Chronic Ingestion of (3R,3′R,6′R)-Lutein and (3R,3′R)-Zeaxanthin in the Female Rhesus Macaque

Abstract

purpose. To investigate how supplementation of the monkey’s diet with high doses of lutein (L), zeaxanthin (Z), or a combination of the two affects the plasma levels and ocular tissue deposition of these carotenoids and their metabolites over time and to determine whether these high doses can cause ocular toxicity.

methods. Eighteen female rhesus monkeys were divided into groups of control (n = 3 control), L-treated (n = 5, 9.34 mg lutein/kg and 0.66 mg zeaxanthin/kg), Z-treated (n = 5, 10 mg zeaxanthin/kg), and L/Z-treated (n = 5, lutein and zeaxanthin, each 0.5 mg/kg). After 12 months of daily supplementation, one control animal, two L-treated animals, two Z-treated animals, and all the L/Z-treated animals were killed. The rest of the monkeys were killed after an additional six months without supplementation. Plasma and ocular tissue carotenoid analyses, fundus photography, and retina histopathology were performed on the animals.

results. Supplementation of monkeys with L and/or Z increased the mean plasma and ocular tissue concentrations of these carotenoids and their metabolites. The mean levels of L and Z in the retinas of the L- and Z-treated animals after 1 year increased significantly over baseline. High dose supplementation of monkeys with L or Z did not cause ocular toxicity and had no effect on biomarkers associated with kidney toxicity.

conclusions. The mean levels of L and Z in plasma and ocular tissues of the rhesus monkeys increase with supplementation and in most cases correlate with the levels of their metabolites. Supplementation of monkeys with L or Z at high doses, or their combination does not cause ocular toxicity.

(3R,3′R,6′R)-lutein (L) and (3R,3′R)-zeaxanthin (Z) are two dihydroxycarotenoids that are found in green and certain yellow-orange fruits and vegetables. ¹ It has been well established that these carotenoids accumulate in the human ocular tissues, particularly in the macula, by way of circulating blood. ^{2 3 4 5 6} Several epidemiologic studies have correlated the high levels of L and Z in the diet and/or serum, with a lower risk of exudative age-related macular degeneration (AMD). ^{7 8} However, high serum levels of L and Z have not been consistently associated with a reduction in the incidence of AMD. ^{9 10 11} For a review of the evidence for protection against AMD by carotenoids and antioxidant vitamins, see the publications by Snodderly ¹² and Schalch et al. ¹³  

Macular carotenoids are thought to function as an optical filter by absorbing short-wavelength visible light and reducing chromatic aberration. ¹³ The absorption of high-energy, short-wavelength light may prevent photochemical damage to cones and retinal pigment epithelium (RPE) in the fovea. ^{13 14} Another mechanism by which macular carotenoids may provide protection against AMD involves their antioxidant function (Mulroy L, IOVS 1998;39:ARVO E-Abstract S129). ^{13 15 16 17 18 19 20} In 1997, we provided preliminary evidence for the photoprotective role of L and Z in the retina as antioxidants by characterizing several oxidative metabolites of these carotenoids. ⁶ Subsequently, a wide spectrum of carotenoids and their metabolites were also identified in the human ocular tissues such as the neural retina, retinal pigment epithelium (RPE/choroid), ciliary body, iris, and lens. ²¹ Another study has also provided support for the protective role of L and Z against oxidative damage in the retina. ²²  

Based on the proposed mechanisms of action of L and Z in the retina, a high macular pigment density (MPD) of these carotenoids would be expected to provide protection against AMD. Snodderly et al. ²³ have shown that supplementation of squirrel monkeys with Z results in an increase in the plasma concentration of this carotenoid without affecting the levels of other carotenoids. In another study, Leung et al. ²⁴ have demonstrated that serum levels of Z in rhesus monkeys can be raised by supplementation with a carotenoid-containing fraction from an extract of Fructus lycii (Gou Qi Zi). In several studies, the relationship between MPD and serum and dietary levels of L and Z has been examined ^{25 26 27 28 29 30} In the mid to late 1990s, several studies of short-term supplementation with the purified or dietary form of L and Z involving a small number of human subjects were conducted to investigate the changes in plasma concentration of these carotenoids. ^{31 32 33 34} In a placebo-controlled, randomized trial, Richer et al. ³⁵ reported an improvement in the visual function when 90 patients with atrophic AMD were supplemented with 10 mg of L alone or in combination with other vitamins and minerals for 12 months. The low-dose human supplementation studies with L and Z conducted to date have not revealed any toxicity and/or side effects. Nonetheless, the safety or lack of ocular toxicity of these carotenoids fed at a high dose would have to be fully established. 

The objectives of the present study were (1) to investigate how a 1-year supplementation of the monkey’s diet with daily chronic doses of L or Z or their combination affects the plasma concentrations of these carotenoids and their metabolites over time; (2) to determine the concentration and tissue deposition of L, Z, and their metabolites in ocular tissues of the animals on supplemented diets; (3) to gain insight into the possible metabolic pathways of L and Z; and (4) to establish whether long-term chronic supplementation with pharmaceutical doses of L or Z can result in ocular toxicity and/or side effects. Chronic doses of L or Z in this study represent an approximately 60-fold increase of the highest dose of L (0.167 mg [0.29 micromoles]/kg body weight per day) administered to human subjects in the study conducted by Richer et al. ³⁵  

Materials and Methods

Animals and Study Design

All procedures were approved by the Institutional Animal Care and Use Committees of the University of Maryland, Comparative Medicine and Veterinary Resources, School of Medicine, Baltimore and the University of Maryland, College Park, and conformed to the NIH guidelines and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 

Eighteen female rhesus macaque (Macaca mulatta) monkeys 1.9 to 3.9 years old and weighing 2.8 to 4.4 kg were ordered from the Republic of China through Shared Enterprises (Richland Town, PA). After arriving in the United States, the animals were kept in quarantine at Covance Research Products (Alice, TX) for 1 month according to Center for Disease Control (CDC, Atlanta, GA) guidelines. The monkeys were then shipped to the animal housing facilities of Veterinary Medicine, University of Maryland (Baltimore, MD) where they spent an additional 10 weeks in quarantine. During this period, all animals received the standardized monkey diet that will be described later. All animals underwent a complete physical examination, and no abnormalities were detected. In addition, complete blood count (CBC) and serum biochemical assays were performed on all monkeys, and the results were found to be within normal limits for age- and sex-matched rhesus macaques. The animals were divided into four groups: C (control), L (L-treated), Z (Z-treated), and L/Z (L/Z-treated). Group C consisted of three control animals, whereas groups L, Z, and L/Z each consisted of five animals that were assigned to treatment. The animals in the group L were supplemented daily with 10 mg/kg body weight of L supplements that contained 6.6% of Z. Therefore the L-treated animals received 9.34 mg (16.42 μM)/kg of L and 0.66 mg of Z (1.16 μM)/kg for 12 months. This is because the L supplements used in this study were isolated and purified from the extracts of marigold flowers that contain approximately 6.6% Z. The removal of Z from L on a large scale that can allow formulation of pure L into supplements cannot be readily accomplished. Further, the presence of small quantities of Z in the L supplements was not deemed to interfere with the main objectives of this study. The animals in group Z received daily supplements of 10 mg (17.60 μM)/kg body weight of Z for 12 months. The monkeys in group L/Z received daily supplements of a combination of L and Z (each at 0.5 mg [0.88 μM]/kg body weight) for 12 months. The objective of the supplementation study with monkeys at a dose of 0.5 mg/kg of each L and Z was to investigate the possible interaction between these carotenoids at a dose that would be three- to sixfold higher than a dose that might be selected in a future human clinical trial. All animals were weighed monthly and received carotenoid doses based on these weights. The supplementation studies with L, Z, and L/Z were run separately, whereas the three control animals were studied at the same time as the L-treated animals. These animals served as controls for all three supplementation studies. The average age (mean ± SEM) of the animals in each group was: control, 3.3 ± 0.3; L-treated, 2.7 ± 0.3; Z-treated, 3.0 ± 0.1; and L/Z-treated, 3.1 ± 0.1 years. After 12 months, one control animal and two L-treated, two Z-treated, and all the L/Z-treated animals were killed. The rest of the animals in the groups L and Z no longer received supplements but were kept under observation for 6 months and then killed. Before euthanasia, animals were first anesthetized with an intramuscular dose of ketamine (10 mg/kg body weight) and then humanely killed with intravenous injection of pentobarbital (100 mg/kg body weight), consistent with the 1993 Report of the American Veterinary Medical Association panel on euthanasia. Throughout these studies, all animals were kept on the same standardized monkey diet, purchased in bulk once every 6 weeks from Harlan Tekland (Madison, WI). Samples of this feed (8775; Harlan Telkand) were extracted and analyzed by HPLC according to our published method, to determine the carotenoid levels of this diet. ^{36 37} The results indicated that, on average, th diet contains 5.49 μg (9.65 nmol)/g feed of total L (all-trans+cis) and 1.47 μg (2.58 nmol)/g feed of total Z (all-trans+cis). Each monkey ate approximately 180 to 240 g/d of the standardized diet. The light cycle for all the animals consisted of 12 hours of light and 12 hours of dark. 

