The Prime Role of HDL to Transport Lutein into the Retina: Evidence from HDL-Deficient WHAM Chicks Having a Mutant ABCA1 Transporter

Abstract

purpose. Lutein and zeaxanthin are largely transported in plasma by high-density lipoprotein (HDL). The Wisconsin hypoalpha mutant (WHAM) chicken has a recessive sex-linked mutation in the ABCA1 transporter gene that results in a severe deficiency of HDL. In this study, the transport and tissue distribution of lutein and zeaxanthin were examined in newly hatched and 28-day-old WHAM chicks compared with control chicks.

methods. One-day-old WHAM and control chicks were randomized to be fed a high-lutein or a control diet for 28 days. The plasma and tissues were analyzed for lutein, zeaxanthin, and lipoproteins on days 1 and 28.

results. The WHAM chicks had very low plasma levels of HDL cholesterol (5.3% of normal). They also had very low concentrations of lutein in the plasma and all other tissues compared with control chicks. The plasma and retina were only 9% and 6% of control levels (P < 0.01), respectively. Zeaxanthin levels were similarly low (9% of control, P < 0.01). The high-lutein diet increased the content of lutein in the plasma and tissues of control chicks (P < 0.01). In contrast, in WHAM chicks, lutein increased greatly in the plasma, liver, and heart, but little in the retina (6% of control).

conclusions. HDL deficiency in the WHAM chicks was associated with a deficiency of lutein and zeaxanthin in the tissues, especially in the retina. The high-lutein diet increased the lutein content of some tissues via LDL and VLDL transport, but retinal lutein remained very low. These data support the prime role of HDL as the specific transporter of lutein and zeaxanthin into the retina. The WHAM chick provides an excellent model for the study of the role of HDL in the retinal uptake of lutein and zeaxanthin.

The Wisconsin hypoalpha mutant (WHAM) chicken has a recessive sex-linked mutation in the ABCA1 transporter that was identified in 1981 at the University of Wisconsin-Madison. ^{1 2 3} As a consequence of this mutation, WHAM chickens have very low levels of HDL, white skin, colorless plasma, and decreased total plasma xanthophyll concentrations. ³ In humans, a mutation in the ABCA1 transporter leads to very low HDL levels in Tangier Disease, ^{4 5 6} which is analogous to the defect in the WHAM chickens. 

Lutein and zeaxanthin are plant pigments obtained from the diet that have important bioactivity in humans and animals. These xanthophylls are concentrated in the retina, especially in the macula, of humans. Although some epidemiologic studies show a protective effect of dietary lutein and zeaxanthin on the prevalence of macular degeneration, ^{7 8} others do not indicate a potential benefit. ⁹ In the findings in the Carotenoids in Age-Related Eye Disease Study, macular pigment density was directly related to the dietary intake of lutein and zeaxanthin, but it was even more strongly related to their serum concentrations. ¹⁰ Dietary intakes of lutein and zeaxanthin may increase macular pigment density. ^{11 12} In both normal and AMD subjects, a diet high in lutein and zeaxanthin increased the plasma levels of lutein and zeaxanthin more than twofold. ¹³ It is worth noting that the National Eye Institute is beginning a large-scale clinical trial of lutein and zeaxanthin as inhibitors of macular degeneration (Age-Related Eye Disease Study 2 [AREDS 2]). This study will further answer questions about the roles of lutein and zeaxanthin in this devastating disease. 

Since lutein and zeaxanthin are primarily transported in plasma by HDL, ^{13 14 15 16} we hypothesized that a deficiency of HDL would be associated with a tissue deficiency of lutein and zeaxanthin. In the present study, we identified specific tissue deficiencies of lutein and zeaxanthin and described the transport and tissue distribution of lutein and zeaxanthin in newly hatched and 28-day-old WHAM and control Leghorn chicks fed control and high-lutein diets. These experiments were designed to assess the importance of HDL in supplying lutein and zeaxanthin to the retina. The retina appears to be especially vulnerable to the effect of a deficiency in HDL on the transport of lutein and zeaxanthin in contrast to other tissues. 

