Absorption and Tissue Distribution of Zeaxanthin and Lutein in Rhesus Monkeys after Taking Fructus lycii (Gou Qi Zi) Extract

Abstract

purpose. To study serum and tissue levels of zeaxanthin and lutein after feeding rhesus monkeys an extract of Fructus lycii (gou qi zi).

methods. A carotenoid-containing fraction (P1) from an extract of F. lycii (equivalent to 2.2 mg zeaxanthin) was fed to three rhesus monkeys for 6 weeks as a daily dietary supplement through a nasogastric tube. Three other monkeys were fed with the vehicle (olive oil) similarly for 4 weeks as a control. Another three animals were fed with normal diet only. All animals were killed 4 hours after the last dose. Samples of serum, liver, spleen, brain, and retina were analyzed for zeaxanthin and lutein by high-pressure liquid chromatography.

results. The basal levels of zeaxanthin and lutein in the monkey sera were 3.0 ± 1.6 ng/ml (range, 2.3–4.8) and 31.5 ± 12.2 ng/ml (range, 22.3–42.5), respectively. Serum levels of zeaxanthin and lutein in the P1-fed group were significantly higher than those of vehicle control (P < 0.05). Besides the retina, the liver had the highest zeaxanthin and lutein levels, whereas the levels in the brain were undetectable. P1 supplement appeared to elevate zeaxanthin levels in liver and spleen. The level of lutein was higher than that of zeaxanthin in the maculae of rhesus monkeys. However, there were no detectable carotenoids in the peripheral and the equatorial regions of the monkey retina. P1 treatment elevated zeaxanthin density but not lutein in the macula.

conclusions. Serum levels and macular density of zeaxanthin was raised by feeding a carotenoid-containing fraction of F. lycii. Therefore, F. lycii is a good dietary source of zeaxanthin supplement.

Zeaxanthin and lutein are two common carotenoids found in plants and are constituents of the yellow macular pigment in the human retina. ¹ The biological functions of these macular pigments are not fully understood, but their absorption spectra enable these pigments to absorb blue light to which the retina is most susceptible for damage. ² In addition, scattering and chromatic aberration of blue light may be minimized by these macular pigments. In vitro and in vivo studies have shown that these carotenoids are also potent antioxidants. ^{3 4 5 6} Therefore, zeaxanthin and lutein may protect the retina against oxidative damage in the macula where free radicals may be generated by lengthy light exposure, high oxygen tension, and high metabolic rate. This hypothesis is supported by an observational study that showed high dietary intake of green vegetables rich in lutein and zeaxanthin correlates with a lower risk of age-related macular degeneration, ⁷ in which oxidative insult to the retina is thought to play an important role. ^{8 9} Because macular zeaxanthin and lutein may be beneficial to the well-being of the retina, attempts to raise serum lutein and macular pigments in humans with lutein supplement ¹⁰ or vegetables ¹¹ have been reported. However, there have been few studies on the effect of dietary supplementation of zeaxanthin, ¹² the predominant carotenoid in the human macula. 

Fructus lycii or gou qi zi, the dried fruit of Lycium barbarum, is a red berry prescribed by Chinese herbalists as a health tonic and a therapeutic agent for a number of eye diseases ranging from cataract to retinitis pigmentosa and glaucoma. ¹³ Chromatographic study of F. lycii indicates a high content of zeaxanthin (300 μg/g) but a negligible amount of lutein (<3 μg/g) in the berries. ^{14 15} Khachik et al. ¹⁶ found elevated serum zeaxanthin levels in three human subjects after taking a zeaxanthin extract from the dried berries of Lycium chinense, a species closely related to L. barbarum. However, the effect on the level of macular pigment in these subjects was not determined. 