Source of L and Z Supplements and Carotenoid Standards

Commercially available L (Kemin Health, Des Moines, IA) contains approximately 6.6% zeaxanthin (Z). Commercially available Z are synthesized by DSM Nutritional Products (Basel, Switzerland) and did not contain any L. The supplemental doses of L and Z were identically formulated by DSM Nutritional Products into 5% water-dispersible beadlets by using their controlled release technology (ActiLease) and provided for the present study. The lutein beadlets (lutein 5%TG) were shown by HPLC to consist of the following geometrical isomers: all-trans- (94.5%), 9-cis- (0.3%), 9′-cis- (0.2%), and 13-cis+13′-cis (5%). In addition, the lutein beadlets contained a total of 93.4% lutein (trans+cis) and 6.6% of zeaxanthin (total L:Z = 14). The zeaxanthin beadlets (5%TG Optisharp; DSM) contained all-trans-Z (84%) as well as its 9-cis- (1%), 13-cis- (13%), and 15-cis- (2%) isomers. All supplements were stored in well-sealed aluminum bags within plastic bags in a refrigerator at 5°C to protect them from moisture, air, and light. The stability and the composition of the L and Z beadlets were monitored by extraction and HPLC analysis throughout the study; no significant changes in quantitative and qualitative profiles of these supplements were found. Daily doses of L and/or Z were mixed with bananas and given to the animals as a treat. Standard samples of L and Z and their metabolites for qualitative and quantitative measurements of these compounds by HPLC were from our large collections of carotenoids that had been either synthesized or isolated from natural sources. ^{36 37 38 39}  

Extraction of Lutein and Zeaxanthin Beadlets

Beadlets of L or Z were extracted and analyzed by HPLC on a silica-based nitrile bonded column according to our published procedures. ^{6 36 37} For spectrophotometric analysis, the concentrations of L (λ_max = 445 nm, E^1% = 2550) and Z (λ_max = 450 nm, E^1% = 2540) in the extracts were measured in ethanol at their corresponding absorption maximum and extinction coefficient. ⁴⁰  

Analysis of Carotenoids and Their Metabolites in Plasma and Ocular Tissues of the Monkeys

Fasting blood samples (2 × 10 mL) were collected from each animal via venipuncture at baseline, months 6, 12, and 18 (follow up animals only) into tubes that were protected from light, and the tubes were centrifuged at 1000g for 20 minutes. The plasma was separated and immediately stored at −80°C until extraction and HPLC analysis. At death, ocular tissues (neurosensory retina [containing no RPE-choroid], ciliary body, iris, and lens) were removed, frozen, and stored at −80°C. Plasma and ocular tissues were extracted for carotenoid analysis by HPLC according to our previously published procedures. ^{6 21 41} all-trans-(3R,3′R,6′R)-L, all-trans-(3R,3′R)-Z, and their cis-geometrical isomers as well as their metabolites, 3′-epilutein and 3-hydroxy-β,ε-carotene-3′-one (3′-oxolutein), were separated by HPLC on a silica-based nitrile bonded column. ^{6 21} In addition, neural retinas from all animals were analyzed on a chiral HPLC column according to our published procedure. This HPLC method allows for the simultaneous separation of (3R,3′R)-Z, (3R,3′S; meso)-Z, (3S,3′S)-Z, (3R,3′R,6′R)-L, and 3′-epilutein. ⁴¹  

One of the animals in the control group was killed after 12 months and the other two after 18 months. Because the animals in this group did not receive supplements of L and/or Z, the average (mean ± SEM) of the carotenoid levels in the ocular tissues of all three animals was used in statistical analysis. Only one eye from the control animal killed at month 12 was available for carotenoid analysis as the other eye was used for retinal histopathology. At month 18, both eyes from the remaining animals in the control group were analyzed for carotenoids and the values were averaged. After 12 months of supplementation, two animals from the L-treated group and two animals from the Z-treated group were killed; one eye from each animal was subjected to histopathology and the other eye was used for carotenoid analysis. The rest of the L- and Z-treated animals were killed at the end of month 18 (6 months after the end of the supplementation period) and both eyes were analyzed for carotenoids. No retina histopathology was performed on the L/Z treated animals and both eyes from all animals were analyzed for carotenoids at death at month 12. In cases in which the right and the left eyes of the monkeys were available, the average levels of L, Z, and their metabolites were used. The Z levels were initially determined from the HPLC analysis of the extracts from ocular tissues on a silica-based nitrile bonded column. Because this HPLC column cannot separate Z and meso-Z, the concentrations of these carotenoids in the retinas of the monkeys were determined by further HPLC analysis on a chiral column. However, in the case of ciliary body, iris, and lens, the levels of Z and meso-Z were too low for chiral HPLC analysis, and consequently only the total concentrations of these carotenoids were measured. 

Urinary Creatinine Analysis and Total Protein Assay

Urine samples (10–20 mL) were collected over a period of 4 to 5 hours from control and treated monkeys at baseline and months 6, 12, and 18 (follow-up animals only). The standard method for collecting urine was to wait until the primates urinated in a clean pan (no fecal matter present) in their respective cages, and the samples were then aspirated using sterile syringes. The samples were centrifuged at 3000g for 10 minutes, and the supernatants were stored at −80°C until analysis. The Sigma Diagnostics (St. Louis, MO) creatinine method was used for quantitative determination of urinary creatinine (milligrams/total volume of urine/animal). ⁴²  

Total protein in urine samples was measured using the bicinchoninic acid (BCA) protein assay (Pierce Biotechnology, Rockford, IL), which is a detergent-compatible formulation based on BCA for the colorimetric detection and quantitation of total protein. ⁴³ The absorbance of all samples was measured at 562 nm within 10 minutes. The standard curve was used to determine the protein concentration of each sample. The excretion of urinary protein was expressed as milligrams of protein per milligram of creatinine. 

