and the very low macula reflecreflec-tion. Individual vignetting was corrected by a shading function to use the approximation of a paraboloid for simulation of the reflective underground below MP. The logarithmic pixel ratio results in the distribution of MPOD. MPOD was measured using the 1-wave-length fundus reflectance method (Visucam 200; Carl Zeiss Med-itec, Milan, Italy). The subject’s pupils were dilated with 1 drop of tropicamide 1%. Fundus color photographs at 45° were obtained, and MPOD was measured in a 30° field of the fundus photograph (flash intensity = 12). MPOD (maximum, mean, and volume) was calculated at 7° of eccentricity, spanning the region where the ma-jority of xanthophylls were concentrated. Maximum/mean MPOD refer to the maximum/mean OD of MP xanthophylls given in OD units (ODU). The unit of OD volume is given as ODU × degrees squared. Automatic corrections for persons >45 years of age were performed, considering the scattering influence of the aging lens. SD-OCT was carried out in all patients (XR Avanti; Optovue Inc., Fremont, CA, USA) and included 19 horizontal lines (6 × 6 mm area), each consisting of 1,024 A-scans per line as a minimum ac-quisition protocol. Further high-resolution 9-mm single B-scans of the foveal choroid (each composed of up to 100 averaged en-hanced depth imaging OCT B-scans) were also obtained. Choroi-dal thickness, ganglion cell complex (GCC) thickness, GCC focal loss volume, and GCC global loss volume were assessed for each eye. All macular imaging examinations were performed indepen-dently by 2 experienced retinal physicians (R.F. and G.C.).
Blood Sampling and Analysis
Blood samples were drawn into test tubes containing EDTA as anticoagulant and subjected to centrifugation at 1,000 rpm for 15
min at 4 ° C to separate plasma from erythrocytes. All procedures
were carried out under subdued light to minimize
photodegrada-tion of xanthophylls. Samples were stored at –80 ° C until analyses.
Extraction of xanthophylls from plasma was performed ac-cording to established procedures [2] . Briefly, plasma proteins were precipitated with ethanol containing 1% butylated hydroxy-toluene (BHT), and xanthophylls were extracted with n-hexane. Extraction of xanthophylls from erythrocytes was performed according to established procedures [28] . Briefly, erythrocytes were washed repeatedly with phosphate-buffered saline (pH 7.4) and centrifuged. The packed cells were suspended in
water/etha-nol/pyrogallol. The mixture was then treated with 1.8 M KOH
fol-lowed by sodium dodecyl sulfate, and xanthophylls were extracted using a mixture of hexane/dichloromethane (1% BHT).
Extraction of xanthophylls from lipoproteins was performed according to established procedures [16, 32] . Briefly, plasma was treated with lipase and cholesterol esterase in the presence of Triton X-100 to release xanthophylls from the total lipoprotein fraction. All xanthophylls released were extracted as described for plasma. To isolate xanthophylls in the HDL fraction, plasma was treated with a reagent precipitating the non-HDL fraction. After centrifugation, the supernatant was processed as described for the total lipoprotein fraction. The concentration of xanthophylls in the non-HDL fraction was determined as the difference between the concentration of xanthophylls in the total lipoprotein fraction and the HDL fraction.
High-performance liquid chromatography of xanthophylls was carried out on an Agilent 1100 series instrument using a 250 × 4.60 mm (5 μm) Sphereclone ODS column (Phenomenex; Castel Maggiore, Italy) at ambient temperature and UV-visible de-tection set to 450 nm. An acetonitrile/methanol/dichloromethane
mixture = 85: 9:6 (v/v) at 0.7 mL/min was used as mobile phase.
Under these conditions, all- E -lutein, epilutein, and 3 ′ -oxolutein were eluted at 11.5, 10, and 9 min, respectively. In this study, lutein (free form) and epilutein were supplement capsules provided by Gupron GmbH (Wels, Austria). Lutein was prepared by saponifi-cation of a marigold (Tagetes erecta ) extract followed by isolation and purification [33] . Epilutein was prepared from lutein by a pro-prietary process [30] . The specified purity of lutein and epilutein were >97%. For lutein and epilutein calibration, internal reference material (identical to the administered test material) was used, and for 3 ′ -oxolutein an internal standard was prepared by oxidation of
lutein with MnO 2 [34] . Analysis of the xanthophylls in the
supple-ment capsules showed that the content of lutein and epilutein ranged from 97 to 102% of the declared content. Zeaxanthin was found to be below the limit of detection.