Materials and Methods

The Incubation of Eggs

Fertilized WHAM eggs were shipped overnight at ambient temperature by The University of Wisconsin. Fertilized control Leghorn eggs were obtained locally. Both groups of eggs were incubated at 99.5° for 21 days in a rocking incubator. A constant humidity was produced by water in the incubator. The newly hatched chicks were incorporated into the experiments, which had been approved by the Animal Care and Use Committee at Oregon Health and Science University and were conducted in compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 

Dietary Groups

One-day-old WHAM (n = 24) and control Leghorn (n = 24) chicks were randomly divided into four groups of six chicks each. One group provided baseline control data at 1 day of age. Two other groups were fed a high-lutein or control diet for 28 days. A fourth group was euthanatized at 14 days for analysis of plasma lipoprotein lipid concentrations. The control diet (Start and Grow Sunfresh) was obtained from Purina Mills, LLC (St. Louis, MO). The high-lutein diet was the same as the control diet, with the addition of lutein. The composition of lutein and zeaxanthin in the diets is given in Table 1 . 

For the high-lutein diet, 420 mg lutein powder (containing lutein 5.2% and zeaxanthin 0.12%; DSM Nutritional Products Ltd., Basel, Switzerland) was dissolved in 10 mL ether ethyl and then mixed thoroughly into the control diet (1000 g), and the ether ethyl was evaporated completely in the hood overnight. 

Procedures

Chicks were euthanatized on days 1 and 28 with 50 mg ketamine+10 mg xylazine/mL. After 5 minutes, the chest was opened, and blood was drawn from the heart. The eyes were removed and the retina dissected. The brain, heart, liver, skin, and adipose tissues also were harvested for analysis. All tissues were washed with saline and stored at −80°C until analysis. 

Plasma and Tissue Analysis for Lutein and Zeaxanthin

A 200-μL aliquot of plasma was diluted with 0.5 mL 0.9% saline solution, and 60 μL BHT (10 mg/mL) was added as an antioxidant. Echinenone, 86 ng in 200 mL ethanol, was added as an internal standard. The mixture was extracted once with 2 mL CHCl3:CH3OH (2:1, vol/vol) and twice with 3 mL hexane. The combined organic solvent phase was evaporated to dryness under nitrogen. The residue was redissolved in 150 μL ethanol. A 50-μL portion was used for HPLC analysis. 

Tissue levels of lutein and zeaxanthin were analyzed by saponifying 30 mg of wet tissue at 37°C for 2 hours in 100 μL 12% pyrogallol in ethanol, 200 μL 30% aqueous KOH, 60 μL BHT (10 mg/mL), and 1 mL ethanol. Echinenone 43 ng in 100 μL ethanol was added as an internal standard. The mixture was extracted twice with 3 mL ether-hexane (2:1, vol/vol). The organic solvent extract was evaporated to dryness under nitrogen. The residue was redissolved in 100 μL ethanol, and a 50-μL sample was used for HPLC analysis. ^{17 18 19}  

Fresh Leghorn and WHAM eggs were boiled and the yolk removed. Lutein and zeaxanthin were measured in the egg yolk and in the diet, as just described. 

HPLC Analysis

The HPLC system consisted of a series 200 pump, autosampler, and UV/V detector plus a C30 carotenoid column (3 μm, 4.6 × 150 mm). The HPLC mobile phase was methanol/methyl-tert-butyl ether/water (83:15:2, vol/vol/vol, solvent A) and methanol/methyl-tert-butyl ether/water (8:90:2, vol/vol/vol, solvent B). The gradient procedure, at a flow rate of 1 mL/min at 16°C, began at 100% solvent A before going to 93% solvent A and 7% solvent B over a 1-minute linear gradient. This was followed by a 3-minute hold at 93% solvent A, followed by a 17-minute linear gradient to 45% solvent A and a 1-minute hold at 45% solvent A, an 11-minute linear gradient to 95% solvent B, a 4-minute hold at 95% solvent B, and finally a 2-minute gradient back to 100% solvent A. The system was held at 100% solvent A for 10 minutes for equilibration, to resolve and return to initial conditions. 

Lutein and zeaxanthin were quantified by determining peak areas in the HPLC chromatograms calibrated against known amounts of standards. They were corrected for losses sustained during extraction and handling, by monitoring the recovery of the internal standards. ¹⁹  

Plasma Lipoproteins

Plasma lipoproteins were isolated by density gradient ultracentrifugation. ^{20 21} Lipoprotein cholesterol was quantified by a cholesterol oxidase colorimetric assay (704 Chemistry Analyzer; Hitachi, Tokyo, Japan). ^{22 23} The carotenoid content of each lipoprotein fraction was measured as described earlier. 