Although plasma or serum levels of carotenoids in several primates such as Saimiri sciureus, Macaca fascicularis, ¹⁷ and others ¹⁸ as well as the relative distribution of macular carotenoids in M. fascicularis ¹⁹ and humans ²⁰ had been studied, the relative distribution of zeaxanthin and lutein in the serum and the macula of rhesus monkeys (Macaca mulatta) has not been determined. There are few reports on the relative distribution of zeaxanthin and lutein in liver or other organ such as spleen in primates. ^{21 22}  

In this study we fed a chromatographic fraction from an extract of F. lycii to three rhesus monkeys for 6 weeks to study the levels of zeaxanthin and lutein in serum, retina, liver, spleen, and brain by high-pressure liquid chromatography (HPLC). 

Materials and Methods

Preparation of Gou Qi Zi Extract

Extraction and chromatographic fractionation of carotenoids in F. lycii were prepared in batches. For each batch, 5 g of the berry were soaked in 30 ml distilled water at 4°C for 16 hours and homogenized at maximum speed for 15 minutes in an electric blender (Waring; Dynamics, New Hartford, CT). An equal volume of ethanol was added, and the mixture was extracted with 4 volumes of hexane. Extraction with hexane was repeated twice. The combined hexane extract was evaporated and lyophilized, and the residue was resuspended in the mobile phase for HPLC fractionation. A semiprepared silica column (19 × 300 mm; Nova-Pak HR; Waters, Milford, MA) eluted with an isocratic mobile phase of 16% dioxane in hexane at 5 ml/min was used to fractionate the sample. The eluant was monitored at 450 nm, the absorption maximum of zeaxanthin, lutein, and other common carotenoids. ²³ Materials coming off the column within the first 25 minutes of elution showing an absorbance higher than 0.01 absorption units (AU) at 450 nm were collected and evaporated to dryness. The residue was reconstituted in 50% ethanol in olive oil. Zeaxanthin concentration was estimated by measuring the absorbance at 450 nm. Ethanol (50%) in olive oil was added to adjust the concentration to 1.1 mg zeaxanthin per milliliter and designated as P1. 

Administration of P1 and Vehicle to the Monkeys

Nine female rhesus monkeys (M. mulatta) were used in this study. They were kept in the Guangdong Shunde Institute of Laboratory Animals, China. All animals were examined and found to be healthy. The study was approved by the Animal Research Ethics Committee of the Chinese University of Hong Kong and followed the guidelines of the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 

The monkeys were fed a normal diet of monkey chow and supplementary fruits before and throughout the experimental period. It was estimated that each monkey consumed approximately 200 g vegetable, 100 g corn, and 100 g monkey chow daily, which provided approximately 2.1 mg of total zeaxanthin and lutein. The age of the animals ranged from 12 to 18 years (15.4 ± 2.2 years; mean ± SD). Monkeys 1, 2, and 3 (mean weight: 4.4 kg; range: 4.3–4.6) were each given a 2-ml mixture of olive oil and ethanol (olive oil:ethanol, 1:1) through a nasogastric tube daily for 4 weeks as vehicle control supplementation. Monkeys 4, 5, and 6 (mean weight: 4.3 kg; range: 3.9- 4.5 kg) were each given P1 in 2 ml olive oil with 50% ethanol (equivalent to 7.3 g F. lycii or 2.2 mg zeaxanthin/day or 0.5 mg zeaxanthin/kg body weight/day) daily through a nasogastric tube for 6 weeks. Another three normal control animals (monkeys 7, 8, and 9; mean weight: 6.3 kg; range: 4.4–6.7 kg) were reared on normal monkey diet for 4 weeks and killed. 

Blood and Tissue Collection

Blood (2-ml aliquots) was taken from the femoral vein of monkeys 1 to 6 four times per month before feeding with vehicle or P1 supplement. After the initiation of the feeding program, blood was taken from each monkey twice each week. All serum samples were stored at −80°C before processing. 

At the end of the feeding period, the monkeys were anesthetized by intramuscular injection of ketamine (10 mg/kg) and killed with a pentobarbital sodium overdose followed by immediate exsanguination. A tissue sample of liver was excised from the inferior part of the right lobe. Samples of spleen and brain were excised from the superior ending closed to the cardiac orifice of the stomach and the visual cortex area 17, respectively. 