Data Analysis
Statistical calculations were performed using the Statistical Package for Social Sciences (version 17.0; SPSS Inc., Chicago, IL, USA). An ANOVA model was used to correct for BL values and to calculate sample size. With a noninferiority hazard ratio margin of 1.4 for between-arm main effects, 80% power, and one-sided 5%
α, a total of 16 events in each group were required to be observed, and, assuming a 20% dropout rate, the target sample size for re-cruitment was 40 participants. The differences between men and women and between smokers and nonsmokers were compared us-ing the t test for 2 independent samples or the Mann-Whitney U test for nonnormally distributed samples. The paired t test was used to assess changes between the measurement time points. Sig-nificance was considered at * p > 0.05, * * p < 0.05, * * * p < 0.01.
Results Demographics
Of the 40 patients with early-stage AMD enrolled in the study, 3 were lost in the run-in period. The 37 remain-ing patients had a mean age of 64 ± 3 years. Twenty-one patients (13 females, 61.9%; mean age 69.4 ± 6.7 years, 35 eyes) were included in group 1. Five patients (23.8%) were smokers. According to BMI, 2 patients (9.5%) were un-derweight, 17 (81%) were healthy, 2 (9.5%) were over-weight, and none was obese. Sixteen patients (9 females, 56.2%; mean age 72.0 ± 6.3 years) were included in group 2. Four patients (25%) were smokers. According to BMI,
Table 1. Best-corrected visual acuity (BCVA), choroidal thickness, ganglion cell complex (GCC) thickness, GCC focal loss volume (GCCFLV), GCC global loss volume (GCCGLV), and retinal nerve fiber layer (RNFL) thickness in both study groups at baseline (BL) and the 2-month visit (M2) (means ± SD)
Group 1 Group 2 BL M2 p B L M2 p BCVA, logMAR 0.08±0.03 0.08±0.02 0.8 0.06±0.02 0.06±0.04 0.8 Choroidal thickness, μm 207.43±66.6 204.2±58.2 0.6 226.6±62.3 201.3±57.2 0.1 GCC thickness, μm 95.14±7.7 90.28±17.2 0.2 86.9±8.2 86.3±14.2 0.7 GCCFLV, % 1.25±0.77 1.47±1.61 0.3 2.29±0.69 2.17±1.3 0.4 GCCGLV, % 3.78±4.17 4.31±3.08 0.2 3.0±3.62 3.9±3.01 0.4 RNFL thickness, μm 110.5±26.4 105.1±17.3 0.1 102.6±16.3 102.1±19.3 0.6
Table 2. Results for mean and maximum macula pigment optical density (in optical density units)
Group 1 Δ vs. BL p value Group 2 Δ vs. BL p value
Mean BL 0.203±0.02 0.215±0.03 Mean M1 0.214±0.04 +0.011 <0.001 0.202±0.03 –0.013 <0.001 Mean M2 0.206±0.03 +0.003 <0.05 0.207±0.02 –0.008 <0.001 Max. BL 0.556±0.07 0.525±0.08 Max. M1 0.571±0.09 +0.015 <0.05 0.513±0.07 –0.012 ns Max. M2 0.559±0.08 +0.003 <0.05 0.522±0.06 –0.003 ns
1 patient (6.3%) was underweight, 13 (81.2%) were healthy, 2 (12.5%) were overweight, and none was obese. No significant differences were found between both groups concerning age ( p = 0.6), sex ( p = 0.3), percentage of smokers ( p = 0.4), and BMI classification ( p = 0.3).
No side effects of treatment were recorded during the study period.
Ophthalmological Results
The results of the ophthalmological examinations for both groups at BL and M2 are shown in Table 1 . The results of MPOD measurements for both groups at BL, M1, and M2 are shown in Table 2 . No differences in BCVA values were observed from BL to M2. Choroidal thickness, GCC thickness, GCC focal loss volume, and GCC global loss vol-ume did not show significant changes from BL to M2. No correlations in both groups were found between MPOD measurements and BMI or sex or smoking status ( p > 0.05).
