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American Journal of Clinical Nutrition, Vol. 79, No. 3, 410-417, March 2004
© 2004 American Society for Clinical Nutrition

ORIGINAL RESEARCH COMMUNICATION

Plasma kinetics of zeaxanthin and 3'-dehydro-lutein after multiple oral doses of synthetic zeaxanthin1,2,3,4

Dieter Hartmann, Petra A Thürmann, Volker Spitzer, Wolfgang Schalch, Birke Manner and William Cohn

1 From the Department of Human Nutrition and Health, Roche Vitamins Ltd, Basel, Switzerland (DH, VS, WS, and WC), and the Institute of Clinical Pharmacology, HELIOS Klinikum Wuppertal, University of Witten/Herdecke, Wuppertal, Germany (PAT and BM).

2 The results of this publication are part of the thesis of Birke Manner submitted in fulfillment of the requirements for the degree of Doctor of Medicine at the University of Witten/Herdecke, Germany.

3 Supported by Roche Vitamins Ltd.

4 Address reprint requests to W Cohn, Department Human Nutrition and Health, Roche Vitamins Ltd, Grenzacherstrasse 124, CH-4070 Basel, Switzerland. E-mail: willy.cohn{at}roche.com.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: Zeaxanthin is hypothesized to reduce the risk of age-related macular degeneration; however, kinetic information is limited.

Objectives: The objective was to investigate the plasma kinetics of synthetic zeaxanthin after repeated oral doses and to assess the possible influence of other carotenoids on plasma zeaxanthin concentrations.

Design: After a run-in of 3 d, 20 healthy volunteers assigned to 2 parallel dose groups received once daily oral doses of either 1 mg (1.76 µmol) or 10 mg (17.6 µmol) zeaxanthin for 42 d. Plasma concentration-time profiles on days 1 and 42, concentrations immediately before zeaxanthin intake during the dosing period, and concentrations after the last dose until day 76 were monitored.

Results: all-E-Zeaxanthin concentrations increased from 0.048 ± 0.026 µmol/L at baseline to 0.20 ± 0.07 and 0.92 ± 0.28 µmol/L with 1 and 10 mg zeaxanthin, respectively. The dose-normalized bioavailability of all-E-zeaxanthin after the10-mg dose was 40% lower (P < 0.001) than after the 1-mg dose. Other kinetic parameters did not differ significantly between groups. After 17 d of dosing, >90% of steady state concentrations were reached, which was compatible with an effective half-life for accumulation of 5 d. The terminal elimination half-life was 12 ± 7 d (n = 20). The time course of plasma all-E-3-'dehydro-lutein concentrations resembled that of all-E-zeaxanthin. The data provided evidence that all-E-3-'dehydro-lutein was derived from all-E-zeaxanthin. Concentrations of other carotenoids were not affected. Zeaxanthin was well tolerated.

Conclusion: Long-term oral intake of 1 and 10 mg zeaxanthin as beadlets increases plasma zeaxanthin concentrations {approx}4- and 20-fold, respectively. Evidence that all-E-3-dehydro-lutein is formed from zeaxanthin was strong.

Key Words: Xanthophyll • carotenoids • zeaxanthin • multiple oral dose kinetics • beadlet formulation • all-E-3'-dehydro-lutein • lutein


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Lutein and zeaxanthin are the carotenoids that constitute the yellow spot (macula lutea) in the center of the macula (1, 2). The preferential accumulation of lutein and zeaxanthin in the macula and the capability of these carotenoids to absorb potentially damaging blue light and to quench reactive oxygen species—a property shared with other carotenoids—have raised expectations that their ingestion may lower the risk of age-related macular degeneration (AMD). AMD is a degenerative disease that can lead to blindness.

Results from 3 epidemiologic case-control studies support these expectations. According to one of these studies, the concentrations of lutein and zeaxanthin in retinas with AMD are lower than in those without AMD (3). Furthermore, plasma concentrations (4) and the dietary ingestion (5) of these carotenoids were inversely correlated with a lower relative risk of AMD.

Fruit and vegetables are natural sources of dietary zeaxanthin (6-8). Estimates of the average total dietary intake of zeaxanthin plus lutein range from 2 to 26 mg/d (9, 10). Together with an estimate of the average ratio of lutein to zeaxanthin in food of 5:1 (11), an average dietary intake of zeaxanthin from 0.4 to 5.2 mg/d can be calculated.