All eyes were enucleated within 1 to 2 hours after exsanguination. Retina samples were taken from the macular, equatorial, and peripheral regions of all the right eyes with trephines of 3- or 6-mm internal diameter. The macular region was excised with the foveola as the center of the trephine after vitrectomy. The equator and peripheral samples were taken from the nasal and the temporal regions, respectively. There was no attempt to separate the retinal pigment epithelium during the dissection. A 3-mm trephine was used in monkeys 4, 5, and 6 accidentally, and the 6-mm trephine was used in all other monkeys. These samples were kept at −80°C until analysis. The whole eyeballs of the left eyes were fixed for histologic examination for a separate study. 

Hexane Extraction of Serum and Tissue Samples

A previously described extraction method was followed. ²⁴ Briefly, an aliquot of 0.2 ml serum sample was mixed with an equal volume of absolute ethanol and subjected to sonic disruption in a Sonifier (Branson Ultrasonics, Danbury, CT) for 30 seconds. The mixture was extracted with 4 volumes of hexane, followed by vigorous mixing for 3 minutes and centrifugation at 2000g for 10 minutes. The organic phase was collected, and the aqueous phase was re-extracted in the same manner twice. The combined organic fraction was evaporated to dryness under nitrogen. 

Approximately 0.5-g tissue samples of liver, spleen, or brain were weighed and minced with razor blades. An aliquot of 2 ml water was added, and the tissue samples were homogenized with a rotating blender for 5 minutes. An equal volume of ethanol was added and mixed with the homogenate. The mixture was extracted with 4 volumes of hexane three times, as described. The organic extracts were combined and evaporated to dryness under nitrogen. 

Samples of macular tissue were thawed and homogenized in 0.2 ml water by sonication for 1 minute. An equal amount of absolute ethanol was added, and the mixture was extracted with 4 volumes of hexane three times, as described. The combined organic extract was evaporated to dryness. 

HPLC Analysis

A previously described HPLC method was used to measure serum and tissue contents of zeaxanthin and lutein. ²⁴ Briefly, all samples were dissolved in appropriate volumes of the mobile phase (16% dioxane in hexane) for injection into a silica column (3.9 × 300 mm; Waters). Analysis was performed with isocratic elution at 2 ml/min. An aliquot of 50 μl specimen was injected. Absorbance at 450 nm was monitored by a photograph-diode array detector (Waters). Zeaxanthin and lutein levels were determined by external standard calibration. Recovery was determined by adding known amounts of zeaxanthin and lutein standards to serum or tissue samples from normal monkeys and found to be 70% to 80% depending on each batch of analysis. The possibility that zeaxanthin metabolite coeluted with the lutein peak was not ruled out. 

Results

Table 1A shows the mean serum levels of zeaxanthin or lutein in each monkey (1, 2, and 3: vehicle control; and 4, 5, and 6: P1 treated) before and during the supplementation period. Basal levels of serum lutein in all monkeys were approximately 10 times higher than those of zeaxanthin. Of the three P1-fed animals, monkey 4 appeared to be a nonresponder, showing insignificant change in serum zeaxanthin and lutein, whereas the other two (monkeys 5 and 6) showed highly significant increases (P < 0.005, t-test) in the serum level of zeaxanthin after P1 treatment. The serum lutein (possibly containing zeaxanthin metabolites) levels in monkeys 5 and 6 were also elevated (P < 0.01). Analysis of variance showed a significant (P < 0.05) effect of feeding P1 (the nonresponder included) on serum zeaxanthin and lutein levels over the vehicle control group. The serum levels of zeaxanthin and lutein (possibly containing zeaxanthin metabolites) before and after feeding of the P1 extract or vehicle were further grouped together and summarized in Table 1B . 