Statistical Analyses

Means and standard deviations were calculated, to characterize the lutein and zeaxanthin content of plasma and tissues of each group (n = 6). Differences between tissues were analyzed by one-way ANOVA, using the Bonferroni adjustment for inequality, to control the overall α-level ²⁴ (SPSS for Windows, ver. 10.0.1; SPSS Inc, Chicago). 

Results

General Appearance of WHAM Chicks and Lutein and Zeaxanthin in WHAM and Control Egg Yolk

The WHAM chicks had white plasma, feet, and heads at birth and subsequently, despite being fed the control diet, which contained lutein and zeaxanthin. They remained white because of the deficiency of lutein and zeaxanthin. Lutein and zeaxanthin are yellow pigments that are responsible for the yellow control chicks. ²⁵  

Despite the white skin of the WHAM chicks, the concentrations of lutein and zeaxanthin in the WHAM and normal egg yolks were similar, which is consistent with the normal yellow of both yolks (Table 2) . 

Lipoprotein Concentrations

The total cholesterol and HDL cholesterol concentrations in WHAM chicks were significantly lower than in control chicks. HDL in WHAM chicks was only 5% of that in control animals (P < 0.01). The VLDL cholesterol concentration was similar in WHAM and control chicks, but the LDL cholesterol concentration in WHAM chicks was higher than that in control chicks (Table 3 , Fig. 1 ). HDL is the predominant lipoprotein of non–egg-laying chickens. 

Lutein and Zeaxanthin in Plasma and Tissues

At 1 day of age, WHAM chicks had very low levels of lutein in the plasma and almost all tissues except the yolk sac and liver, compared with control chicks, but the most dramatic differences were in the plasma and retina (Table 4 , Fig. 2 ). The lutein concentration in plasma of WHAM chicks (45 μg/dL) was only 9% of the level in control chicks (504 μg/dL, P < 0.01). The lutein levels in retina, heart, skin, and adipose tissue of WHAM chicks also were only 6%, 47%, 55%, and 42% of the same tissues of control chicks, respectively. Brain tissue had very low concentrations of lutein and zeaxanthin in both control and WHAM chicks. 

The zeaxanthin content was similarly low in plasma and tissues, including the retina of WHAM chicks (Table 5 , Fig. 3 ). Zeaxanthin in the plasma of WHAM chicks (1 day old) was only 6% of content levels in the plasma of control chicks (P < 0.01). Zeaxanthin levels in retina, heart, and skin of WHAM chicks (1 day old) also were only 9%, 71%, and 28% of the same tissues of control chicks (Table 5 , Fig. 2 ). 

Effects of the High-Lutein Diet at 28 Days

The high-lutein diet significantly increased the plasma and tissue content of lutein in both WHAM and control chicks (Table 4) , but the lutein content in the plasma and tissues of WHAM chickens remained significantly lower than the content in the control Leghorn chickens. The lutein content in plasma of control chicks fed the high-lutein diet increased from 504 to 1148 μg/dL (Δ = 644 μg/dL) at 28 days, but increased only from 46 to 238 μg/g in the plasma of WHAM chicks. Lutein levels in retinas of control chicks increased from 4.5 to 11.0 μg/g after 28 days of the high-lutein diet compared with an increase from 0.39 to only 0.71 μg/g in WHAM chicks. In WHAM chicks fed the high-lutein diet, lutein levels increased significantly from 2.0 to 8.2 μg/g in the liver and 1.1 to 3.8 μg/g in the heart. The levels in liver and heart after the high-lutein diet for 28 days were similar in control and WHAM chicks. 

The lutein content of plasma and lipoprotein HDL, LDL, and VLDL increased four- to fivefold in WHAM chicks fed the high-lutein diet for 28 days (Table 6) . Fifty-three percent of the lutein was transported in HDL. The zeaxanthin content of the lipoproteins after the control and high-lutein diets was very low and not different in the WHAM chicks. Why the high lutein content of HDL did not increase the lutein content in the retina is intriguing and will be discussed later. 