In contrast with lutein, for which plasma dose-response data are available for several formulations at different doses (12, 13), the plasma kinetics of supplemental zeaxanthin have been investigated only in one previous study; in that study, <=30 mg/d was administered for {approx}4 mo (14). This lack of data is probably related to the fact that formulated zeaxanthin has only recently been introduced into the market. The availability of synthetic zeaxanthin formulated as water-dispersible beadlets allowed for a more in-depth kinetic study of this carotenoid.

The objective of the present study was to acquire more kinetic data on zeaxanthin after multiple, once daily, supplemental oral doses of 1 and 10 mg of the synthetic carotenoid in beadlet formulations. Data to be generated were the responses of plasma zeaxanthin concentrations after the first dose and at steady state, the time required to attain steady state, accumulation properties, effective half-life for accumulation (teff), terminal elimination half-life, and dose proportionality. The possible influence of zeaxanthin on plasma concentrations of other carotenoids, carotenes, retinol, {alpha}-tocopherol and lipids, and the safety of zeaxanthin at these doses, were also to be assessed. In particular, the plasma kinetics of all-E-3'-dehydro-lutein (3R,6R-3'-hydroxy-ß,{epsilon}-carotene-3'-one) and its possible origin from lutein or zeaxanthin were additionally investigated. The structural formula of all-E-zeaxanthin, all-E-3'-dehydro-lutein, and all-E-lutein are shown in Figure 1Go.



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  		FIGURE 1.. Structural formula and nomenclature of all-E-zeaxanthin, all-E-3'-dehydro-lutein, and all-E-lutein.

 

   SUBJECTS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Subjects
Ten men and 10 women participated in the study. Demographic data and baseline concentrations (Cb) of zeaxanthin, lutein, and all-E-3'-dehydro-lutein are given in Table 1Go. As part of a prestudy screening, a medical history, a physical examination, and clinical laboratory tests (including a clinical chemistry panel, hematologic tests, a test for hepatitis B, and serology tests for HIV-1 and HIV-2) showed no evidence of preexisting clinically relevant medical disorders. Women were neither pregnant nor breastfeeding and reported using a safe method of contraception. The subjects were requested to avoid excessive intakes of food items rich in zeaxanthin (eg, corn, spinach, broccoli, and dates) and to maintain their usual lifestyle activities.


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  		TABLE 1. Baseline demographic characteristics and plasma carotenoid concentrations of subjects1

 
Written informed consent was obtained from each subject after they received an adequate explanation of the aims, methods, objectives, and potential hazards of the study. The study protocol was in accordance with the Declaration of Helsinki and was approved by the local ethics committee of the University of Witten/Herdecke, Wuppertal, Germany.

Study design
This was a single-center, open-label, randomized, parallel-group study. The study included 3 phases: a run-in period of 3 d (days -3 to -1), a dosing period of 42 d (days 1-42), and a postdosing period of 34 d (days 42-76). After the prestudy screening, 20 subjects were randomly allocated (block randomization) to 2 treatment groups (A or B), each of which comprised 5 men and 5 women. On days 1-42 (dosing phase), the subjects received once daily doses of either 1 mg (1.76 µmol; group A) or 10 mg (17.6 µmol; group B) of zeaxanthin. The beadlet formulation was incorporated into hard gelatin capsules. The beadlets contained 5.3% total zeaxanthin (80% all-E, 0.4% 9-Z, 17.5% 13-Z, and 2.2% 15-Z), the carotenoid being finely dispersed in a cornstarch-coated matrix of bovine gelatin and sucrose with all-rac-{alpha}-tocopherol, sodium ascorbate, and ascorbyl palmitate added as antioxidants. 3-Dehydro-lutein could not be detected in the zeaxanthin beadlets (limit of detection for the 10-mg dose: <0.4% of the zeaxanthin contents.

On days 1 and 42, the capsule was ingested in the clinical center together with 200 mL water during a standardized breakfast (one roll with cheese and coffee or tea). On days 2-41, the subjects took their zeaxanthin capsule at home. To ensure compliance with the study protocol, the subjects were reminded to take the capsule and to confirm the intake in the subject diary each morning at 0800 by sending an SMS (short message system) via a mobile phone, which was provided for this purpose. At each visit to the clinical center, the remaining capsules were counted.