Table 2 depicts the zeaxanthin and the lutein contents in liver and spleen tissues of the monkeys. The liver showed the highest content of zeaxanthin and lutein, whereas the brains of all animals showed no detectable level of those carotenoids. In both liver and spleen, the lutein levels were consistently higher than those of zeaxanthin. Supplementation with P1 appeared to elevate tissue zeaxanthin in both tissues (liver and spleen). However, the differences in zeaxanthin or lutein between the treated and the control groups were not statistically significant in liver or spleen because of the large SD (P > 0.05). 

Zeaxanthin and lutein content in the maculae are tabulated in Table 3 . Monkeys fed with P1 showed elevated zeaxanthin level (approximately twofold when compared with the vehicle treated) in spite of a smaller area of tissue used for analysis (Table 3A) . There was a significant difference (P < 0.05) in the density (in nanograms per square millimeter) of zeaxanthin in the maculae in P1-treated versus the vehicle control animals. Although monkey 4 had a low serum zeaxanthin concentration (Table 1A) , the amount of zeaxanthin in its macula was the highest among all the animals given P1 (Table 3A) . Lutein levels (total amount) in the maculae of the monkeys treated with vehicle, P1, or normal diet were all comparable. Retinas taken from peripheral and equatorial regions of all three groups fed with P1, vehicle, or normal diet showed undetectable lutein or zeaxanthin (data not shown). In the vehicle-treated and the normal control maculae, the average ratios of zeaxanthin to lutein were approximately 1:2, whereas that of the P1-treated retinas was 1:1. Similar results were also obtained when Hammond’s exponential distribution equation of macular pigment ²⁵ was used to correct the differences in the diameter of the trephines used for foveal sampling (Table 3B) . 

Discussion

In this study, we noted a rapid and significant increase in zeaxanthin and lutein levels in the serum as well as a significant increase in zeaxanthin density in maculae of rhesus monkeys after dietary supplementation with a fraction of a lipid extract of F. lycii. Zeaxanthin contents in spleen and liver also ap-peared to increase, although the increase was not statistically significant. Our observation confirmed other studies ^{16 26} that suggested that serum zeaxanthin could be elevated by dietary supplementation. Our findings also suggest that there was a selective uptake of zeaxanthin in the macular tissue in these monkeys. Contrary to the studies in humans ²⁷ and M. fascicularis, ²⁸ which indicate a higher level of zeaxanthin than lutein in the central retina, we found that there was more lutein than zeaxanthin in the maculae in rhesus monkeys (M. mulatta), suggesting interspecies differences in macular zeaxanthin and lutein contents. 

Basal serum zeaxanthin and lutein levels in our rhesus monkeys were 10 and 3 times lower than those reported in M. fascicularis and S. sciureus, respectively. ¹⁷ It was unlikely that the low serum zeaxanthin and lutein levels in our rhesus monkeys were due to a difference in dietary carotenoid contents because analysis of monkey diet (including monkey chows and fruit supplement) showed a daily intake of 2.1 mg of total zeaxanthin and lutein in our vivarium, which was comparable to the monkey diet in the study by Snodderly et al. ²⁶ Slifka et al. reported the combined zeaxanthin and lutein levels in the sera of different primates varied from nondetectable level in the golden lion tamarin to an average of 1017 ng/ml in the sooty mangabey with most of the animals having a level between 28 and 170 ng/ml. ¹⁸ The combined levels of zeaxanthin and lutein in our animals ranged from 26 to 47 ng/ml (mean, 35) and remained in the lower end of the spectrum reported by Slifka et al. Therefore, different primates may have varying ability to absorb and metabolize various carotenoids, resulting in differences in serum levels of these carotenoids. 