The zeaxanthin concentration in the plasma and extrahepatic tissues of WHAM chicks was also lower than or similar to the level in chicks fed the control and high-lutein diets for 28 days (Table 5) . The high-lutein diet contained proportionately less zeaxanthin than in the control diet. The lutein-zeaxanthin ratio was 3 in the control diet, and this ratio increased to 12 after the high-lutein diet. The zeaxanthin content of the plasma of control chicks fed the high-lutein diet decreased from 235 (day 1) to 61 (day 28) μg/g, but it did not change significantly in the WHAM chicks (15 μg/g on day 1 vs. 20 μg/g on day 28; P > 0.05). In the retina of WHAM chicks, the level of zeaxanthin was significantly lower than that in control chicks, but it did not change after either the control or high-lutein diets (0.36 μg/g vs. 0.52 μg/g; P > 0.05 and 0.36 μg/g vs. 0.48 μg/g; P > 0.05). In the livers of WHAM chicks fed the high-lutein diet, the zeaxanthin level was significantly higher than that in the control chicks fed the high-lutein diet (0.95 μg/g vs. 0.64 μg/g; P < 0.01). In the skin of WHAM chicks fed the high-lutein diet, the zeaxanthin level remained significantly lower than that of the control chicks (0.25 μg/g vs. 0.55 μg/g; P < 0.05). The zeaxanthin concentrations in brain, heart, and adipose tissue were not significantly different between WHAM and control chicks after the normal and high-lutein diets for 28 days (Table 5) . 

Discussion

The WHAM chicken has a sex-linked ABCA1 mutation associated with a 90% reduction in the plasma concentrations of HDL cholesterol and apoA-I. ^{2 3} ApoA-I synthesis and secretion by the liver and intestine are normal, but HDL cholesterol levels are low because the ABCA1 mutation blocks the apoA-I-mediated efflux of cholesterol and phospholipid from the peripheral tissues into nascent HDL. The lipid-poor apoA-I is rapidly degraded. ²⁶ In the present study, as anticipated, the total plasma cholesterol concentration in WHAM chicks was one third the level in control chicks (52 mg/dL vs. 165 mg/dL; P < 0.01), but the HDL cholesterol level in WHAM chicks was only 5.5% of the level in control chicks (8 mg/dL vs. 145 mg/dL; P < 0.01). Levels of LDL and VLDL lipoproteins in WHAM chickens were normal or even increased (LDL). 

Both lutein and zeaxanthin are carotenoid pigments that are soluble in organic solvents and fat but are insoluble in water. In the present study, the dietary fat content was sufficient to facilitate normal absorption of carotenoids in WHAM chicks, as reflected by normal accumulation of these carotenoids in the liver. Lutein and zeaxanthin are absorbed from micelles formed in the intestinal lumen and are transported in chylomicrons in lymph and then in the blood to the liver. ²⁷ Levels of lutein and zeaxanthin in the liver and yolk sac of WHAM chicks were also normal. Yolk levels were normal because they were maternally derived. The reason for normal hepatic levels is that the transport mechanism from the enterocyte to the liver is mediated by chylomicrons and VLDL. Non-HDL mediated transport systems appear to be intact in the WHAM chick. Subsequent transport of lutein and zeaxanthin from the liver to other tissues appears to occur predominantly via HDL, the principal lipoprotein of control chickens. In the WHAM chicks, this process was inhibited presumably because of HDL deficiency caused by the block in formation of nascent HDL. Accordingly, lutein and zeaxanthin were poorly distributed to most extrahepatic tissues in WHAM chicks, resulting in lower concentrations of lutein and zeaxanthin in the retina, skin, and adipose tissue, despite normal hepatic level levels of these carotenoids. 

It is hypothesized that specific transport mechanisms facilitate the incorporation of lutein and zeaxanthin into the retina. The retina is part of the central nervous system and is protected by the blood–brain barrier. However, the content of lutein and zeaxanthin in the brain, per se, is infinitesimal. In control 28-day-old chicks, there was 0.11 μg of lutein per gram of tissue in the brain and 0.14 μg of zeaxanthin. The comparative amounts in the retina were 4.33 μg lutein and 7.11 μg zeaxanthin per gram of tissue. Hence, there was almost 40 times more lutein and zeaxanthin in the retina than in the brain. Clearly, the penetration of the blood–retinal barrier by lutein and zeaxanthin was far greater than that of the blood–brain barrier. The differences between retina and brain transport of lutein and zeaxanthin may be accounted for by a specific retinal receptor for HDL that then releases lutein and zeaxanthin into retina. Then, a xanthophyll binding protein may transport lutein and zeaxanthin to photoreceptor cells. ^{28 29} Several proteins have been suggested. Bernstein et al. ³⁰ reported that tubulin is major soluble carotenoid-binding protein in bovine and human retinal tissues. Lutein and zeaxanthin were found to copurify with tubulin. The results of their recent study indicate that the Pi isoform of human glutathione S-transferase (GSTP1) is also a specific xanthophyll-binding protein (XBP) in the human macula that interacts with meso-zeaxanthin and dietary zeaxanthin. ³¹ Thus, there is evidence of a specific xanthophyll transport protein in the retina. 

