The Journal of Nutrition Vol. 128 No. 10 October 1998, pp. 1802-1806

Dietary Lutein Absorption from Marigold Extract is Rapid in BALB/c Mice^1,2,3

Jean Soon Park^*, Boon P. Chew^{*, , 4}, and Teri S. Wong

^* Program in Nutrition and  Department Animal Sciences, Washington State University, Pullman, WA 99164-6351

	ABSTRACT

Abstract Introduction Methods Results Discussion References

Even though lutein can stimulate immunity and decrease cancer growth, no systematic studies are available on the uptake of lutein in mice. We studied the uptake of lutein in 8-wk-old female BALB/c mice fed a diet containing 0, 0.05, 0.1, 0.2 or 0.4% lutein. Mice were killed on d 0, 3, 7, 14, 21 and 28 (n = 6/period), and blood, spleen and liver were collected. Food intake and body, liver and spleen weights did not differ among treatment groups. Lutein + zeaxanthin were not detectable in the plasma, liver and spleen of unsupplemented mice. Mice fed lutein showed very rapid lutein + zeaxanthin absorption. On d 3, concentrations of plasma lutein + zeaxanthin had rapidly increased (P < 0.05) in lutein-fed mice and no further increases were observed. Plasma lutein + zeaxanthin concentrations did not differ among lutein-fed mice by d 7 (2.58 ± 0.2 µmol/L). Even though maximal uptake of plasma lutein + zeaxanthin was observed by d 3, uptake of lutein + zeaxanthin by the liver and especially by the spleen generally continued to increase (P < 0.05) through d 28 to reach concentrations of 0.11 ± 0.001 (spleen) and 0.71 ± 0.0002 (liver) nmol/g. Therefore, dietary lutein is readily absorbed into the plasma and taken up by liver and spleen of mice. Plasma lutein + zeaxanthin concentrations were higher than in human studies; however, mice were fed lutein at a level several hundredfold greater than in humans. The liver is a major storage organ for lutein + zeaxanthin in mice. Uptake of lutein + zeaxanthin by the spleen suggests a role for lutein in modulating immunity.

	INTRODUCTION

Abstract Introduction Methods Results Discussion References

Carotenoids represent a group of fascinating, abundant and widely distributed natural pigments. Carotenoids' possible role in reducing cancer in humans was first proposed by Peto et al. (1981). Since then, numerous epidemiological studies have suggested one or more carotenoids from fruits and vegetables may decrease the incidence of major chronic diseases such as lung, breast and prostate cancers (Flagg et al. 1995, Harris et al. 1991, Poppel and Goldbohm 1995). Optimism about the anticancer activity of -carotene was dampened by recent studies that showed an increased incidence of lung cancer with -carotene intake among high-risk populations of smokers and asbestos workers (Albanes et al. 1995, Omenn et al. 1996) and no effect against cancer and heart disease (Hennekens et al. 1996). Studies with other carotenoids have begun to emerge.

The xanthophylls, lutein and zeaxanthin, are nonprovitamin A carotenoids and have specific biological functions in decreasing cancer development, in enhancing immune function (Chew et al. 1996) and in protecting against age-related macular degeneration (Snodderly 1995). For instance, mice fed high levels of lutein had slower growth of a transplantable mammary tumor (Chew et al. 1996). Furthermore, dietary lutein enhanced lymphocyte proliferation response (Chew et al. 1996). In humans, high dietary lutein is correlated with greater expression of estrogen receptors in breast cancer cells and consequently greater survival rates and better response to hormone therapy (Rock et al. 1996).

Lutein possesses potent antioxidant activity. In cultured cells, lutein is more effective than -carotene in inhibiting the auto-oxidation of cellular lipids (Zhang et al. 1991) and in protecting against oxidant-induced cell damage (Martin et al. 1996). In humans, lutein and zeaxanthin are absorbed more efficiently than -carotene (Gartner et al. 1996). Low dietary lutein increased, whereas high dietary lutein disrupted -carotene absorption in rats and humans (High and Day 1951, Kostic et al. 1995). In our continued effort to study the role of dietary lutein in modulating immunity and in inhibiting mammary cancer in the BALB/c mice, it was necessary to understand the absorption and tissue uptake of dietary lutein in this species. Therefore, our objective was to study the uptake of dietary lutein into the plasma, liver and spleen of BALB/c mice fed different levels of lutein.