Contrary to the study by Snodderly et al. ²⁶ showing no change in the concentration of serum lutein after supplementation of zeaxanthin to squirrel monkeys, we noted a significant increase in lutein levels in the serum after P1 supplementation. Because F. lycii has a very low content of lutein (less than 1%), it is an unlikely source of serum lutein. It is possible that zeaxanthin can be converted to lutein, but there is no evidence in the literature to suggest that zeaxanthin is converted into lutein in serum or retina, whereas the reverse has been proposed by Bone et al. ²⁹ and Khachik et al. ³⁰ However, lutein and other carotenoids are present in the monkey chows and daily fruits given to the animals. It is possible that zeaxanthin or other components in the P1 extract may enhance the absorption of lutein. It is also possible that there is a metabolite of zeaxanthin coeluting with lutein that cannot be separated by the present methodology. 

Lutein, cryptoxanthin, and β-carotene are the most common carotenoids found in fruits or vegetables, while zeaxanthin is present only in minute quantities in most fruits and vegetables. ^{30 31 32} Therefore, dietary zeaxanthin intake is very low. In addition, feeding corn and spinach, which are relatively rich in zeaxanthin, does not alter serum zeaxanthin levels. ¹¹ Recently, egg yolk was suggested to be a good dietary source of zeaxanthin. ³² Plasma lutein and zeaxanthin levels were elevated by 39% and 128%, respectively, after a dietary supplement of 1.3 egg yolks daily for 4.5 weeks. ³³ However, the constant consumption of egg yolk would increase the risk of cardiovascular diseases by increasing low-density lipoprotein cholesterol. In our study, serum zeaxanthin concentration increased by a factor of 2.5 with feeding of an extract from 7.3 g F. lycii (equivalent to 0.55 mg zeaxanthin per kilogram body weight per day) for 6 weeks in two monkeys. The dosage is relatively low compared with the study by Snodderly et al. ²⁶ in which 2.2 mg zeaxanthin was fed each day to squirrel monkeys with body weights from 0.7 to 1.1 kg. ²⁶ Therefore, F. lycii is a good dietary source of zeaxanthin. 

In a previous study, zeaxanthin was extracted from the dried fruits of L. chinense (closely related to L. barbarum) and orally taken by three human subjects as dietary supplements. A rapid elevation of serum zeaxanthin was noted. ¹⁶ In spite of the low basal levels of zeaxanthin and lutein in our rhesus monkeys, we also noted a rapid increase in zeaxanthin levels in the sera of two monkeys, but not in monkey 4 which also had a low level of zeaxanthin in liver and spleen. We deemed monkey 4 a nonresponder that showed poor absorption of zeaxanthin. Nonresponders such as monkey 4 have previously been reported. ^{11 34} We speculate that the absence of sufficient carotenoid-carrying proteins may explain the low levels of serum carotenoids irrespective of dietary intake. However, the rapid increase in serum zeaxanthin levels in the responders, even in those with low basal zeaxanthin levels, suggests that the serum level of zeaxanthin may be easily modulated by supplementation and does not necessarily depend on the basal level. However, the level of macular zeaxanthin, but not lutein, of the nonresponding monkey, was high after supplementation. Therefore, the uptake of zeaxanthin into the retina may be highly specific. 

Landrum et al. ¹⁰ and Bone et al. ³⁵ reported a gradual increase in macular pigment (MP) with a noninvasive psychophysical method after dietary supplementation of zeaxanthin or lutein in human subjects. It was assumed that the increase in MP corresponded to the increase in serum zeaxanthin or lutein. Our HPLC measurement showed that zeaxanthin levels in the maculae were elevated by feeding a dietary supplement rich in zeaxanthin. This observation supports the conclusion of Bone et al. The average zeaxanthin content in the maculae of the P1-treated group is two times higher than those of the normal and the vehicle-treated groups. Therefore, the uptake of zeaxanthin from serum into the macula was highly effective. 