Blood samples (8 mL) were drawn in the morning (after an overnight fast) of days -3 to -1 (baseline). Samples collected on 3 consecutive days were considered to be sufficient to establish a stable zeaxanthin concentration at baseline. On days 1 and 42, after the subjects had fasted overnight, blood specimens were drawn with an indwelling canula before and 2, 4, 6, 8, 12, 15, and 24 h after zeaxanthin administration. Additional samples were collected (in the morning after an overnight fast) by venipuncture on days 7, 14, 21, 28, 35, 38, 39, 40, and 41 (dosing period) and on days 44, 48, 53, 58, 64, 70, and 76 (postdosing period). Blood samples were collected into precooled Monovettes (Sarstedt, Nuembrecht, Germany) containing EDTA as anticoagulant. Within 30 min after collection, the samples were centrifuged at 4 °C for 10 min at 2000 x g. The resulting plasma was immediately transferred into labeled polypropylene tubes. Plasma samples were stored at -35 °C or below until analyzed. Blood collection and handling of samples were done under light protection (no direct sunlight, no strong fluorescent light). The same tests for safety as performed at the prestudy screening were performed on day -1, predose on day 42, and on day 76. Adverse events were monitored over the whole study period.

Analytic assays
Plasma samples were analyzed for all-E-zeaxanthin and for the sum of 3-Z-zeaxanthin isomers (13-Z-zeaxanthin, 9-Z-zeaxanthin, and 15-Z-zeaxanthin); for E- and Z-isomers of lutein, {alpha}-carotene, ß-carotene, lycopene, and ß-cryptoxanthin; and for all-E-3'-dehydro-lutein, retinol, {alpha}-tocopherol, and lipids (total cholesterol and triacylglycerols).

The xanthophylls were extracted from plasma (100 µL) with a 20% mixture of n-hexane and chloroform (1100 µL) after dilution with water (100 µL) and proteins precipitation with ethanol (200 µL). After centrifugation, an aliquot (800 µL) of the clear supernatant fluid was dried under nitrogen at room temperature. The dried residue was quantitatively redissolved in the mobile phase (200 µL n-hexane and acetone; 19%, by vol). The resulting solution was injected (100 µL) into a normal-phase HPLC system equipped with an autosampler (15 °C), a column oven (40 °C), an HPLC pump, and an ultraviolet-visible detector. Data were analyzed with a multichannel data acquisition system (Multichrom; Thermo Electron, Altrincham, United Kingdom).

The separation was done on a polar column (Lichrosorb, Si60, 5 µm, 250 x 4 mm; Stagroma, Reinach, Switzerland) with a mixture of n-hexane and acetone (19%, by vol) at a flow rate of 1 mL/min. Xanthophylls were detected at a wavelength of 452 nm. Tentative identification of the individual isomers of lutein was made by comparing the HPLC elution pattern of the plasma extracts with the HPLC pattern of authentic E-lutein (Roche Vitamins Ltd, Basel, Switzerland) and a Z-isomer mixture of lutein obtained after heat isomerization. The zeaxanthin isomers and 3-dehydro-lutein were identified by comparing the HPLC retention data of synthetic E- and Z-isomers (Roche) and synthetic 3-dehydro-lutein (provided by J Landrum, Miami), respectively. The identity of 3-dehydro-lutein was further confirmed by its ultraviolet-visible spectrum compared with the reference compound and additionally by comparing the retention times on 2 different HPLC systems (including normal phase and C30-phase) and spiking experiments.

The intraday variation was 5.6% for zeaxanthin and 4.8% for lutein. Reproducibility was 6.8% for zeaxanthin and 2.6% for lutein. The lower limit of quantification was 0.0070 µmol/L (4 µg/L) for zeaxanthin and lutein. The lower limit of detection was 0.0018 µmol/L (1 µg/L). The efficiency of plasma extraction was 99% and 100% for lutein and zeaxanthin, respectively.

Other carotenoids in plasma were assessed according to published procedures (15). The lower limit of quantification was 0.0186 µmol/L for lycopene and carotenes, 0.036 µmol/L for ß-cryptoxanthin, 0.0698 µmol/L for retinol, and 0.0464 µmol/L for {alpha}-tocopherol. Cholesterol and triacylglycerol concentrations in plasma were measured according to published methods (16-18) by using the CHOD-PAP method (Merck AG, Dietikon, Switzerland), adapted to a centrifugal analyzer (CobasBio; Roche Diagnostics, Basel, Switzerland).