Ophthalmic Examination and Fundus Photography

All animals were subjected to ophthalmic examination at baseline and at 6, 12, and 18 months (follow-up animals only), which provided comparison with the photographic assessment. Anterior segment evaluations and direct ophthalmoscopy were performed and an overall stereoscopic view of the fundus and vitreous including the posterior pole and anterior view extending to the equator in all quadrants was obtained. At baseline examinations, special attention was paid to macular abnormalities. Color fundus photographs of each eye were obtained from all animals at baseline, and at months 6, 12, and 18. Initially, fundus photography was performed with the animals under ketamine-xylazine anesthesia, with a digital hand-held nonmydriatic fundus camera (Nidek, Gamagori, Japan), which produced a digital image density of 2 megapixels. However, most of the photographs were taken with another fundus camera (Topcon, Tokyo, Japan) and slide film (Kodachrome 100 plus; Eastman Kodak, Rochester, NY) that produced a higher-resolution image than the Nidek. Adequate dilation of the pupil was usually achieved with 1 drop of 1.0% tropicamide and 1 drop of 2.5% phenylephrine hydrochloride. This was followed by a second set of drops 10 minutes later. If adequate dilation was not achieved with this regimen, 1 drop of 1% atropine was added. Fundus photographs were taken through the dilated pupil of each eye of each monkey, concentrating on the posterior pole. 

Retina Histopathology

One of the eyes from each monkey in groups C (control), L, and Z that were killed after 12 months was subjected to complete retinal histopathology, and the other eyes were used for extraction and carotenoid analysis. Eyes for histopathology were enucleated and immediately placed in one-quarter strength Karnovsky fixative at room temperature after making a slit at the limbus. Whole eyes were fixed overnight before the anterior segment was removed. The tissues were trimmed to include the posterior pole region of the eye, which contain the optic disc and the macula, and returned to fresh fixative until processing. The tissues were then washed, dehydrated, infiltrated, and embedded in glycol methacrylate (JB-4; Polysciences, Warrington, PA). Cured blocks were trimmed and sectioned along an axis from the nasal disc to the temporal macula. Serial sections containing disc and macula were collected until the foveal center was reached. 

Sections were stained with thionin or periodic acid-Schiff and hematoxylin and examined by light microscopy. 

Statistical Analyses

Statistical analyses of the concentrations of carotenoids in the plasma and eye tissues (neural retina, ciliary body) were performed by analysis of covariance (ANCOVA) with repeated measurements (SAS ver. 8.2; SAS Institute Inc., Cary, NC). For statistical analysis, body weight was used as the covariate. The protected least significant difference (PLSD) test was used to determine whether the plasma concentrations of L, Z, and their metabolites were significantly different at baseline and at months 6, 12, and 18 and between supplement groups. The same statistical analyses were performed for the levels of L, Z, and their metabolites in the eye tissues (retina and ciliary body) at months 12 and 18. 

The PLSD test was only performed when the ANCOVA was significant. Goodness-of-fit statistics were used to select an appropriate variance-covariance structure for the repeated measures. After selecting the variance-covariance structure, nonsignificant higher sources of variation were removed one at a time from the initial full model until the model contained only the significant variable and the covariate as well as their significant interactions. P < 0.05 was considered statistically significant. 

Results

Nomenclature

Lutein (L) and zeaxanthin (Z) refer to (3R,3′R,6′R)-lutein and (3R,3′R)-zeaxanthin, respectively; these are the only stereoisomers of these carotenoids found in the diet. 3′-Epilutein and 3′-oxolutein are the common names for (3R,3′S,6′R)-lutein and (3R,6′R)-3-hydroxy-β,ε-carotene-3′-one, respectively. meso-Zeaxanthin or meso-Z refers to (3R,3′S;meso)-zeaxanthin, a nondietary stereoisomer of Z (Fig 1) . 

Plasma Carotenoid Analysis

The major carotenoids in the plasma of the animals in this study were identified as L and Z, and their cis-geometrical stereoisomers as well as their metabolites, 3′-oxolutein and 3′-epilutein. meso-Zeaxanthin was absent in plasma and was detected only in the ocular tissues of the monkeys. The plasma concentrations (mean ± SEM, μM) of L (trans+cis) and Z (trans+cis) in the L-treated (n = 5), Z-treated (n = 5), and L/Z-treated animals (n = 5) in comparison with the control subjects (n = 3) at baseline and at months 6, 12, and 18 are listed in Table 1 . It must be noted that the all-trans- and the cis-isomers of L (9-cis, 9′-cis, 13-cis, 13′-cis, and 15-cis) and Z (9-cis, 13-cis, and 15-cis) were simultaneously separated by HPLC according to our published methods. ^{6 36 37 44} However, because HPLC analyses of the plasma extracts of the animals at various time points revealed no significant changes in the ratio of the trans- to cis-isomers of L and Z, the plasma concentrations of trans- and cis-isomers of these carotenoids were combined. 

The plasma carotenoid data for L-treated and control monkeys were analyzed by ANCOVA with repeated measurements. The assumptions for normality of variances were met. When lutein was the dependent variable, the first-order autoregressive structure presented a better fit based on the goodness-of-fit statistics and was chosen as the variance-covariance structure. The covariate body weight was nonsignificant (P = 0.5100). The mean plasma L level at baseline for monkeys in the control group compared with the mean baseline level in the monkeys in the L-treated group was not significantly different (P = 0.8418). In the control group, the mean plasma L level at baseline was not significantly different from the mean levels at the end of months 6 (P = 0.8861) and 12 (P = 0.8389). In the L-treated group, the mean plasma L levels increased 2.6-fold over baseline at 6 months of supplementation (P < 0.0001) and increased further by 12 months (P < 0.0122). In the three animals remaining in this group after 12 months, plasma L returned to baseline levels by 18 months, after 6 months without supplementation. It should be noted that the supplemental L also contained approximately 6.6% of Z. Therefore, the monkeys in the L-treated group in addition to receiving a daily dose of 9.34 mg (16.42 μM) L/kg also received 0.66 mg (1.16 μM) Z/kg. The mean plasma Z level at baseline in monkeys in the control group compared with the mean value at baseline in the monkeys in the treatment group was not significantly different (P = 0.7991). In the control group, the mean plasma Z level at baseline was not significantly different than the mean levels at 6 (P = 0.0541) and 12 (P = 0.9203) months. The mean plasma Z level in the L-treated group did not increase over baseline at month 6 of supplementation (P = 0.0933), but after 12 months the level was 1.4-fold higher than baseline (P = 0.0005). In the three animals remaining in this group after 12 months, the mean plasma Z returned to baseline by 18 months, after 6 months without supplementation. 