The location of zeaxanthin and lutein in the retina is very unique. In humans, zeaxanthin is highest in the center of the fovea, whereas lutein is relatively abundant in the perifoveal region. Although the level of lutein is higher than zeaxanthin in the central maculae in our rhesus monkeys, the elevated level of zeaxanthin after P1 supplementation showed that the preferential absorption of zeaxanthin also occurs in the central macula, independent of the relative distribution of zeaxanthin and lutein. The absence of any carotenoids in peripheral or equatorial regions in our study further suggests specific uptake mechanisms at the center of the retina. 

F. lycii, the red berry of L. barbarum is an important ingredient in Chinese herbal medicine for cataract, glaucoma, and retinitis pigmentosa. ¹³ Whether F. lycii is beneficial in the treatment of these ocular diseases remains to be studied. However, the abundant zeaxanthin in this berry and the ready absorption of its zeaxanthin into serum and the macula of primates may be beneficial in protecting the retina against free radicals and blue light damages. 

In summary, we noted significant levels of zeaxanthin and lutein in the maculae of rhesus monkeys with a 1:2 ratio (zeaxanthin to lutein). We also recorded elevated levels of zeaxanthin and lutein in the sera and the maculae of rhesus monkeys after feeding an extract of F. lycii. Therefore, F. lycii is a good source of zeaxanthin to increase circulating and macular zeaxanthin. 

 Submitted for publication March 27, 2000; revised August 25, 2000; accepted September 20, 2000.

 Commercial relationships policy: N.

Table 1.

 

 

Mean Zeaxanthin and Lutein Contents in the Serum of Rhesus Monkeys

Table 1.

 

Mean Zeaxanthin and Lutein Contents in the Serum of Rhesus Monkeys

A. Serum levels in each monkey
Monkey	Treatment	Zeaxanthin									Lutein
				Before Treatment	n	After Treatment	n	P *	Before Treatment	n	After Treatment	n	P *
1	Vehicle	2.5 ± 0.5	4	2.8 ± 0.8	8	0.514	33.5 ± 5.2	4	22.3 ± 5.0	8	0.05
2	Vehicle	3.2 ± 2.9	3	5.8 ± 1.4	8	0.067	34.7 ± 17.5	3	62.9 ± 14.8	8	0.024
3	Vehicle	2.3 ± 1.5	4	5.0 ± 1.4	8	0.012	23.6 ± 13.2	4	41.4 ± 14.4	8	0.065
4	P1	2.5 ± 1.7	4	2.3 ± 1.2	12	0.797	22.3 ± 10.2	4	19.4 ± 9.2	12	0.602
5	P1	4.8 ± 1.1	4	10.3 ± 2.1	12	<0.001	42.5 ± 11.6	4	77.1 ± 15.6	12	0.001
6	P1	3.0 ± 0.5	4	8.2 ± 2.7	12	0.002	33.4 ± 8.4	4	53.9 ± 11.6	12	0.006

B. Serum levels in all monkeys
	Zeaxanthin	n	Lutein	n
Pretreatment	3.0 ± 1.6	23	31.1 ± 12.8	23
After vehicle treatment	4.6 ± 1.7	24	42.3 ± 20.5	24
After P1 treatment	6.9 ± 4.0	36	50.1 ± 26.9	36

 Data are expressed as mean nanograms per millimeter ± SD.

*  P obtained by comparing the serum carotenoids before and after supplementation. Because of the large individual variation of the serum zeaxanthin and lutein, P < 0.01 was considered significant.

Table 2.

 

 

Zeaxanthin and Lutein Contents in Liver and Spleen of Rhesus Monkeys after Supplementation

Table 2.