To assess the daily and long-term laboratory performance of the HPLC plasma analytics, dedicated control plasma was used. The control samples were analyzed >=2 times/d during the study. In addition, the methods for the analysis of vitamins and carotenoids were monitored through participation in external quality-assurance programs (eg, National Institute for Standards and Technology, Gaithersburg, MD).

Kinetic analysis
Because plasma zeaxanthin concentrations were essentially reflected by all-E zeaxanthin, accounting for 84-95% of total zeaxanthin concentrations, only all-E-zeaxanthin time-concentration profiles were subjected to kinetic analysis. For all-E-zeaxanthin, the following parameters were determined: mean Cb, baseline-corrected maximum concentrations on days 1 and 42 ({Delta}Cmax1, {Delta}Cmax42), times to reach maximum concentrations on days 1 and 42 (tmax1, tmax42), baseline-corrected areas under the concentration-time curve over 24 h on days 1 and 42 ({Delta}AUC1, {Delta}AUC42), the apparent terminal elimination half-life (t1/2) after day 42, the average steady state concentration (Css), R, the teff, and the corresponding time to reach >=90% of steady state concentrations (tss).

Cb was estimated as the mean of concentrations on days -3 to -1. Cmax and tmax were read directly from the plasma concentration-time curves. {Delta}Cmax was assessed after subtraction of Cb (baseline correction). {Delta}AUC was estimated by the linear trapezoidal rule after subtraction of Cb at each time point. Css was calculated as AUC42/24. Assessment of t1/2 was based on baseline-corrected concentrations in the terminal phase of the concentration-time curve after dosing on day 42 (17). R was determined by dividing {Delta}AUC42 by {Delta}AUC1. teff was calculated by iteration with the use of Equation 1 for R:

(1)
where n is the number of doses of zeaxanthin (n = 42) and {tau} denotes the dosing interval (24 h).

tss was determined as follows:

(2)
Analogous equations valid for n approaching infinity are given elsewhere (19).

Because plasma concentrations of all-E-3'-dehydro-lutein appeared to be related to concentrations of all-E-zeaxanthin, the plasma kinetics of all-E-3'-dehydro-lutein was also evaluated in more detail. The parameters Cb, Cmax42, tmax42, AUC42, Css, and t of all-E-3'-dehydro-lutein were determined in the same manner as for those of all-E-zeaxanthin. Furthermore, baseline-corrected plasma concentrations of all-E-3'-dehydro-lutein were related to baseline-corrected concentrations of all-E-zeaxanthin by using the following differential equation:

(3)
where CDH and CZ are the baseline-corrected concentrations of all-E-3'-dehydro-lutein and all-E-zeaxanthin, respectively; kf is the rate constant for the formation of all-E-3'-dehydro-lutein; and ke denotes the elimination rate constant. The baseline-corrected plasma concentrations of all-E-zeaxanthin as a function of time were described by linear interpolation between experimental data points.

Concentrations of lutein, ß-carotene, lycopene, and ß-cryptoxanthin were represented as the sum of their E- and Z-isomers. The following evaluation was used for these carotenoids: the mean Cb was estimated as the mean of concentrations on days -3 to -1. The time-averaged concentrations over 24 h were not baseline-corrected and were calculated as C1 = AUC1/24 (day 1) and C42 = AUC42/24 (day 42). The AUC was assessed by using the linear trapezoidal rule.

The parameters kf and ke of all-E-3'-dehydro-lutein were estimated by nonlinear least-squares regression (weight = 1) based on Equation 3 (MicroMath Scientific Software, Salt Lake City). All other kinetic parameters were calculated by using Excel 5.0 (Microsoft, Redmond, WA).