In group 1, a significant increase in mean MPOD was observed at M1 and M2 compared to BL. Likewise, the
change in maximum MP density was significant at M1 and M2 compared to BL ( Fig. 2 ). Changes in MP volume were significant at M1 compared to BL ( p = 0.001). Cho-roidal thickness, GCC thickness, GCC focal loss volume, and GCC global loss volume did not show significant changes from BL to M2.
a b c
d e f
Fig. 2. Right eye of a patient with early-stage AMD treated with a combination of 8 mg epilutein and 2 mg lutein
(group 1). Color fundus images ( a , d ), fundus autofluorescence ( b , e ), and macular pigment optical density
(MPOD) evaluation ( c , f ) at baseline (BL) ( a–c ) and at the 2-month visit (M2) ( d–f ). Maximum MPOD value was
0.520 optical density units (ODU) at BL, 0.526 ODU at the 1-month visit (M1), and 0.566 ODU at M2. Mean MPOD value was 0.193 ODU at BL, 0.199 ODU at M1, and 0.212 ODU at M2. Maximum and mean macular pigment density was increased.
Table 3. Results for mean macula pigment optical density (in optical density units) in patients with plasma lutein concentration <300 ng/mL at baseline Group 1 (n = 9) Δ vs. BL Group 2 (n = 5) Δ vs. BL Mean BL 0.218±0.04 0.216±0.06 Mean M1 0.220±0.06 +0.002 0.184±0.02 –0.032 Mean M2 0.222±0.05 +0.004 0.188±0.02 –0.028
In group 2, a significant decrease in mean MPOD was observed at M1 and M2 compared to BL. For both groups, changes in maximum MP density and MP volume from BL to M2 were not significant.
Table 3 lists the results of the mean MPOD measure-ments in patients with a plasma lutein concentration <300 ng/mL at BL. Due to the small sample size (9 and 5 patients in groups 1 and 2, respectively) and the high in-terindividual variations, no statistically significant differ-ence could be found.
Pharmacokinetic Results
Plasma concentrations of total xanthophylls increased significantly at M1 and M2 in group 1 and at M1 in group 2 compared to BL ( Fig. 3 ). At M1, significantly increased lutein concentrations were observed for groups 1 and 2 compared to BL. At M1 and M2, significantly increased epilutein concentrations were observed for group 1 and at M1 for group 2 compared to BL.
The 3 ′ -oxolutein plasma concentrations did not
change significantly over time in both groups.
In erythrocytes, lutein was observed in the majority of patients at BL, but only in 5 patients (group 1) and 2 patients (group 2) epilutein was observed ( Fig. 4 ). In 16 patients (group 1) and 6 patients (group 2), epilutein was observed at M1. In group 2, lutein was significantly increased at M1 and M2 compared to BL. Significant increases in epilutein were noted at M1 and M2 in group
1 compared to BL. The 3 ′ -oxolutein concentrations did not change significantly over time in both groups.
In the HDL fraction, lutein was observed in the major-ity of patients at BL, but only in 4 patients (group 1) and 3 patients (group 2) epilutein was observed. In 13 patients (group 1) and 6 patients (group 2), epilutein was observed at M1. A significant increase in epilutein at M1 and M2 was noticed in group 1 and at M1 in group 2 compared to BL. The 3 ′ -oxolutein concentrations did not change sig-nificantly over time in both groups.
In the non-HDL fraction, lutein was found in 16 pa-tients (group 1) and 8 papa-tients (group 2) at BL; only in 1 patient (group 1), epilutein was demonstrated. In 13 pa-tients (group 1) and 6 papa-tients (group 2), epilutein was detected at M1. A significant increase in lutein at M1 and M2 and epilutein at M1 was discovered in group 1 com-pared to BL. In group 2, a significant increase in epilutein was recognized at M1 and M2 compared to BL. The 3 ′ -oxolutein concentrations did not change significantly over time in both groups ( Fig. 5 ).