The design of the Z supplementation study (n = 5, 10 mg Z/kg per day) was identical with that of the 12-month L supplementation study. Because no control animals were used with the Z-fed monkeys, the plasma concentrations of L and Z in the Z-fed monkeys were compared with those in the three control animals described earlier. The plasma concentrations of Z were the dependent variable in the repeated measures analysis, and the compound symmetry was chosen as the variance-covariance structure. The covariate body weight was significant (P = 0.0025) as was the weight-treatment interaction (P = 0.0340); both were included in the final model. It should be noted that the mean plasma concentration of Z at baseline in the Z-treated animals was nearly double the mean plasma concentration of this carotenoid in the control animals at baseline. In the Z-treated group, the mean plasma Z level increased 3.6-fold over baseline at 6 months of supplementation (P = 0.0002) and increased further by 12 months (P < 0.0001). In the three animals remaining in this group after 12 months, plasma Z was 1.8-fold lower than baseline by 18 months, after 6 months without supplementation. The mean plasma levels of Z after 6 and 12 months in the Z-fed animals were nearly 12 and 8 times the mean levels of this carotenoid in the control group at these time points, respectively. After 6 months without supplementation (month 18), the mean plasma concentrations of Z in the Z-fed and the control animals were not significantly different (P = 0.8242). Plasma L concentrations in the Z-fed monkeys did not differ across the different time points. 

The mean plasma level of L in the L/Z-treated animals (n = 5) at month 6 was 2.4-fold higher (P = 0.0001) than baseline. However, despite the fact that supplementation with L and Z continued between months 6 and 12, there was no significant difference (P = 0.8856) between the mean plasma level of L at month 12 in comparison with baseline. The mean plasma levels of L in the L/Z-fed animals were not significantly different from those in the control subjects at baseline (P = 0.8814) and 12 months (P = 0.6813), whereas at 6 months this level in the treated animals was 2.5-fold higher than in the control monkeys (P = 0.0004). 

The mean plasma Z level of the L/Z-fed monkeys at month 6 was also significantly higher (3-fold, P < 0.0001) than baseline. Similar to L, the mean Z plasma concentration after 12 months of supplementation was not significantly different (P = 0.4778) in comparison with baseline. Although the mean plasma levels of Z in the L/Z-fed animals were not significantly different from those of the control animals at baseline (P = 0.8098) and 12 months (P = 0.6342), this level in the treated animals at 6 months was 5-fold higher than that in the control animals (P < 0.0001). 

The mean plasma level of L in the L-fed animals at 12 months was 2.6-fold higher than that in the L/Z-fed group (P < 0.0001), whereas this level was not significantly different between the two groups at baseline (P = 0.6719) and 6 months (P = 0.6256). The mean plasma levels of Z in the Z-fed animals at baseline, 6 months, and 12 months were 2-fold (P = 0.0123), 2.5-fold (P < 0.0001), and 6.6-fold (P < 0.0001) higher than those in the L/Z-fed group, respectively. 

Plasma Analysis of L and Z Metabolites

The concentrations of 3′-epilutein and 3′-oxolutein in the plasma of the animals in the control group (n = 3) and the five animals in each of the groups supplemented with L, Z, or L/Z are summarized in Table 2 . For the statistical analysis, where 3′-epilutein was the dependent variable, the first autoregressive was chosen as the variance-covariance structure, whereas for 3′-oxolutein the compound symmetry structure was chosen. The covariate weight was nonsignificant for 3′-epilutein (P = 0.6278) and 3′-oxolutein (P = 0.3930). The mean plasma level of 3′-epilutein at baseline in monkeys in the control group was not significantly different from that of the baseline level in the monkeys in the L-treated group (P = 0.9215). In the control group, the mean plasma level of 3′-epilutein did not differ across the different time points In the L-treated group, the mean plasma level of 3′-epilutein did not change significantly at 6 months versus baseline (P = 0.5980); however, at 12 months, this level was 3.2-fold higher than baseline (P = 0.0124). The mean plasma levels of 3′-epilutein in the L-fed monkeys at 12 months was 4.7-fold higher than those in the control group (P = 0.0003), whereas there were no significant differences in the level of this metabolite among the two groups at baseline (P = 0.9302) and 6 months (P = 0.2039). Meantime, The mean plasma concentration of 3′-oxolutein at baseline for monkeys in the control group compared with the baseline level in the monkeys in the L-treated group was not significantly different (P = 0.4887). In the L-treated group, the mean plasma concentration of 3′-oxolutein increased 2.3-fold over baseline at 6 months of supplementation (P = 0.0010) and increased 2.9-fold over baseline by 12 months (P < 0.0010). However, the increase between months 6 and 12 was not significant (P = 0.1870). The mean plasma levels of 3′-oxolutein in the L-fed monkeys at 6 and 12 months were 2-fold (P = 0.0193) and 2.6-fold (P = 0.0019) higher than those in the control animals at these time points, respectively. 

Supplementation of monkeys with Z for 1 year did not alter the plasma concentration of 3′-epilutein. In addition, no significant difference in the mean plasma levels of 3′-epilutein was observed among the Z-fed and the control group across the different time points. The mean plasma concentration of 3′-oxolutein in the Z-fed monkeys increased 1.2-fold over baseline at 6 months of supplementation (P = 0.0448) and increased 1.6-fold over baseline by 12 months (P = 0.0132). In comparison with the control subjects, a 2.4-fold increase in the mean plasma levels of 3′-oxolutein in the Z-fed animals was noted at 12 months (P = 0.0018). 

Daily supplementation of the monkeys with a combination of L and Z (each 0.5 mg/kg) for 12 months did not result in a significant change in the mean plasma concentration of 3′-epilutein from baseline in comparison with months 6 (P = 0.2167) and 12 (P = 0.5914). The mean plasma level of 3′-epilutein in the L/Z-fed animals at 6 months was 4.3-fold higher than that in the control animals (P = 0.0101). In contrast to 3′-epilutein, the mean plasma concentration of 3′-oxolutein at month 6 in the L/Z-fed monkeys was 1.8-fold higher than baseline (P = 0.0033). At month 12, the mean plasma level of 3′-oxolutein had declined and was no longer different from baseline (P = 0.6853). The mean plasma level of 3′-oxolutein in the L/Z-fed monkeys at 6 months was nearly 2-fold higher than that of the control animals, whereas no significant difference among the two groups was observed at baseline (P = 0.6966) and 12 months (P = 0.8641). 

The mean plasma level of 3′-epilutein in the L-fed monkeys at month 12 was 4.7-fold and 2-fold higher than those of the Z-fed (P < 0.0001) and L/Z-fed (P = 0.0052) groups, respectively. The mean plasma concentration of 3′-oxolutein in the L-, Z-, and L/Z-fed animals at 6 months was not significantly different. However, at 12 months, the level of this metabolite in the L- and Z-fed groups was 2.8-fold higher than that of the L/Z-fed animals (both P = 0.0003). 

Ocular Tissue Analysis of Carotenoids and Their Metabolites

The ocular tissue concentrations (mean ± SEM, nanograms total tissue content) of L, Z, and their metabolites in the eyes (neurosensory retina, ciliary body, iris, lens) of the monkeys with L, Z, or L/Z supplementation for 12 months in comparison with control animals are shown in Table 3 . Supplementation of the monkeys with L for 12 months resulted in a 3.7-fold increase in the mean level of this carotenoid in the retina in comparison with that of the control group (P < 0.0001). In the three remaining animals in the L-fed group, the mean level of L in the retina was not significantly different from that of control animals by 18 months, after 6 months without supplementation (P = 0.2830). The mean level of the Z in the retinas of the L-fed monkeys at month 12 was not significantly different from those of the control animals (P = 0.3545) and the 18-month animals (P = 0.0952). The mean level of meso-Z in the retinas of the two L-treated monkeys that were killed after 12 months was not significantly different from that of the control animals (P = 0.1092) but was 3.4-fold higher (P = 0.0041) than that of the remaining three monkeys in the same group that were killed after 18 months. The mean level of 3′-oxolutein in the retinas of the L-treated monkeys at 12 months was 3.2- and 6-fold higher than those of the control animals (P = 0.0017) and the remaining three animals at month 18 (P = 0.0003), respectively. Meanwhile, the mean level of L in the ciliary body of the L-treated monkeys after 12 months was not significantly different from those of the control subjects (P = 0.2237) and the 18-month animals (P = 0.1749). The mean concentration of L in the iris of the L-treated monkeys at month 12 was 4.2- and 2.9-fold higher than levels in the control subjects and the 18-month animals, respectively. The mean level of L in the lens of the L-treated monkeys at 12 months was not significantly different from that in the control subjects but was 1.8-fold lower in comparison with the 18-month animals. 