Statistical analysis
The kinetic parameters Cb, {Delta}Cmax1, tmax1, {Delta}AUC1, {Delta}Cmax42, tmax42, {Delta}AUC42, Css, t1/2, R, teff, and tss of all-E-zeaxanthin and all-E-3'-dehydro-lutein (where appropriate) and the all-E-3'-dehydro-lutein specific parameters kf and ke are presented as means ± SDs (version 6.12; SAS Institute Inc, Cary, NC) by treatment group. Arithmetic means and the corresponding SDs were calculated for Cb, tmax, t1/2, teff, and tss. Assuming a logarithmic normal distribution, {Delta}Cmax, {Delta}AUC, Css, R, kf, and ke were analyzed after logarithmic transformation, and, accordingly, geometric means and the corresponding SDs were calculated (20).

Further to descriptive statistics, the all-E-zeaxanthin parameters {Delta}Cmax, {Delta}AUC, and Css (after dose-normalization and logarithmic transformation); R (after logarithmic transformation); and Cb, teff, tss, and t1/2 (untransformed) were subjected to an analysis of variance (ANOVA) with the use of the general linear models procedure (version 6.12; SAS Institute Inc). In a first step, treatment group and sex were used as effects. Because none of the parameters showed a significant sex effect, the ANOVA was subsequently performed with treatment group as the only effect parameter. The two-sided 95% CI for the ratio (group A/group B) of geometric means ({Delta}Cmax, {Delta}AUC, Css, and R) and for the difference (group A - group B) of arithmetic means (Cb, teff, tss, and t1/2) were estimated by using the residual error of the ANOVA. The parameters tmax1 and tmax42 were analyzed with the use of the Mann-Whitney U test (Wilcoxon's scores). {Delta}Cmax42, {Delta}AUC42, Css, and tmax42 for all-E-3'-dehydro-lutein were analyzed in the same manner as for all-E-zeaxanthin. Furthermore, a linear regression analysis of the {Delta}AUC42 of all-E-3'-dehydro-lutein versus the {Delta}AUC42 of all-E-zeaxanthin was performed (REG procedure, version 6.12; SAS Institute Inc).

Cb, C1, and C42 of lutein, ß-carotene, lycopene, and ß-cryptoxanthin were evaluated by descriptive statistics (means, SDs) and, furthermore, were subjected to an ANOVA with group as effect. The 95% CIs for the difference of means (group A - group B) were estimated by using the residual error of the ANOVA. The level of significance was set to 0.05 in all tests.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Subjects
All 20 subjects completed the study according to the protocol. There were no clinically relevant abnormalities in laboratory indexes, vital signs, or electrocardiograms observed during the study that could be related to the compound under investigation.

Plasma kinetics of all-E-zeaxanthin
all-E-Zeaxanthin concentrations in plasma at baseline and on day 1 accounted for 95% of total zeaxanthin concentrations but decreased to 86% on day 42. The remaining concentrations were determined as the sum of 3-Z-zeaxanthin isomers (typically, 85% 13-Z-zeaxanthin in addition to minor amounts of 9-Z-zeaxanthin and 15-Z-zeaxanthin). 13-Z-Zeaxanthin was also contained in the zeaxanthin beadlets as outlined in Study design. The kinetic parameters of all-E-zeaxanthin are summarized in Table 2Go. Plasma concentration-time profiles of all-E-zeaxanthin over the entire study period are presented in Figure 2Go. Kinetic profiles over dosing intervals (24 h) on days 1 and 42 are shown in Figure 3Go.


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  		TABLE 2. Kinetic parameters of plasma all-E-zeaxanthin1

 


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  		FIGURE 2.. Mean (±SD) plasma concentrations of all-E-zeaxanthin. For clarity, only concentrations immediately before the intake of zeaxanthin in the dosing period (days 2-42) and during the postdosing period (days 43-76) are given. Group A ({blacksquare}; n = 10) received 1 mg zeaxanthin/d, and group B ({blacktriangleup}; n = 10) received 10 mg zeaxanthin/d.

 


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  		FIGURE 3.. Mean (±SD) plasma concentrations of all-E-zeaxanthin on day 1 (A) and day 42 (B). Group A ({blacksquare}; n = 10) received 1 mg zeaxanthin/d, and group B ({blacktriangleup}; n = 10) received 10 mg zeaxanthin/d.

 
The mean Css values in groups A and B, respectively, were 3.9 times and 20 times those of the corresponding Cb values. The dose-normalized Css in group A was significantly higher than that in group B. The pertaining two-sided 95% CI for the ratio of geometric means (group A/group B) ranged from 1.57 to 2.87.