	MATERIALS AND METHODS

Abstract Introduction Methods Results Discussion References

Animals and diet.  Eight-week-old female BALB/c mice (n = 180) were fed a semisynthetic diet (Dyets, Bethlehem, PA) containing 0, 0.05, 0.1, 0.2 or 0.4% of lutein (Table 1). Animals were housed in plastic cages (3 mice/cage) in a light- (12 h light) and temperature-controlled (23°C) room. The lutein was from marigold extract (INEXA C. A., Quito, Ecuador) and contained (per 100 g) 37 g of lutein esters and 0.5 g of zeaxanthin esters, with the rest mainly consisting of fatty acid esters of high molecular weight alcohols (Chew et al. 1996). The lutein esters (per 100 g) were 56 g of lutein dipalmitate, 36 g of lutein dimyristate and 8 g of lutein monomyristate. The marigold extract (hereafter referred to as lutein) was first mixed with the safflower oil portion (heated to 60°C) of the diet before being gradually added to the rest of the dietary ingredients. The prepared diets were kept at 4°C until used, and at weekly intervals, a portion of each diet was mixed with agar and allowed to solidify (Park et al. 1993). The solidified diet was freely available to the mice. Food intake was measured daily and body weight was recorded weekly. Animals had free access to clean water.

Six mice in each treatment group were killed on d 0, 3, 7, 14, 21 and 28 after the initiation of dietary lutein supplementation. Blood plasma, liver and spleen were obtained and liver and spleen weights recorded. All samples were frozen at 20°C under a layer of nitrogen. The protocol was approved by the Washington State University Animal Care and Use Committee (Pullman, WA).

Plasma and tissue extraction and high performance liquid chromatography (HPLC)⁵ analysis.  Analyses of plasma and tissues were performed using HPLC (Waters, Milford, MA) as previously described (Chew et al. 1996). Plasma protein was precipitated by adding an equal volume of ethanol containing 0.1% butylated hydroxytoluene (BHT) (Aldrich Chemical Co., Milwaukee, WI). The mixture was extracted with 10 vol of a 1:1 mixture of anhydrous diethyl ether/petroleum ether and the residue was resuspended in mobile phase consisting of a 47:47:6 (v/v/v) mixture of HPLC-grade acetonitrile/methanol/chloroform (Fisher Scientific, Fair Lawn, NJ).

Liver and spleen (0.3 g) were homogenized in 5 mL of ethanol containing 0.1% BHT. The homogenate was saponified by adding 2 mL of 10 mol/L KOH and incubating the mixture at 60°C for 45 min. Subsequently, 2 mL of cold deionized H₂O was added and the mixture extracted as described for plasma.

Residues from plasma and tissue extracts were redissolved in the mobile phase (47:47:6 v/v/v mixture of acetonitrite, methanol and chloroform) and eluted on a reversed-phase 5-µm spherical, C-18 column (Resolve, Waters; 3.9 × 150 mm) at a flow rate of 1 mL/min. Lutein and zeaxanthin could not be clearly resolved and results are subsequently presented as a single peak of lutein + zeaxanthin. Lutein + zeaxanthin was detected at 450 nm with a limit of detection of 0.09 nmol/L.

Statistical analysis.  Data were analyzed by analysis of variance (ANOVA) using the General Linear Models Procedure of SAS (SAS 1991). All studies were performed with n = 180. Statistical significance was accepted at the level of P = 0.05. Treatment differences in lutein and zeaxanthin concentrations, food intake and body weight were analyzed by repeated measures ANOVA using the following statistical model: Y_ij = µ + treatment_i + mice(treatment)(error A used to test the effects of treatment) + period_j + treatment × period_ij + error_ij. Treatment differences in the lutein + zeaxanthin concentration in plasma, liver and spleen were compared using the Student's t test (Steel and Torrie 1980).

	RESULTS

Abstract Introduction Methods Results Discussion References

Body weight and feed intake.  Body, liver and spleen weights were not significantly different among treatment groups during the study period (Table 2). Similarly, there was no significant treatment difference in food intake throughout the experimental period. Therefore, total lutein intake among the treatment groups reflected the level of dietary supplementation (P < 0.05) (Table 2).

Plasma lutein.  Lutein + zeaxanthin were the only carotenoids found in the plasma of mice fed lutein in the form of lutein esters. Concentrations of plasma lutein + zeaxanthin were not detectable in unsupplemented mice during the 4-wk study period (Fig. 1). In contrast, plasma lutein + zeaxanthin in all lutein-supplemented mice increased (P < 0.01) dramatically to reach concentrations of over 2.5 µmol/L on d 3. At this period, lutein + zeaxanthin absorption into the plasma was dose-dependent (P < 0.05) in mice fed up to 0.2% of lutein. No further increase in plasma lutein + zeaxanthin was observed in mice fed 0.4% of lutein. Concentrations of plasma lutein + zeaxanthin plateaued throughout the rest of the study period in all groups.