The important role of HDL in the transport and retinal incorporation of lutein and zeaxanthin is further emphasized in the WHAM chick, which has a very low concentration of HDL but fairly normal amounts of the other lipoproteins LDL and VLDL. For example, the ratio of lutein in the retinas of the WHAM chicks compared with the control chicks was 0.063, whereas the ratio in the heart tissue was 0.47 at 1 day and 1.0 after 28 days of the high-lutein diet. The heart of the WHAM chick had 5.4 times more lutein than did the retina after the high-lutein diet. This suggests that the heart may derive its lutein mainly from LDL and VLDL, which are not suppressed in WHAM chicks, whereas the retina acquires its lutein and zeaxanthin mainly from the HDL transport mechanism. 

Furthermore, the important role of HDL in transporting lutein and zeaxanthin into the retina is illustrated by the results of feeding of the high-lutein diet to WHAM chicks and the responses of the plasma and various tissues, including the retina. The plasma, heart, and adipose tissue of WHAM chicks had increases in lutein content of 388%, 248%, and 220%, whereas the retinal lutein increased only 69% and remained low at 0.71 μg/g versus control retinas of 11.0 μg/g. We surmise that LDL and VLDL transported lutein normally from the liver to the heart and adipose tissue, but severe HDL deficiency prevented effective transport of lutein to the retina despite the high-lutein diet. Thus, HDL appears to be essential for transporting lutein to the retina. 

The administration of a high-lutein diet to the WHAM chicks did indeed increase the lutein in the plasma, from 54 to 238 μg/dL. The lutein of the HDL increased from 28.6 to 127.5 μg /dL after the high-lutein diet. Although the lutein content of HDL increased almost fivefold, the diet did not seem to affect the retinal lutein content very much. Retinal levels remained quite low at 0.71 μg /g of retina in contrast to the retinal values of 11.0 μg /g in the control chicks in which HDL concentration was normal. These data emphasized that increasing the lutein content of HDL did not correspondingly increase the lutein content of the retina, because the number of HDL molecules remained the same although supersaturated with lutein. The low-HDL concentration simply prevented the entrance of much more lutein into the retina. To increase retinal lutein, two components are necessary: a normal HDL level and adequate lutein and zeaxanthin transported by HDL. 

The preference of the retina for zeaxanthin over lutein is illustrated in the control chicks fed the high-lutein diet, which contained only a small amount of zeaxanthin. The zeaxanthin content of the plasma and most tissues declined during the high-lutein diet, but the content of zeaxanthin in the retina increased to 6.8 from 5.6 μg/g at day 1. Others have also noted the retinal preference for zeaxanthin. ³² However, levels of zeaxanthin in the WHAM retina remained very low (0.48 μg/g). 

Tangier disease in humans, analogous to the WHAM chick, is characterized by a defect in the ABCA1-mediated cellular efflux of free cholesterol and phospholipid. ^{5 6} This results in a greatly reduced level of plasma HDL. Mutations in ABCA1 cause a severe HDL deficiency syndrome characterized by accumulation of cholesterol in tissue macrophages and sometimes accelerated atherosclerosis. ^{4 5} Because of its ability to deplete macrophages of cholesterol and to raise plasma HDL levels, ABCA1 has become a promising therapeutic target for preventing cardiovascular disease. ^{33 34 35} WHAM chicks have a gene mutation similar to that in Tangier disease and have a severe deficiency of plasma HDL. The potential role of ABCA1 in the acquisition of HDL-derived carotenoids in the retina is unknown. The usual role of ABCA1 in peripheral tissues is to facilitate transport of sterols from cells to nascent HDL particles. 