The mean concentrations of Z, meso-Z, and 3′-oxolutein in the retinas of the monkeys increased significantly over control levels after 12 months of Z supplementation (P = 0.0002, 0.0014, and 0.0021, respectively), whereas the concentration of L did not (P = 0.1188). At 18 months, levels of all carotenoids had decreased and were no longer different from those in control subjects The mean level of Z in the ciliary body of the Z-treated monkeys after 12 months was 7.7-fold (P = 0.0008) and 3.7-fold (P = 0.0031) higher than those of the control subjects and the 18-months animals, respectively. After 12 months of supplementation with Z, the mean levels of L and 3′-oxolutein in the ciliary body of the monkeys did not change in comparison with those in the control and the 18-month animals. Although no detectable amounts of Z were found in the iris of the animals in the control group, measurable amounts of this carotenoid were detected in the iris of the Z-fed monkeys after 12 months. The mean concentration of Z in the lens of the Z-treated monkeys at 12 months was not significantly different from that in the control subjects, and at 18 months this carotenoid was no longer detectable. 

The mean concentrations of L and Z in the retinas of the L/Z-treated group after 12 months of supplementation with 0.5 mg/kg of each of these carotenoids were not found to be significantly different from those of L (P = 0.9080) and Z (P = 0.7081) of the animals in the control group. Similarly, a comparison of the mean levels of the two metabolites of L and Z in the retinas of the monkeys at 12 months with those of meso-Z (P = 0.0845) and 3′-oxolutein (P = 0.0698) in the control group revealed no significant difference The mean concentrations of L, Z, and 3′-oxolutein in the ciliary body of the L/Z-treated animals after 12 months were not significantly different from those of the control subjects. Only a significant increase in the mean levels of L and Z in the iris of the monkeys was noticeable after 12 months of L/Z treatment in comparison with those in control subjects Supplementation of the monkeys with L and Z for 12 months was not accompanied by a significant change in the mean concentrations of these carotenoids in the lens when compared with those of the control subjects. 

Fundus Photography and Retina Histopathology

No abnormalities were observed in any of the fundus photographs (baseline and months 6 and 12) in animals with L (10 mg/kg) or Z (10 mg/kg) supplementation for 12 months, with the exception of the presence of small, yellow refractile spots in the center of the macula of one of the monkeys in the control group; however, histopathology did not reveal the presence of any yellow crystalline deposits. Before the monkeys were killed at month 12, fundus photographs of one control monkey and two L-fed (10 mg/kg) and two Z-fed (10 mg/kg) monkeys were taken. The eyes of these monkeys were then subjected to histopathology. Gross examination of the posterior pole of the eyes of these monkeys revealed clear vitreous and an unremarkable fundus in all animals with the exception of some scattered refractile spots in the parafoveal region of the monkey in the control group and one of the L-treated monkeys. In histologic sections of the macular region, the neural retinas were unremarkable in all animals, with no evidence of ganglion cell, inner nuclear layer, or outer nuclear layer cell loss or degeneration. The photoreceptor inner and outer segments were intact but outer segments showed some evidence of processing artifact (stretching and separation from the RPE). 

Excretion of Urinary Creatinine and Protein

As shown in Table 4 , there were no significant differences in urinary creatinine excretion in the L- or Z-fed groups compared with control subjects. In contrast, a significant decrease in urinary creatinine excretion was found in the L/Z-fed group. Because the monkeys in the L/Z-treated group were killed at month 12, the samples were unavailable at month 18. Table 5shows that the total urinary protein excretion was not affected by supplementation with L, Z or L/Z. 

Discussion

Plasma Levels of L, Z, and Their Metabolites in Response to Supplementation

To date, several dietary supplementation studies with L and Z involving various species of monkeys have been conducted. ^{23 24 45 46} As our study was in its final phases, Neuringer et al. ⁴⁶ reported a study in which they elegantly demonstrated that rhesus (Macaca mulatta) monkeys respond to either dietary L or Z supplementation, with increases in the serum and macular pigment concentration of these carotenoids, even after life-long xanthophyll deficiency. This well-designed study was particularly significant, because the investigators separated and measured the concentrations of L, Z, and their geometrical isomers in the serum of the monkeys. In the present study, we also selected rhesus monkeys because of their relatively low serum xanthophyll concentrations in comparison with those in other monkey species that have been studied. ^{45 47 48} Therefore, we anticipated that it would be a challenging task to increase the plasma concentrations of L and Z in rhesus monkeys by supplementation with high doses of these carotenoids. In the dietary supplementation studies conducted by Leung et al. ²⁴ and Neuringer et al. ⁴⁶ in which rhesus monkeys were fed a stock diet, the mean serum concentrations of L were in the range of 54 to 74 nM, whereas the mean serum concentrations of Z were in the range of 5 to 58 nM. The results of our study revealed much higher mean plasma concentrations of L (240 ± 20 nM) and Z (120 ± 10 nM) in the control monkeys at baseline than those reported by Leung et al. ²⁴ and Neuringer et al. ⁴⁶ In our study, each animal received approximately 0.99 to 1.32 mg/d (1.74–2.32 μM) L and 0.26 to 0.35 mg/d (0.46–0.62 μM) Z from the standardized monkey diet, regardless of their treatment. Comparison between the L (5.49 μg/g) and Z (1.47 μg/g) content of the diet given to the monkeys in our study with the L (4 to 6 μg/g) and Z (4 to 5 μg/g) content of the stock diet given to the monkeys in the study conducted by Neuringer et al. ⁴⁶ does not provide a reasonable explanation of these differences. However, these differences may be related to the age (3.3 ± 0.3 years) and weight (3.25 ± 0.06 kg) of the control animals in our study compared with the age (11.9 ± 1.3 years) and weight (8.1 ± 0.8 kg) of the monkeys with nonsupplemented diets in Neuringer et al. ⁴⁶ Because the primary focus of our study was to assess the safety of long-term supplementation with L and Z at high doses, these carotenoids were each fed separately at the dose of 10 mg/kg body weight/d (17.58 μM/kg) for 1 year. This dose is approximately five times the dose of these carotenoids in the study conducted by Neuringer et al. In their study, the mean serum concentration of L or Z in the xanthophyll-free rhesus monkeys supplemented with these carotenoids, each at a dose of 2.2 mg/kg per day (3.9 μM), exceeded the levels in monkeys fed a stock diet (4–6 μg/g each of L and Z) by 2 weeks. ⁴⁶ After this time, the concentrations were approximately 10 times as high for L and 10 to 20 times as high for Z. Our results are difficult to compare with those of Neuringer et al. because the study design, age, weight, dose, and duration and frequency of supplementation with L and Z in the studies were quite different. In Neuringer et al., after supplementation with 2.2 mg/kg per day of either L or Z, the mean plasma concentrations of both carotenoids were approximately 0.8 μM after 6 months and 0.5 μM after 12 months. In our study, after 6 months of supplementation of monkeys with 10 mg/kg per day of L or Z, the mean plasma level of these carotenoids were 0.57 and 0.82 μM, respectively (Table 1) . After 12 months, the mean plasma level of L increased to 0.71 μM in the L-fed group and that of Z increased to 0.92 μM in the Z-fed group. Despite the fact that our doses of L and Z were fivefold higher than those in Neuringer et al., the mean blood levels of these carotenoids in the animals in the two studies are within a close range. 