Discussion
The rationale to conduct the current study was to in-vestigate whether epilutein, a stereoisomer of all- E -lutein, plays a role in the supply of xanthophylls to MP. As out-lined before, epilutein is found in nutritional xanthophyll
6,000 4,000 2,000 Plasma concentration, ng/mL 0 BL
ଶ Lutein ଶ Epilutein ଶ 3-Oxolutein
M1 ** Group 1 M2 6,000 4,000 2,000 Plasma concentration, ng/mL 0 BL M1 Group 2 M2 * * * * ** ** ** **
Fig. 3. Mean plasma concentrations of lutein, epilutein, and 3 ′ -oxolutein in both treatment groups. Significance is given for each compound and for all the xanthophylls versus baseline (BL). M1, 1-month visit; M2, 2-month visit. * p > 0.05, * * p < 0.05.
sources after cooking of vegetables and in fish skin [18, 35] . Epilutein was shown to occur in human plasma and MP. Moreover, a correlation between the plasma concen-trations of epilutein and the stage of AMD has been
ob-served [23] . As of today, epilutein is considered a “non-dietary xanthophyll” and sometimes even a “degradation product” of lutein. We hypothesized that epilutein may support the lutein supply to MP. To the best of our
knowl-2,000 1,500 1,000 500 Concentration in er ythr ocyt es, ng/mL 0 BL
ଶ Lutein ଶ Epilutein ଶ 3-Oxolutein
M1 Group 1 M2 6,000 4,000 2,000 Concentration in HDL, ng/mL 0 BL M1 Group 1 M2 8,000 6,000 4,000 2,000 Concentration in non-HDL, ng/mL 0 BL M1 Group 1 M2 2,000 1,500 1,000 500 Concentration in er ythr ocyt es, ng/mL 0 BL M1 Group 2 M2 6,000 4,000 2,000 Concentration in HDL, ng/mL 0 BL M1 Group 2 M2 8,000 6,000 4,000 2,000 Concentration in non-HDL, ng/mL 0 BL M1 Group 2 M2 *** ** ** * *** ** * ** *** ** *** ***
Fig. 4. Mean concentrations of lutein, epilutein, and 3 ′ -oxolutein in erythrocytes and HDL- and non-HDL fractions of lipoproteins for both treatment groups. Significance is given compound specific versus baseline (BL). M1, 1-month visit; M2, 2-month visit. * p > 0.05, * * p < 0.05, * * * p < 0.01.
edge, this study is the first to evaluate the administration of epilutein in humans. Epilutein is found in nutritional xanthophyll sources and was shown to occur in the plas-ma and MP [18, 36, 37] . Moreover, a correlation between
the plasma concentrations of epilutein and the stage of AMD has been observed [23] .
Oral administration of xanthophylls to the eye is as-sociated with their absorption into the blood, uptake into
600 400 200 Concentration in er ythr ocyt es, ng/mL 0 BL ଶ Lutein ଶ Epilutein M1 Group 1 M2 1,000 750 750 500 Concentration in HDL, ng/mL 0 BL M1 Group 1 M2 1,000 750 500 250 Concentration in non-HDL, ng/mL 0 BL M1 Group 1 M2 600 400 200 Concentration in er ythr ocyt es, ng/mL 0 BL M1 Group 2 M2 1,000 750 250 500 Concentration in HDL, ng/mL 0 BL M1 Group 2 M2 1,000 750 500 250 Concentration in non-HDL, ng/mL 0 BL M1 Group 2 M2
Fig. 5. Mean concentrations of lutein and epilutein in erythrocytes and HDL- and non-HDL fractions of lipo-proteins in patients with plasma lutein concentration <300 ng/mL at baseline (BL) in group 1 ( n = 9) and group 2 ( n = 5). M1, 1-month visit; M2, 2-month visit.
lipoproteins and absorption by erythrocytes. The study at hand investigated the concentrations of lutein, epilutein, and 3 ′ -oxolutein, a central metabolite of the xanthophyll turnover, in plasma, erythrocytes, and lipoproteins after oral administration of either a combination of 8 mg epi-lutein and 2 mg epi-lutein or 10 mg epi-lutein alone for 8 weeks. A daily dose of 10 mg xanthophylls is within the usual dose range for dietary intervention studies. The combina-tion of 8 mg epilutein and 2 mg lutein is based on epide-miological evidence that 2 mg lutein are the average daily nutritional supply in humans so that 8 mg epilutein can be considered as dietary intervention [38, 39] .
Several studies showed that MPOD is related to the nutritional xanthophyll status and that dietary treatment with xanthophylls increases MPOD [6, 14, 40] . Epidemi-ological studies indicate that a higher MPOD may be as-sociated with a lower risk to develop AMD. Results from relevant studies are still controversial although there are more studies pointing to a link between MPOD and AMD [41–43] than those that deny a link [44, 45] .