WHAM chicks provide an animal model that can be applied to the study of HDL-mediated xanthophyll transport and to the study of the effects of very low concentrations of lutein and zeaxanthin in the retina and other tissues. This avian model is similar to humans in that HDL is the chief transporter of lutein and zeaxanthin, ¹³ but it is different in that lutein and zeaxanthin are present in the avian photoreceptors of the retina as esters in oil droplets. ^{36 37} The avian retina differs also in not having a macula. 

In summary, the mutation in the ABCA1 transporter and the very low levels of HDL in the WHAM chicks were associated with normal intestinal absorption and hepatic uptake of lutein and zeaxanthin, but a dramatic lack of HDL-mediated transport of lutein and zeaxanthin to many extrahepatic tissues. The content of lutein and zeaxanthin in the retina was barely detectable (6% of control). Levels in heart and adipose tissue were 60% and 32% of normal, respectively, but only 18% of normal in skin. The high-lutein diet fed to WHAM chicks increased the content of lutein in the plasma and other tissues, but its content in the retina remained low. These data emphasize the unique role of HDL in the transport of lutein and zeaxanthin into the retina. VLDL and LDL appeared to transport lutein and zeaxanthin into other tissues, but apparently could not supply the retina even after the high-lutein diet. The WHAM chick provides an excellent genetic model for the study of transport mechanisms and tissue deficiencies of lutein and zeaxanthin. These physiologic processes may be involved in the pathophysiology of age-related macular degeneration, a disease that most studies have suggested is related to low concentrations of lutein and zeaxanthin in the diet and in the plasma and retina. 

 Supported by the Foundation Fighting Blindness.

 Submitted for publication October 23, 2006; revised February 27 and May 23, 2007; accepted July 5, 2007.

 Disclosure: W.E. Connor, None; P.B. Duell, None; R. Kean, None; Y. Wang, 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.

 

Table 1.

 

Compositions of the Chicken Diets

Nutrient	High-Lutein (%)	Control Diet (%)
Protein	18	18
Lysine	0.88	0.88
Methionine	0.32	0.32
Fat	3	3
Fiber	5	5
Calcium	1.25	1.25
Phosphorus	0.6	0.6
NaCl	0.85	0.85
Vitamin A (IU/lb)	5000	5000
Vitamin E (IU/lb)	14	14
Lutein (mg/kg)	27.2	5.2
Zeaxanthin (mg/kg)	2.2	1.7
Lutein-zeaxanthin ratio	12	3

Table 2.

 

Lutein and Zeaxanthin Concentrations in WHAM and Control Egg Yolks

	Lutein (μg/g)	Zeaxanthin (μg/g)
WHAM egg yolk*	5.69 ± 1.32	2.50 ± 1.05
Control egg yolk A*	5.21 ± 1.47	1.94 ± 0.69
Control egg yolk B, †	6.00 ± 2.03	2.84 ± 0.70

 Mean ± SD. n = 6.

*  From Department of Animal Science, University of Wisconsin.

†  Obtained locally.

Table 3.

 

Cholesterol Concentrations in the Plasma Lipoproteins of 14-Day-Old WHAM and Control Chicks

	Total	VLDL	LDL	HDL
WHAM	52.0 ± 3.5*	7.7 ± 2.9	36.7 ± 2.1*	7.7 ± 5.5*
Control	164.5 ± 30.4	7.0 ± 9.9	25.0 ± 1.4	145.0 ± 1.5

 Data are expressed as mean milligrams per deciliter ± SD. n = 6.

*  Compared with control, P < 0.01.

Figure 1.

 

Cholesterol concentration in the plasma lipoproteins of WHAM and control chicks (*P < 0.01).

Table 4.

 

Plasma and Tissue Lutein Levels after Control and High-Lutein Diet at 28 Days in Control and WHAM Chicks