As shown in Table 1 , the mean plasma levels of L in the animals with 9.34-mg/kg (L-fed group) and 0.5-mg/kg (L/Z-fed group) supplementation of this carotenoid after 6 months were not significantly different (P = 0.6256) and increased by 2.6- and 2.4-fold over baseline, respectively. Meanwhile, when Z was fed to the monkeys at a dose of 0.66 mg/kg in combination with a high dose of 9.34 mg/kg L (L-fed group), after 6 months the mean plasma concentration of Z did not increase over baseline (Table 1) . In contrast, when the animals were fed a slightly lower dose of 0.50 mg/kg of each L and Z (L/Z-fed group), a threefold increase in the mean plasma level of Z was achieved after 6 months. These results suggest that when L and Z are both given to the monkeys at comparable doses, L does not affect the mean plasma concentration of Z. On the contrary, a high dose of L fed with a relatively low dose of Z appears to prevent and/or delay the absorption of Z into the plasma at least within the first 6 months. However, in the absence of the plasma Z response of the monkeys supplemented with only a low dose of Z (0.5–0.66 mg/kg), the observed interaction between L and Z remains uncertain. Of interest, after 12 months of supplementation with these doses of Z (0.66 and 0.50 mg/kg), the mean plasma levels of Z in the two treatment groups (L- and L/Z-fed groups) were not significantly different (P = 0.8220). However, between months 6 and 12, whereas the mean level of Z in the L-fed group increased by 1.3-fold over the 6-month level, that of Z in the L/Z-fed group decreased by 2.4-fold. As mentioned earlier, this was also the case with the mean plasma level of L in the L/Z-fed monkeys, which also decreased between months 6 and 12 by 2.3-fold over the 6-month level and returned to baseline by month 12. When Z was fed to the animals alone at a high dose of 10 mg/kg, the mean plasma levels of this carotenoid at months 6 and 12 were 2.5- and 6.6-fold higher than those fed a low dose of L+Z (L/Z-fed group), respectively. When L and Z were fed separately at a high dose of nearly 10 mg/kg, the mean plasma level of Z after 12 months increased by 4-fold over baseline, whereas that of L increased by 3.2-fold (Table 1) . 

Before discussing the changes in the mean plasma concentrations of 3′-epilutein and 3′-oxolutein in the animals with L or Z supplementation, the possible metabolic transformation that can yield these carotenoids must be clarified. Based on evidence from our earlier studies, we proposed that 3′-oxolutein is most likely formed from allylic oxidation of dietary L. ^{6 31 32 41} 3′-Oxolutein can then be reduced to form either 3′-epilutein or revert back to L (Fig. 1) . Alternatively, dietary Z can undergo double-bond isomerization to 3′-epilutein that can be oxidized to form 3′-oxolutein. Therefore, in an overall scheme in which all these possibilities are considered, 3′-oxolutein and 3′-epilutein can be formed from L and/or Z. Although the true nature of these metabolic transformations can only be unequivocally established by supplementation studies with isotopically labeled L or Z, the studies described herein provide an insight into our proposed pathways. 

The mean plasma concentration of 3′-oxolutein in the L-fed monkeys increased by month 6, and, even though this level continued to increase between months 6 and 12, the change was not significant (Table 2) . Meanwhile, an opposite effect with 3′-epilutein was observed, as the mean level of this metabolite in the L-fed animals did not increase significantly from baseline at month 6, but at month 12 the level was significantly higher. These observations are consistent with the oxidation of L to 3′-oxolutein within the first 6 months to build up the level of this metabolite in the plasma to a point where the stereo-controlled reduction of 3′-oxolutein to 3′-epilutein becomes significant (months 6–12). The mean plasma level of 3′-oxolutein in the Z-fed monkeys did increase within the first 6 months of supplementation but by month 12 it was significantly higher than baseline and reached nearly the same concentration achieved in the L-fed monkeys at month 12. Meanwhile, supplementation with Z did not affect the mean plasma level of 3′-epilutein at various time points. These findings suggest that in Z-fed monkeys, 3′-epilutein is not necessarily involved in the formation of 3′-oxolutein, and this metabolite most likely originates from Z by a direct mechanism. 

Carotenoids in the Ocular Tissues of the Monkeys

The major carotenoids in the ocular tissues (retina, ciliary body, iris, lens) of the animals were identified as L and Z. In addition, the retina and the ciliary body of the animals also revealed the presence of 3′-oxolutein and meso-Z. We were interested in the carotenoid profile of the ciliary body because this tissue in humans accumulates a wide range of carotenoids and their metabolites, which may protect against glaucoma and/or presbyopia by an antioxidant mechanism of action. ²¹ As we have proposed earlier, 3′-oxolutein can be formed from L and/or Z and its presence in the ocular tissues may be due to its transport from the circulating blood or the in vivo enzymatic and light-induced metabolic transformation of L and/or Z in the eye. ^{6 21 31 32 36 41 44} However, we have previously shown that the nondietary meso-Z is absent in the human plasma and liver but present in the human retina. ⁴¹ Therefore, although the metabolic origin of 3′-oxolutein in the ocular tissues is not known at present, meso-Z is most likely formed in the retina from dietary L. In a similar study of rhesus monkeys with L and Z supplementation as conducted by Neuringer et al., ⁴⁶ Johnson et al. ⁴⁹ provided additional support for metabolic transformation of L to meso-Z. They demonstrated that meso-Z, which was absent in the retinas of xanthophyll-free and Z-fed monkeys, was present in the retinas of xanthophyll-free monkeys after supplementation with L. In addition, Johnson et al. ⁴⁹ reported that meso-Z was present only in the macular region but not outside the 4-mm area of the macula. To ensure the detection and identification of 3′-oxolutein and meso-Z that are normally present at low concentrations, we analyzed the neurosensory retina for carotenoids and did not dissect and examine the fovea separately. 

Table 3clearly shows a significant increase of 3.7-fold in the mean concentration of L in the retina of the L-fed monkeys in comparison with that in the control subjects, whereas supplementation with low doses of L/Z (L/Z-fed group) did not alter the mean levels of L or Z in this tissue. This may be because the mean concentrations of these carotenoids in plasma of the monkeys peaked on month 6 and thereafter declined to their baseline values. Therefore, it is likely that L and Z are initially incorporated into the retinas of the monkeys in the first 6 months; however, since the plasma levels of these carotenoids decreased significantly between months 6 and 12, the retina levels may follow the same pattern. The significant increase in the mean level of 3′-oxolutein in the retina of the L-fed monkeys after 12 months in comparison with that of control subjects (by 3.2-fold) and the 18-month animals (by sixfold) may be indicative of the in vivo oxidation of L into this metabolite. The mean level of meso-Z in the retina of the L-fed monkeys at month 12 is not significantly increased when compared with that of the control subjects but is 3.4-fold higher than that of the 18-month animals. Because of this large interindividual variability, the 18-months data may be a better measure of the mean baseline level of meso-Z. 