Patients diagnosed with early-stage AMD according to the AREDS criteria were chosen for this study as this pop-ulation constantly shows a low MPOD, which is consid-ered a possible target for treatment to prevent and/or delay disease progression [38] .
It is currently accepted that MPOD is correlated with the xanthophyll status of the blood [46] and that an ade-quate MPOD is maintained only with adeade-quate nutrition-al xanthophyll supply [8] . Therefore, a thorough ophthnutrition-al- ophthal-mological investigation including MPOD assessment was part of this study to preliminarily assess a potential clini-cal efficacy of the treatment.
MPOD was measured using fundus reflectance, which is a reliable, objective methodology with high accuracy. In 2010, Schweitzer et al. [47] reported the reproducibil-ity of all parameters obtained by this technique to <6%. This method has been validated by similar results in other studies [48–51] .
MOPD measurements revealed a significant increase in the epilutein/lutein supplementation group but a sig-nificant decrease in the lutein supplementation group. Due to the short treatment period, these results should be considered cautiously, particularly because the absolute changes in MPOD observed are not clinically relevant compared to long-term supplementation studies. On the other hand, these results may point to a different short-term response in both supplementation groups. Although MPOD values seem to be significantly different between groups 1 and 2 at BL, and some reports suggested that subjects with low MPOD tend to show a larger increase
than subjects with high MPOD [52] , analysis of all the individual increments revealed that in group 1 a signifi-cantly higher number of subjects experienced an incre-ment than in group 2, with BL MPOD values being equal.
The study results confirmed that an 8-week supple-mentation with lutein/epilutein or lutein alone yield a substantial increase in these compounds in plasma, eryth-rocytes, and HDL- and non-HDL fractions.
The most striking results observed is that supplemen-tation with lutein alone yielded over time substantial concentrations of epilutein in plasma, erythrocytes, and HDL- and non-HDL fractions. This finding strongly sug-gests that lutein is converted to epilutein in the liver and/ or blood. It should be emphasized that the appearance of epilutein after lutein supplementation is not accompa-nied by significant increases in 3 ′ -oxolutein. Interesting-ly, another substantial difference in the pharmacokinetic response was observed between both treatment groups. Supplementation with lutein alone yielded higher lutein concentrations in plasma, erythrocytes, and HDL- and non-HDL fractions at M2 than at BL, but lower concen-trations compared to M1. Considering that lutein is con-verted to epilutein, it can be speculated that this decrease is due to the conversion to epilutein. In support of this interpretation, the corresponding concentrations after supplementation with epilutein/lutein were found to keep its increase from BL more stable.
In a subset of patients with plasma concentrations of lutein <300 ng/mL at BL, the results for MPOD develop-ments and the correlation with xanthophyll concentra-tions is worth investigating. The limit of 300 ng lutein/mL plasma was chosen as most epidemiological studies re-port 150–300 ng lutein (or xanthophylls) as the “normal range” observed in untreated populations [53] . In this subset of patients, a slight increase in the epilutein/lutein supplementation group and a decrease in the lutein sup-plementation group was observed, too. When linking these results with the concentrations of epilutein and lu-tein observed in erythrocytes, and HDL- and non-HDL fractions, it could tentatively be hypothesized that higher epilutein concentrations, especially in erythrocytes and the HDL-fraction, might be causally related to increased MPOD.
Limitations of the present study are the limited num-ber of patients and the short treatment period. The pilot study was planned and performed to assess the potential of epilutein as a new dietary source of eye xanthophylls on the basis of the pharmacokinetic responses and the ef-fect on MPOD. Differences in MPOD between group 1 and group 2 are higher in M1 than in M2, and, therefore,
the necessity for a longer study period is indicated. Based on the promising results observed, further studies evalu-ating the efficacy and safety of epilutein are necessary.
In conclusion, this study provided for the first time evidence that epilutein is not only a degradation product of dietary lutein but also plays a role in the supply of xan-thophylls after oral administration, which warrants fur-ther investigation. It could be demonstrated that epilu-tein is transported in the blood and lipoproepilu-teins and is also absorbed by erythrocytes and that lutein is converted
to epilutein in the blood circulation. The effects on MPOD in this short-time study in a small population indicate that epilutein is at least equivalent to lutein, especially re-garding short-term responses of MPOD in patients with early-stage AMD.
Disclosure Statement
The authors declare no conflicts of interests.
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