View larger version (24K): [in this window] [in a new window]   Fig 1. Concentrations of plasma lutein + zeaxanthin in mice fed diets containing 0 (control), 0.05, 0.1, 0.2 and 0.4% lutein for 28 d. Values are means ± SEM, n = 6. Plasma lutein + zeaxanthin concentrations were not detectable in mice fed the control diet. Concentrations were significantly higher in all lutein-supplemented mice at all sampling periods (d 3-28) compared to control (P < 0.05, repeated measures analysis of variance). Means at a time point with different letters differ significantly, P < 0.05.

Liver lutein.  As with plasma, lutein + zeaxanthin were not detectable in the liver of unsupplemented mice (Fig. 2). Concentrations of lutein + zeaxanthin in the liver of lutein-fed mice increased (P < 0.01) rapidly by d 3 and generally increased (P < 0.05) dose-dependently through d 7. Liver lutein + zeaxanthin concentrations in mice fed 0.4% lutein continued to increase throughout the study period while those fed lower levels of lutein plateaued after d 14. Unlike in plasma, the concentration of lutein + zeaxanthin in the liver was highest (P < 0.05) in mice fed 0.4% of lutein throughout the study (P < 0.01) although there were no significant treatment differences in liver lutein + zeaxanthin on d 14 (Fig. 2).

View larger version (25K): [in this window] [in a new window]   Fig 2. Concentrations of liver lutein + zeaxanthin in mice fed diets containing 0 (control), 0.05, 0.1, 0.2 and 0.4% lutein. Values are means ± SEM, n = 6. Mice fed the control diet did not have detectable lutein + zeaxanthin in the liver. Concentrations were significantly higher in all lutein-supplemented mice at all sampling periods (d 3-28) compared to control (P < 0.05, repeated measures analysis of variance). Means at a time point with different letters differ significantly, P < 0.05.

Total lutein + zeaxanthin content in the liver generally reflected changes in liver lutein concentrations, particularly during the first week of supplementation (Table 3). Liver lutein + zeaxanthin content showed a dose-related increase through d 28 (P < 0.05). Lutein uptake by the liver reached the steady state by d 14 in mice fed 0, 0.05 and 0.1% of lutein, whereas liver uptake by mice fed 0.2 and 0.4% of lutein continued to increase lutein uptake through d 28. 

Spleen lutein.  Concentrations of lutein + zeaxanthin in the spleen of mice fed lutein increased (P < 0.01) rapidly by d 3 and continued to increase, even though more gradually, throughout the rest of the study period (Fig. 3). This was in contrast to unsupplemented mice that had nondetectable amounts of splenic lutein + zeaxanthin. Concentrations of lutein + zeaxanthin in the spleen generally showed a dose- and time-dependent increase, with mice fed 0.4% of lutein having significantly higher (P < 0.05) concentrations on d 3-28 than those fed lower amounts of lutein. In the spleen, differences in total lutein + zeaxanthin (Table 3) generally reflected differences observed in lutein + zeaxanthin concentrations (Fig. 3).

View larger version (27K): [in this window] [in a new window]   Fig 3. Concentrations of lutein + zeaxanthin in the spleen of mice fed diets containing 0 (control), 0.05, 0.1, 0.2 and 0.4% lutein. Values are means ± SEM, n = 6. Mice fed the control diet did not have detectable lutein + zeaxanthin in the spleen. Splenic lutein concentrations were significantly higher in all lutein-supplemented mice at all sampling periods (d 3-28) when compared to control (P < 0.05, repeated measures analysis of variance). Means at a time point with different letters differ significantly, P < 0.05.

	DISCUSSION

Abstract Introduction Methods Results Discussion References

Numerous species including humans (Kostic et al. 1995, Parker 1989), birds (Schaeffer et al. 1988, Tyczkowski and Hamilton 1986a and 1986b), calves (Bierer et al. 1995), ferrets (Tang et al. 1995), rats (High and Day 1951) and mice (Chew et al. 1996) can absorb dietary lutein. Lutein is one of the major carotenoids in the human body (Parker 1989) and has been hypothesized to be interconverted to zeaxanthin in biological systems (Bone et al. 1993, Khachik et al. 1995).