When L and Z were fed to the monkeys separately at a high dose (∼10 mg/kg), Z was slightly better absorbed by the retina than L, as the increase in the mean levels of these carotenoids over baseline were 4.3-fold and 3.7-fold, respectively. 

Between months 12 and 18, during which animals were no longer receiving the supplements, the mean concentrations of L and Z in the retinas of the monkeys were significantly reduced at about the same rate and on month 18 the levels were not significantly different from those in the control subjects. The mean concentration of meso-Z in the retina of the Z-fed monkeys after 12 months was significantly higher than that in the control subjects and the animals killed after 18 months. This is contrary to the finding by Johnson et al. ⁴⁹ who did not detect meso-Z in the retina of Z-fed monkeys. However, it should be noted that the studies conducted by Johnson et al. have clearly demonstrated that meso-Z in the retina of the L-fed monkeys must have originated from L, because the animals in that study were raised on an L- and Z-free diet and received only supplements of pure L that contained no Z, whereas this was not the case in our studies. The mean concentrations of meso-Z and 3′-oxolutein in the retinas of the L/Z-treated animals after 12 months appear to correlate directly with those of L and Z in this tissue and were not significantly different from those in the control subjects. Metabolic studies with isotopically labeled L and Z, can unequivocally establish the origin of meso-Z. Of note, supplementation of monkeys with Z for 12 months resulted in significant increases in the mean levels of this carotenoid in the ciliary body, iris, and lens, whereas in the L-fed monkeys, only a significant increase in the mean level of L in the iris was recorded. 

Safety of Long-Term Supplementation of Monkeys with High Doses of L and Z

The results of fundus photography and histopathology of retinas revealed no abnormalities in any of the animals who received daily high-dose supplements of L or Z for 12 months. The RPE of all the monkeys appeared normal and showed a normal distribution of melanosomes and lipofucsin granules. Bruch’s membranes were normal and free of deposits. The choroids and their cellular components were also normal. In addition, there were no signs of inflammation or an abnormal number of circulating leukocytes in retinal or choroidal blood vessels. Therefore supplementation with L or Z at high doses would not be expected to cause ocular toxicity. Overall, the excreted urinary creatinine (Table 4)and protein (Table 5)data suggest that the various L and Z treatments do not produce any clinical renal damage to the monkeys. 

Conclusions

Daily supplementation of monkeys with L (9.34 mg/kg) for 1 year resulted in a 3.2- and 3.7-fold increase in the mean concentrations of this carotenoid in plasma and retina, respectively. Similarly, supplementation of monkeys with Z (10 mg/kg/d) for 1 year raises the mean levels of Z in plasma and retina by 4.0- and 4.3-fold, respectively. The mean levels of meso-Z and 3′-oxolutein in the retinas of the L- and the Z-fed monkeys also increased significantly. Supplementation of monkeys with L/Z (each at a dose of 0.5 mg/kg per day) for 1 year increased the mean plasma concentration of these carotenoids within the first 6 months, but thereafter the levels returned to baseline. This result was also reflected in the mean levels of L and Z in the retinas of the L/Z-fed monkeys after 12 months. Supplementation of monkeys with L or Z for 1 year at a dose of approximately 10 mg/kg body weight did not cause ocular toxicity and had no effect on the biomarkers associated with kidney toxicity. In the 60-kg human, this dose is equivalent to 600 mg/d of L or Z. Future long-term human supplementation studies with L and Z, which are designed to investigate the efficacy of these carotenoids in the prevention of AMD at a much lower dose (e.g., 0.5 mg/kg or lower), should not present any problems associated with toxicity. 

²  Present affiliation: Department of Nutrition, University of California, Davis, California;

⁷  ATSDR, Division of Toxicology and Environmental Medicine, Atlanta, Georgia.

 Presented at the 14th International Symposium on Carotenoids, Edinburgh, Scotland, UK, July, 2005.

 Supported by the National Eye Institute Grant EY12658–01A1 (FK).

 Submitted for publication February 22, 2006; revised June 9 and August 9, 2006; accepted October 11, 2006.

 Disclosure: F. Khachik, (P); E. London, None; F.F. de Moura, None; M. Johnson, None; S. Steidl, None; L. DeTolla, None; S. Shipley, None; R. Sanchez, None; X.-Q. Chen, None; J. Flaws, None; G. Lutty, None; S. McLeod, None; B. Fowler, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Figure 1.

 

Chemical structures of dietary (3R,3′R,6′R)-lutein and (3R,3′R)-zeaxanthin and their metabolites identified in humans.

Table 1.

 

Changes in the Mean Concentrations of L and Z in the Plasma of Animals in the Control Group and the Groups Receiving L, Z, or L/Z Supplementation for 12 Months

Animals	Lutein							Zeaxanthin
		Baseline	6 Months	12 Months	18 Months	Baseline	6 Months	12 Months	18 Months
Control	0.24 ± 0.02x a	0.24 ± 0.02z a	0.23 ± 0.04y a	0.21 ± 0.05x a	0.12 ± 0.01y a	0.07 ± 0.01z a	0.12 ± 0.03y a	0.12 ± 0.03x a
L treated	0.22 ± 0.03x c	0.57 ± 0.08x b	0.71 ± 0.10x a	0.23 ± 0.05x c	0.11 ± 0.01y b	0.12 ± 0.02z b	0.15 ± 0.02y a	0.11 ± 0.02x b
Z treated	0.25 ± 0.06x a	0.35 ± 0.07z a	0.38 ± 0.07y a	0.29 ± 0.04x a	0.23 ± 0.03x c	0.82 ± 0.07x b	0.92 ± 0.10x a	0.13 ± 0.02x c
L/Z treated	0.26 ± 0.03x b	0.61 ± 0.06x a	0.27 ± 0.04y b		0.11 ± 0.02y b	0.33 ± 0.04y a	0.14 ± 0.02y b

 Data are the mean ± SEM of plasma concentrations of L and Z (trans + cis) (μmol/L). Three animals were in the control group (receiving no supplement), and five animals each were in the L-, Z-, and L/Z-treated groups. At the end of the supplementation period (12 months), one control animal, two L-fed, two Z-fed, and all L/Z-fed animals were killed, and the rest of the animals were kept under observation for 6 months without supplementation and then killed at 18 months. For each carotenoid, mean data followed by different superscript letters (a, b, c) within the same row are significantly different at the 5% level of significance; data followed by different subscript letters (x, y, z) within the same column are significantly different at the 5% level of significance.

Table 2.

 

Changes in the Mean Concentrations of L and Z Metabolites in the Plasma of Animals in the Control Group and the Groups Receiving L, Z or L/Z Supplementation for 12 Months

Animals	3′-Epilutein					3′-Oxolutein
		Baseline	6 Months	12 Months	Baseline	6 Months	12 Months
Control	0.012 ± 0.001x a	0.007 ± 0.002y a	0.009 ± 0.002y a	0.040 ± 0.012x,y a	0.033 ± 0.010y a	0.033 ± 0.010y a
L treated	0.013 ± 0.003x b	0.018 ± 0.004x,y b	0.042 ± 0.011x a	0.029 ± 0.004y b	0.067 ± 0.012x a	0.085 ± 0.012x a
Z treated	0.011 ± 0.001x a	0.009 ± 0.001y a	0.009 ± 0.001y a	0.050 ± 0.010x b	0.060 ± 0.01x a,b	0.080 ± 0.010x a
L/Z treated	0.019 ± 0.004x a	0.030 ± 0.010x a	0.021 ± 0.004y a	0.034 ± 0.005x,y b	0.062 ± 0.008x a	0.030 ± 0.002y b,c

 Data are the mean ± SEM plasma concentration of L and Z metabolites (μmol/L), and are as described in Table 1 .