 

Zeaxanthin and Lutein Contents in Liver and Spleen of Rhesus Monkeys after Supplementation

Monkey	Treatment	Liver			Spleen
				Zeaxanthin	Lutein*	Zeaxanthin	Lutein*
;l>1	Vehicle	44.9	334.0	2.0	14.9
2	Vehicle	42.8	387.3	1.9	26.1
3	Vehicle	24.0	138.6	ND	4.2
	Mean ± SD	37.3 ± 9.42	286.6 ± 106.9	1.9 ± 0	15.1 ± 8.9
4	P1	18.4	150.5	2.1	17.9
5	P1	87.0	670.5	12.2	114.0
6	P1	94.3	423.6	5.2	34.3
	Mean± SD	66.6 ± 34.2	414.9 ± 212.4	6.5 ± 4.2	55.3 ± 42.0
7	Null	ND	ND	ND	8.6
8	Null	37.9	310.2	1.3	21.5
9	Null	33.7	213.7	5.6	46.6
	Mean± SD	35.8 ± 2.1	216.9 ± 48.3	3.5 ± 2.2	25.6 ± 15.8

 Data are expressed in nanograms per gram of tissue. Zeaxanthin and lutein were undetectable in all brain tissues. ND, not detectable.

*  The possibility of a zeaxanthin metabolite coeluting with lutein cannot be ruled out.

Table 3.

 

 

Zeaxanthin and Lutein Contents in Monkey Maculae

Table 3.

 

Zeaxanthin and Lutein Contents in Monkey Maculae

Animal	Treatment	Diameter of Trephine (mm)	Area of Retina (mm2)	Zeaxanthin			Lutein*
								(ng)	(ng/mm2)	(ng)	(ng/mm2)
A. Before adustment
1	Vehicle	6	28.3	0.44	0.016	0.78	0.028
2	Vehicle	6	28.3	0.77	0.027	1.77	0.063
3	Vehicle	6	28.3	0.44	0.016	0.71	0.025
	Mean± SD			0.55 ± 0.19	0.020 ± 0.006	1.09 ± 0.59	0.039 ± 0.021
4	P1	3	7.07	1.28	0.181	0.40	0.057
5	P1	3	7.07	0.7	0.099	1.06	0.150
6	P1	3	7.07	1.16	0.164	1.56	0.221
	Mean± SD			1.05 ± 0.31	0.148 ± 0.043	1.01 ± 0.58	0.143 ± 0.082
7	Null	6	28.3	1.19	0.042	1.99	0.070
8	Null	6	28.3	0.42	0.015	0.35	0.012
9	Null	6	28.3	0.47	0.017	1.28	0.045
	Mean± SD			0.69 ± 0.43	0.025 ± 0.015	1.21 ± 0.82	0.042 ± 0.029
B. After adjustment, †
1	Vehicle			0.44	0.016	0.78	0.028
2	Vehicle			0.77	0.027	1.77	0.063
3	Vehicle			0.44	0.016	0.71	0.025
	Mean± SD			0.55 ± 0.19	0.020 ± 0.006	1.09 ± 0.59	0.039 ± 0.021
4	P1			1.41	0.050	0.44	0.016
5	P1			0.77	0.027	1.17	0.041
6	P1			1.28	0.045	1.72	0.061
	Mean± SD			1.16 ± 0.34	0.041 ± 0.012	1.11 ± 0.64	0.039 ± 0.023
7	Null			1.19	0.042	1.99	0.070
8	Null			0.42	0.015	0.35	0.012
9	Null			0.47	0.017	1.28	0.045
	Mean± SD			0.69 ± 0.43	0.025 ± 0.015	1.21 ± 0.82	0.042 ± 0.029

*  The possibility of coelution of a zeaxanthin metabolite with lutein cannot be ruled out.

†  The calculation was based on the exponential equation derived by Hammond et al, ²⁵ and there is an assumption that the total amount of macular pigment from 0 to 1.5 mm and 1.5 mm to 3 mm from the center was proportional to the area under the exponential curve.

 

The authors thank Josephine Ngai for technical assistance in preparing the P1 extracts and HPLC analysis, Zhang Chun for surgical removal of monkey tissues, Ngai Nga Man Laura for the mathematical conversion of macular pigment in the 3-mm punches to that in 6-mm punches, and Albert Cheung of the Center for Clinical Trials and Epidemiologic Research, the Chinese University of Hong Kong, for statistical analysis.