No significant differences in Cb (see Table 1Go for total zeaxanthin and Table 2Go for all-E-zeaxanthin), R, tss, teff, or t1/2 were found between the groups. The 95% CI for the difference of arithmetic means (group A - group B) was -0.019 to 0.031 µmol/L for Cb, -6.1 to 6.8 d for tss, -1.9 to 2.0 d for teff, and -5.4 to 2.0 d for t1/2; the corresponding CI for the ratio of geometric means (group A/group B) ranged from 0.70 to 1.38 for R. The parameters tmax1 and tmax42 did not differ significantly between groups.

Significant differences in the dose-normalized parameters {Delta}Cmax1, {Delta}AUC1, {Delta}Cmax42, and {Delta}AUC42 were found between the groups. The 95% CI for the ratio of geometric means (group A/group B) was 1.32 to 2.52 for {Delta}Cmax1, 1.19 to 2.54 for {Delta}AUC1, 1.32 to 2.24 for {Delta}Cma42, and 1.30 to 2.26 for {Delta}AUC42. Thus, {Delta}Cmax and {Delta}AUC were significantly greater in group A than in group B on both day 1 and day 42.

Plasma kinetics of all-E-3'-dehydro-lutein
The kinetic parameters of all-E-3'-dehydro-lutein are summarized in Table 3Go. The relatively high Cb values of all-E-3'-dehydro-lutein (approaching those of all-E-zeaxanthin; Table 1Go) led to large fluctuations in baseline-corrected concentrations, particularly in the low concentrations in group A and generally on day 1. Therefore, t could only be evaluated in 3 subjects in group A; {Delta}Cmax1, {Delta}AUC1, and tmax1 could not be determined in either dose group.


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  		TABLE 3. Kinetic parameters of plasma all-E-3'-dehydro-lutein1

 
The mean Css values of all-E-3'-dehydro-lutein in groups A and B were significantly (P < 0.05) increased by factors of 1.7 and 5.1, respectively, when compared with mean Cb values. The dose-normalized {Delta}Cmax42 and {Delta}AUC42 values in group B were {approx}60% of those in group A (P < 0.05). The parameter tmax42 was highly variable in both groups and occurred significantly later in group B than in group A (Table 3Go).

The CV of nonlinear regression estimates of kf and ke in group A (n = 4 subjects) and group B (n = 10 subjects) ranged from 7% to 23% (median: 19%) and from 20% to 35% (median: 27%), respectively. A comparison of the experimental and calculated plasma concentrations of all-E-3'-dehydro-lutein in both groups is provided in Figure 4Go. There was a highly significant relation between the {Delta}AUC42 of all-E-3'-dehydro-lutein and the {Delta}AUC42 of all-E-zeaxanthin (Figure 5Go).



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  		FIGURE 4.. Comparison of mean (±SD) baseline-corrected experimental and calculated plasma concentrations of all-E-3'-dehydro-lutein. Calculated concentrations ({blacksquare}, {blacktriangleup}) were obtained by nonlinear regression with the use of linearly interpolated baseline-corrected all-E-zeaxanthin concentrations as input function. The SDs of the experimental concentrations are given. The SDs of the calculated concentrations are not shown; they were 10% smaller than those of the experimental concentrations. Group A ({square}; n = 4) received 1 mg zeaxanthin/d, and group B ({triangleup}; n = 10) received 10 mg zeaxanthin/d.

 


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  		FIGURE 5.. Linear regression of baseline-corrected areas under the concentration-time curve over 24 h on day 42 ({Delta}AUC42) of all-E-3'-dehydro-lutein versus the {Delta}AUC42 of all-E-zeaxanthin. Slope = 0.18 (P = 0.0001), intercept = -0.084 (P = 0.687), and r2 = 0.92. Group A ({blacksquare}; n = 10) received 1 mg zeaxanthin/d, and group B ({blacktriangleup}; n = 10) received 10 mg zeaxanthin/d.

 
Other carotenoids, retinol, {alpha}-tocopherol, and lipids
Plasma concentrations of all-E-lutein accounted for >91% of total lutein concentrations, and all-E-ß-carotene concentrations accounted for 83% of total ß-carotene concentrations. Only results of the sum of E- and Z-isomers of lutein, ß-carotene, lycopene and ß-cryptoxanthin are presented. Mean Cb values and time-averaged (over 24 h) concentrations C1 (day 1) and C42 (day 42) of lutein, ß-carotene, lycopene, and ß-cryptoxanthin, and differences of means (group A-group B) with corresponding 95% CIs, are summarized in Table 4Go. Plasma lutein concentrations versus time are shown in Figure 6Go.