	Control Chicks					WHAM Chicks
		1 Day Old	28 Days Old			1 Day Old	28 Days Old
				Control Diet	High-Lutein Diet			Control Diet	High-Lutein Diet
Yolk sac (μg/g)	23.5 ± 6.7			22.2 ± 4.5
Plasma (μg/dL)	504 ± 153	651 ± 66	1148 ± 290*	45.6 ± 13.1, †	54.4 ± 32.3, †	238 ± 141* , †
Liver (μg/g)	2.71 ± 0.69	5.84 ± 0.83	8.30 ± 2.52*	2.03 ± 0.54	3.03 ± 0.46, †	8.16 ± 2.99*
Retina (μg/g)	4.48 ± 1.71	4.33 ± 1.81	11.0 ± 3.26*	0.39 ± 0.07, †	0.42 ± 0.15, †	0.71 ± 0.20* , †
Heart (μg/g)	2.41 ± 0.91	1.83 ± 0.15	3.85 ± 0.69*	1.14 ± 0.23, †	1.10 ± 0.18, †	3.84 ± 1.72*
Skin (μg/g)	1.79 ± 0.39	2.19 ± 0.19	6.48 ± 1.55*	0.98 ± 0.24, †	0.39 ± 0.07, †	0.87 ± 0.20* , †
Adipose (μg/g)	1.59 ± 0.62	2.25 ± 0.61	4.29 ± 1.54*	0.66 ± 0.13, †	0.74 ± 0.14, †	2.38 ± 0.88* , †
Brain (μg/g)	0.16 ± 0.04	0.11 ± 0.02	0.27 ± 0.07*	0.20 ± 0.01	0.25 ± 0.03, †	0.26 ± 0.03

 Data are expressed at the mean ± SD. n = 6.

*  Compared with control diet, P < 0.01,

†  Compared with control chicks, P < 0.01

Figure 2.

 

Plasma and tissue lutein levels in 1-day-old WHAM and control chicks (*P < 0.01).

Table 5.

 

Plasma and Tissue Zeaxanthin Levels after the Control and High-Lutein Diets at 28 Days in Control and WHAM Chicks

	Control Chicks					WHAM Chicks
		1 Day Old	28 Days Old			1 Day Old	28 Days Old
				Control Diet	High-Lutein Diet			Control Diet	High-Lutein Diet
Yolk sac (μg/g)	6.2 ± 1.3			6.7 ± 1.4
Plasma (μg/dL)	235.3 ± 65	192 ± 18	61 ± 11*	14.5 ± 3.7, †	22.0 ± 12.7, †	20.3 ± 12.9, †
Liver (μg/g)	1.23 ± 0.33	2.79 ± 0.31	0.64 ± 0.21*	1.09 ± 0.30	0.70 ± 0.12, †	0.95 ± 0.34, †
Retina (μg/g)	5.60 ± 1.61	7.11 ± 1.23	6.76 ± 2.58	0.36 ± 0.13, †	0.52 ± 0.19, †	0.48 ± 0.27, †
Heart (μg/g)	0.38 ± 0.17	0.83 ± 0.08	0.31 ± 0.1*	0.27 ± 0.15	0.53 ± 0.11, †	0.39 ± 0.22
Skin (μg/g)	0.72 ± 0.12	1.04 ± 0.13	0.55 ± 0.12*	0.20 ± 0.22, †	0.27 ± 0.10, †	0.25 ± 0.02, †
Adipose (μg/g)	0.46 ± 0.06	0.73 ± 0.14	0.40 ± 0.16*	0.55 ± 0.10	0.50 ± 0.10, †	0.48 ± 0.1
Brain (μg/g)	0.10 ± 0.02	0.14 ± 0.04	0.10 ± 0.02	0.10 ± 0.01	0.17 ± 0.03	0.18 ± 0.1

 Data are expressed as the mean ± SD. n = 6.

*  Compared with control diet, P < 0.01.

†  Compared with control chicks, P < 0.01.

Figure 3.

 

Plasma and tissue zeaxanthin levels in 1-day-old WHAM and control chicks (*P < 0.01).

Table 6.

 

Lutein and Zeaxanthin Content of the Lipoproteins in WHAM Chicks Fed the High-Lutein and Control Diets for 28 Days

	Lutein			Zeaxanthin
		Control Diet	High-Lutein Diet	Control Diet	High-Lutein Diet
Plasma	54.4 ± 32.3	238 ± 141*	22.0 ± 12.7	20.3 ± 12.9
HDL	28.6 ± 5.4	127.5 ± 58.8*	13.2 ± 8.4	11.6 ± 6.4
LDL	20.4 ± 4.5	104.7 ± 24.4*	4.3 ± 1.6	5.1 ± 1.9
VLDL	2.8 ± 0.2	3.6 ± 1.0	3.8 ± 1.3	3.1 ± 1.2

 Data are expressed as mean micrograms per deciliter ± SD. n = 6.

*  Compared with the control diet, P < 0.01.

The authors thank Christian Fizet and Joseph Schierle (DSM Nutritional Products Ltd.), who provided the carotenoid standards.