This study demonstrates that the uptake of dietary lutein by plasma and tissues in mice is very rapid (within 3 d of initiation of lutein feeding). However, differences exist between plasma and tissues in the rate of lutein + zeaxanthin uptake. Concentrations in plasma were maximal by d 3 in all lutein-supplemented mice and were not significantly influenced by the level of dietary lutein starting by d 7. In contrast, lutein + zeaxanthin concentrations generally continued to accumulate in the liver and, especially, in the spleen even though the most rapid increase was observed during the first 3 d of lutein supplementation. Uptake of lutein + zeaxanthin by the spleen differed (P < 0.05) among treatment groups; levels were highest in mice fed 0.4% lutein. As with canthaxanthin (Tang et al. 1995), the liver appears to be a major storage organ for lutein + zeaxanthin in mice.

Studies using a single dose of oral lutein showed peak concentrations in the plasma at 16 h in humans (Kostic et al. 1995) and at 8 to 12 h in cattle (Bierer et al. 1995). In humans, dietary lutein is readily absorbed and, furthermore, is preferentially taken up by chylomicra compared to -carotene (Gartner et al. 1996). Humans supplemented with 10 mg of lutein daily for 18 d showed a continuous increase in plasma lutein concentrations (Khachik et al. 1995). However, it is not known from the study when saturation in the plasma would occur. In the present study, plasma lutein + zeaxanthin saturation was observed by d 7 of feeding. Plasma lutein concentrations in humans (Khachik et al. 1995) are lower (1.4 µmol/L) than observed in mice in the present study (2.5 to 3.0 µmol/L). This difference is likely a reflection of the level of lutein supplementation. When lutein consumption was calculated based on body weight, mice in the present study were supplemented with lutein at a level several hundred times greater than that used in the human study (Khachik et al. 1995). Mice fed the lowest level of lutein (0.05%) consumed 70 mg of lutein/kg body weight compared to an estimated dose of 0.14 mg of lutein/kg body weight in the human study (Khachik et al. 1995).

It is interesting to note in this study that mice fed 0.2% of lutein generally showed numerically higher concentrations of plasma lutein + zeaxanthin than those fed 0.4% of lutein (P < 0.10). This supports an earlier study (Chew et al. 1996) in which mice fed 0.1% of lutein for 70 d had higher concentrations of plasma lutein than mice fed 0.4% of lutein (P < 0.05). These studies suggest decreased intestinal absorption or increased tissue uptake or metabolism of circulating lutein + zeaxanthin in mice fed 0.4% of lutein. In spite of slightly lower plasma concentrations, mice fed 0.4% of lutein had lower incidence and growth of mammary tumors than those fed the lower amount of lutein (Chew et al. 1996). Unfortunately, in the latter study, no data were available on liver or spleen uptake of lutein. In the present study, mice fed 0.4% of lutein had the highest concentration of lutein + zeaxanthin in the spleen. Therefore, it would seem that splenic lutein + zeaxanthin concentration is more indicative of the anticancer activity of lutein + zeaxanthin against mammary tumor growth than is the concentration in plasma. In the spleen, lutein may modulate immune function (Chew et al. 1996).

There were no significant treatment differences in changes in body, liver or spleen weights or feed intake. Furthermore, daily food intake in all mice was as expected (NRC 1978). This indicates a lack of a toxic effect of dietary lutein from marigold extract and is in agreement with other reports (CARIG 1996, Chew et al. 1996, Gartner et al. 1996).

Studies with lutein must take into consideration its possible interaction with other lipid-soluble compounds including other carotenoids. In humans, lutein intake affects -carotene absorption (High and Day 1951, Kostic et al. 1995). Furthermore, there is a preferential uptake of lutein + zeaxanthin over that of -carotene by blood chylomicron (Gartner et al. 1996). Tissue uptake of carotenoids, retinoids and tocopherol is decreased with increased dietary canthaxanthin feeding (Tang et al. 1995).

Mice, like humans, can absorb lutein from the diet and the lutein is rapidly taken up by the plasma, liver and spleen. The concentration of lutein in plasma is more readily saturable than that in liver or spleen. Therefore, plasma lutein concentration reflects short-term feeding while concentrations of liver and spleen reflect long-term feeding. Furthermore, lutein concentrations in the spleen may more accurately reflect its activity since splenic uptake of lutein + zeaxanthin (this study) correlates with the activity of lutein + zeaxanthin in stimulating immunity (Chew et al. 1996) and in inhibiting the growth of mammary tumors (Park et al. 1998).

	FOOTNOTES

Manuscript received 16 December 1997. Initial reviews completed 10 March 1998. Revision accepted 13 July 1998.

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