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  		TABLE 4. Kinetic parameters of selected plasma carotenoids1

 


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  		FIGURE 6.. Mean (±SD) plasma concentrations of lutein (sum of E- and Z-isomers). For clarity, only baseline concentrations (days -3 to -1), ie, the concentrations immediately before the intake of zeaxanthin in the dosing period (days 2-42), and concentrations during the postdosing period (days 43-76) are given. Group A ({blacksquare}; n = 10) received 1 mg zeaxanthin/d, and group B ({blacktriangleup}; n = 10) received 10 mg zeaxanthin/d.

 
There were neither significant differences between Cb and C1 or C42 for both lutein and lycopene nor between dose groups. Zeaxanthin dosing did not affect concentrations of retinol, {alpha}-tocopherol, total cholesterol, or triacylglycerols, which remained essentially unchanged during the whole study period. Furthermore, concentrations of these plasma constituents did not differ between dose groups (data not shown). There was a significant difference between dose groups in Cb values of ß-carotene and ß-cryptoxanthin, which was reflected in significant differences on day 1 and day 42. After correction for baseline values, the differences disappeared.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
all-E-Zeaxanthin
For both dose groups, maximum plasma concentrations of all-E-zeaxanthin were achieved on average at a tmax of 10-15 h after dosing (Figure 3Go); tmax values >12 h were also observed for {alpha}-tocopherol, carotenes, and vitamin A esters. These highly lipophilic compounds enter the systemic circulation by the lymphatic pathway, and transport via this pathway could, at least in part, account for the slow accumulation of all-E-zeaxanthin in plasma. However, plasma concentrations of carotenoids are also critically dependent on redistribution processes via VLDL and subsequent transport in intermediate-density lipoprotein, LDL, and HDL; therefore, hepatic recirculation might also cause the slow build-up of plasma zeaxanthin concentrations. On both day 1 and day 42, the mean dose-normalized {Delta}Cmax and {Delta}AUC values in group B were significantly lower and amounted only to {approx}60% of those in group A. Because relevant disposition parameters (R, tss, and t1/2) did not differ significantly between groups (Table 2Go), the observed nonlinearity was not related to dose-dependent disposition kinetics of all-E-zeaxanthin. Similarly, as discussed for other carotenoids (21), the capacity for intestinal absorption of zeaxanthin may become limiting with increasing doses. However, alternative explanations, such as capacity limitation of hepatic zeaxanthin recirculation in VLDL, cannot be excluded.

For all-E-zeaxanthin, t1/2 values of {approx}12 d were observed in the present study, whereas t1/2 values of >30 d were reported by others (12, 22). Therefore, an additional elimination phase from plasma at concentrations below the limit of quantification cannot be excluded.

The accumulation factor R is primarily a kinetic parameter that expresses the extent of accumulation by relating the increase in exposure over dosing intervals between the first dose and a dose at steady state. R for all-E-zeaxanthin was {approx}7.5 (Table 2Go), which is much lower than that expected for a t1/2 of {approx}300 h. After attaining maximal concentrations at 10-12 h after dosing, plasma concentrations decayed in a biphasic fashion (Figure 3Go). In the case of a multiphasic plasma concentration-time profile (absorption and elimination phases), the accumulation is determined by a teff, which is a weighted average of the absorption half-life and the half-lives describing the disappearance from plasma (23). Thus, the teff is always shorter than the t1/2. The mean teff values for zeaxanthin were 5.2 and 5.1 d in groups A and B, respectively. On the basis of this teff value, a time of {approx}17 d was calculated to be required to reach a >90% fraction of plasma steady state concentration (Figure 2Go).

all-E-3'-Dehydro-lutein
A new observation in this study was that zeaxanthin dosing resulted in a considerable accumulation of all-E-3'-dehydro-lutein above Cb values in plasma and that the resulting concentration-time profile resembled that of all-E-zeaxanthin (Figures 2 and 4). Because lutein concentrations remained unaffected by zeaxanthin dosing (Figure 6Go), the increase in all-E-3'-dehydro-lutein was postulated to be derived from zeaxanthin. To delineate this further, plasma all-E-3'-dehydro-lutein concentrations were coupled to linearly interpolated all-E-zeaxanthin concentrations, which served as input function. This approach allowed us to approximate the time course of plasma concentrations of all-E-3'-dehydro-lutein under the assumption that the formation and the elimination of all-E-3'-dehydro-lutein obey linear kinetics and that plasma all-E-3'-dehydro-lutein concentrations can be described by a one-compartment model. However, this approach did not require a kinetic model for all-E-zeaxanthin to be specified.