Table 3.

 

Ocular Tissue Contents of L, Z, meso-Z, and 3′-Oxolutein in Monkeys in the Control Group and the Groups with Diets Supplemented with L, Z, and L/Z

Animals	Retina (ng/tissue) (picomol/tissue)							Ciliary Body (ng/tissue)					Iris (ng/tissue) Lutein Zeaxanthin, §	Lens (ng/tissue) Lutein Zeaxanthin, §
		Lutein	Zeaxanthin	meso-Zeaxanthin	3′-Oxolutein	Lutein	Zeaxanthin	3′-Oxolutein
Control* , † (n = 3)	11.05 ± 2.19b,c (19.4 ± 3.8)	4.78 ± 0.62b (8.4 ± 1.1)	3.20 ± 0.30b,c (5.6 ± 0.5)	1.24 ± 0.10b,c (2.2 ± 0.2)	4.03 ± 0.17a	1.67 ± 0.38b	0.60 ± 0.06a	0.40 ± 0.01 ND	0.60 ± 0.10 0.18 ± 0.20
L treated
12 mo L, † (n = 2)	40.4 ± 1.41a (71.0 ± 2.5)	7.56 ± 0.71b (13.3 ± 1.3)	4.87 ± 0.70b (8.6 ± 1.2)	3.97 ± 0.55a (7.0 ± 1.0)	7.12 ± 0.22a	1.84 ± 0.58b	1.09 ± 0.06a	1.68 ± 0.91 ND	0.76 ± 0.08 ND
12 mo L + 6 mo L− free, ‡ (n = 3)	6.20 ± 0.70c (10.9 ± 1.2)	2.34 ± 0.78b (4.1 ± 1.4)	1.45 ± 0.44c (2.5 ± 0.8)	0.66 ± 0.08c (1.2 ± 0.1)	3.64 ± 1.20a	1.39 ± 0.36b	NDb	0.59 ± 0.19 ND	1.36 ± 0.33 ND
Z Treated
12 mo Z, † (n = 2)	19.2 ± 9.07b (33.8 ± 16)	20.4 ± 3.88a (35.9 ± 6.8)	7.20 ± 1.37a (12.7 ± 2.4)	3.87 ± 0.82a (6.8 ± 1.5)	4.97 ± 2.55a	12.9 ± 4.28a	1.34 ± 0.50a	0.26 ± 0.05 0.40 ± 0.01	0.34 ± 0.01 0.37 ± 0.05
12 mo Z + 6 mo Z− free, ‡ (n = 3)	7.84 ± 1.03c (13.8 ± 1.8)	4.45 ± 0.42b (7.8 ± 0.7)	1.82 ± 0.45c (3.2 ± 0.8)	0.84 ± 0.27c (1.5 ± 0.5)	7.18 ± 1.04a	3.53 ± 0.62b	0.92 ± 0.19a	0.64 ± 0.01 ND	0.63 ± 0.10 ND
L/Z Treated
12 mo L/Z, ‡ (n = 5)	11.5 ± 2.53b,c (20.2 ± 4.5)	5.66 ± 2.02b (10.0 ± 3.6)	1.75 ± 0.54c (3.1 ± 0.9)	2.37 ± 0.33b (4.2 ± 0.6)	4.90 ± 1.66a	3.92 ± 1.59b	1.17 ± 0.25a	0.77 ± 0.18 0.52 ± 0.15	0.56 ± 0.07 0.39 ± 0.06

 Mean tissue contents (ng/tissue) of L, Z, meso-Z, and 3′-oxolutein in the ocular tissues of monkeys after 12 months of supplementation and 6 months of follow-up. For each carotenoid data followed by different superscript letters (a, b, c) within the same column are significantly different at the 5% level of significance. Values for retinas are also expressed in picomoles/tissue in parentheses. ND, not detected.

*  Mean ± SEM for one control animal killed after 12 months and two control animals killed after 18 months.

†  Only one eye of each animal was available for analysis at month 12, as the other eye was used for histopathology, whereas at month 18, both eyes were analyzed for carotenoids and the values were averaged (data expressed as mean ± SEM).

‡  Includes average results for the left and the right eyes in all animals.

§  Combined data for Z and meso-Z.

Table 4.

 

Excretion of Urinary Creatinine from the Animals in the Control Group and the Groups Supplemented with L, Z, and L/Z for 12 Months

Animals	Baseline	6 Months	12 Months	18 Months
Control	2.79 ± 0.52 (3)	5.87 ± 6.01 (3)	6.80 ± 2.38 (3)	4.34 ± 0.33 (2)
L Treated	2.88 ± 1.46 (5)	6.79 ± 3.49 (5)	6.77 ± 3.37 (5)	3.16 ± 1.92 (3)*
P-value	0.924	0.788	0.987	0.472
Z Treated	5.59 ± 4.32 (5)	4.96 ± 3.01 (5)	8.16 ± 6.38 (5)	4.14 ± 4.68 (3)*
P-value	0.320	0.780	0.741	0.959
L/Z Treated	2.60 ± 1.03 (5)	3.47 ± 1.34 (5)	0.96 ± 0.17 (4)	—, †
P-value	0.783	0.402	0.001, ‡	—, †

 Data are the mean ± SD concentration (mg/total volume of urine/animal) of urinary creatinine excreted by monkeys at various intervals throughout the supplementation studies. All animals received a standardized monkey diet, and urine samples were collected over a period of 4 to 5 hours. The number of samples is shown in parentheses.

*  Data for monkeys after 12 months of supplementation and 6 months of treatment-free follow up.

†  L/Z-treated animals were all killed at 12 months and treatment-free follow-up samples at 18-month were therefore not available.

‡  Significant difference versus baseline.

Table 5.

 

Excretion of Urinary Protein from the Animals in the Control Group and the Groups with L, Z, and L/Z Supplementation for 12 Months

Animals	Baseline	6 Months	12 Months	18 Months
Control	5.89 ± 0.32 (3)	6.96 ± 3.76 (3)	4.38 ± 0.04 (3)	5.08 ± 0.66 (2)
L treated	5.01 ± 1.22 (5)	6.03 ± 1.34 (5)	5.06 ± 1.17 (5)	4.04 ± 2.13 (3)*
P-value	0.280	0.621	0.364	0.567
Z treated	5.20 ± 0.92 (5)	6.08 ± 1.41 (5)	3.73 ± 1.26 (5)	3.46 ± 0.81 (3)*
P-value	0.268	0.644	0.423	0.102
L/Z treated	7.19 ± 1.41 (5)	6.44 ± 0.99 (5)	9.63 ± 7.03 (4)	—, †
P-value	0.176	0.770	0.262	—, †

 Data are the mean ± SD concentration of urinary protein (mg protein/mg creatinine) excreted from monkeys at various intervals throughout the supplementation studies. All animals received a standardized monkey diet. The number of samples is shown in parentheses.

*  Data for monkeys after 12 months of supplementation and 6 months of treatment-free follow-up.

†  L/Z-treated animals were all killed at 12 months, and treatment-free follow-up samples at 18 months were therefore not available.

The authors thank Regina Goralczyk, DSM Nutritional Products Ltd., Human Nutrition and Health Division, Basel, Switzerland, for providing the beadlets of lutein and zeaxanthin.