The predicted concentrations were not significantly different from the experimental plasma concentrations of all-E-3'-dehydro-lutein (Figure 4Go). The model parameters kf and ke were associated with mean half-lives (both groups pooled) of 14 and 2.7 d, respectively. The half-life associated with kf was in the range of the t1/2 value for all-E-zeaxanthin, which indicates that the elimination of all-E-3'-dehydro-lutein was limited by the rate of formation. The {Delta}AUC42 of all-E-3'-dehydro-lutein in group B was only 60% of that in group A, which agreed with the percentage found for all-E-zeaxanthin (see Results). These results provide strong evidence that the increase in all-E-3'-dehydro-lutein above baseline was closely related to the plasma zeaxanthin concentration and, therefore, was a consequence of zeaxanthin dosing. The highly significant linear relation between the {Delta}AUC42 of all-E-3'-dehydro-lutein and the {Delta}AUC42 of all-E-zeaxanthin (Figure 5Go) with an intercept close to zero supports this view.

all-E-3Dehydro-lutein in human plasma was identified previously (24). Formation of all-E-3'-dehydro-lutein was previously assumed to be the result of lutein oxidation. The Css values of all-E-3'-dehydro-lutein were significantly higher than Cb values, by 65% after the 1-mg dose and by 440% after the 10-mg dose of zeaxanthin. The ratio of mean baseline-corrected plasma all-E-3'-dehydro-lutein concentrations over the corresponding all-E-zeaxanthin concentrations was {approx}0.2, but the ratio of mean Cb values was {approx}0.8. Taken together, these results suggest that all-E-3'-dehydro-lutein under normal dietary conditions is predominantly formed from other sources, most likely from lutein, rather than from dietary zeaxanthin.

However, formation of 3-dehydro-lutein from zeaxanthin was previously reported. After chickens were fed 15,15-[3H]zeaxanthin, {approx}5-10% of the recovered radioactivity in the egg yolk was identified as 3-dehydro-lutein (25). To the best of our knowledge, no studies have shown the formation of all-E-3'-dehydro-lutein as a consequence of zeaxanthin dosing to humans. The formation of all-E-3'-dehydro-lutein from zeaxanthin cannot be accounted for by a one-step conversion but rather by a sequence of reactions.

In summary, the present results show that long-term oral intakes of 1 and 10 mg zeaxanthin elevate mean plasma zeaxanthin concentrations {approx}4- and 20-fold, respectively. It takes {approx}17 d of daily dosing to attain zeaxanthin concentrations corresponding to 90% of the steady state concentration. Plasma concentrations of all-E-3'-dehydro-lutein increased in parallel with those of all-E-zeaxanthin, and the increase in all-E-3'-dehydro-lutein concentration indicates that this was clearly related to all-E-zeaxanthin dosing. Other carotenoids, retinol, {alpha}-tocopherol, total cholesterol, and triacylglycerols remained unaffected by zeaxanthin dosing.


   ACKNOWLEDGMENTS
 
DH analyzed the kinetic and statistical data and prepared the draft manuscript. PAT conducted the clinical portion of the study and was involved in the design of the protocol (Principal Investigator according to Good Clinical Practice guidelines). VS was responsible for the carotenoid and lipid analyses. WS initiated and supervised the project. BM performed the clinical investigations and supervised the dietary instructions (clinical coinvestigator). WC designed the study and supervised the data analysis and the preparation of the manuscript.

PAT received research grants from Roche Vitamins Ltd to conduct the clinical portion of the study. At the time of the study, DH was a consultant to Roche Vitamins Ltd, and VS, WS, and WC were employees of Roche Vitamins Ltd.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
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Received for publication May 6, 2003. Accepted for publication July 10, 2003.




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