- Email: [email protected]
Lutein as a Model Dihydroxycarotenoid for the Study of Pigmentation in Chickens1,2
Lutein as a Model Dihydroxycarotenoid for the Study of Pigmentation in Chickens1'2 JULIUSZ K. TYCZKOWSKI and PAT B. HAMILTON3 Department of Poultry Science, North Carolina State University, Raleigh, North Carolina 27695-7608 (Received for publication September 16, 1985) ABSTRACT A model for the study of pigmentation in young chickens is described in which a white corn-soy based diet supplemented with varying amounts of free lutein (0, 5, 10, 20, 40, and 80 Mg/g diet) was fed from hatching until 3 weeks of age. The carotenoid content of tissues dissected from chicks of the various groups was measured by high pressure liquid chromatography. In intestinal contents, three forms of lutein were found, with lutein monoester > free lutein > lutein diester. In the serum, free lutein (96%) and lutein monoester (4%) were found. In the liver, free lutein (80%), monoester (20%), and traces of diester were found. In the integument (toe web), diester > monoester s free alcohol were found. In each tissue, the concentrations were directly proportional to the dietary concentration of free lutein. The simplest explanation of the data appears to be that part of the free lutein in the diet is esterified during its passage down the intestinal tract and, regardless of its status when absorbed, it is transported in the body as the free alcohol. When it enters depot sites such as the integument, lutein is deposited mainly as esters, presumably as the result of local enzymatic activity. (Key words: lutein, chickens, pigmentation, carotenoids, absorption, transport, deposition) 1986 Poultry Science 65:1141-1145 INTRODUCTION The profile of carotenoids in corn, the primary source of economically desirable pigments in eggs and broiler chickens, is very complex (Marusich and Bauernfeind, 1981). Grogan and Blessin (1968) included four single oxycarotenoids in two classes among the major carotenoids in corn. The use of high pressure liquid chromatography (HPLC) revealed that even white corn had at least 12 detectable carotenoids (Tyczkowski and Hamilton, unpublished data). This complexity of the carotenoid profile of corn and the even greater complexity of the profile in tissues of birds fed yellow corn-soy diets (e.g., 20 peaks were seen on HPLC analysis of livers) renders the study of carotenoid metabolism in poultry difficult, unless simplification can be achieved. The major (>50%) carotenoid in corn (Marusich and Bauernfeind, 1981; Grogan and
1 Paper Number 10028 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC. 2 The use of trade names in this publication does not imply endorsement by the North Carolina Agricultural Research Service, nor criticism of similar ones not mentioned. 3 To whom correspondence should be addressed.
Blessin, 1968; Quackenbush et al, 1961) and a very effective pigmenter of chickens and their products is lutein, a dihydroxycarotenoid (Fritz et al, 1957; Williams et al, 1963; Kuzmicky et al, 1968; Marusich and Bauernfeind, 1981). As a class, monohydroxycarotenoids, such as cryptoxanthin, which occurs in corn (8 to 10% of total carotenoids), are less effective pigmenters than dihydroxycarotenoids. Carotenes, the other main class of carotenoids in corn, scarcely pigment at all (Borenstein and Bunnell, 1967; Quackenbush, 1970; Marusich and Bauernfeind, 1981). It appears that lutein, a dihydroxycartenoid, has the essential characteristics of a model for the study of pigmentation in chickens. Briefly, lutein is readily absorbed from the gastrointestinal tract and transported to depot sites, where it is deposited in tissues. Also, there are reliable techniques for identifying and quantitating low levels of lutein in diets and tissues; it is the major carotenoid in corn and poultry tissues and is available in a purified form. The present communication describes the development and characterization of a model, which utilizes a white corn-soy based diet supplemented with known amounts of purified lutein available commercially or by laboratory procedures, for the study of carotenoid metabolism in chickens.
1141
1142
TYCZKOWSKI AND HAMILTON MATERIALS AND METHODS
Poultry Husbandry. Day-old male broiler chickens (Arbor Acre X Arbor Acre) were obtained from the university farm and housed in electrically heated batteries under continuous illumination with feed and water available ad libitum. The diet was a commercial type based on corn and soy bean meal with white corn substituted for the customary yellow corn ; it did not contain corn gluten meal or alfalfa meal. The basal diet contained 1.35 Mg total carotenoids/g diet. Four groups of 10 birds/level of lutein (0, 5, 10, 20, 40, and 80 £(g/g diet) were fed for 3 weeks, at which time the experiment was terminated. In an initial experiment, lutein (91% free alcohol, 8% monoester, and 1% diester) was prepared by saponification of lutein diester (Xantho-Glow, A. L. Laboratories, Englewood Cliffs, NJ), extraction with hexane, and stabilization with butylated hydroxytoluene (Marusich and Bauemfeind, 1981). Thereafter, lutein was added to the diet as Oro-Glo (Kemin Industries, Inc., Des Moines, I A), zeaxanthin (3%), lutein monoester (3%), and lutein diester (<1%). Both Xantho-Glow and Oro-Glo are from marigold (Tagetes erecta) concentrates. The birds were bled for serum collection and killed by cervical dislocation prior to removing contents of the jejunum and large intestine, liver, and feet which were combined on a group basis and stored at —20 C until analyzed for carotenoid content. Extraction of Samples. The samples were thawed and extracted with 30 ml hexane: acetone: toluene: ethanol (10:7:7:6) overnight at room temperature. The amounts of tissues extracted were 2 ml of serum, 2 ml of liver homogenate (1:5 ratio of liver to deionized water), 20 punches of toe web (obtained with a paper punch of 6-mm diameter from which the area was calculated), or 1-g intestinal contents. When the diet or ingredients were analyzed, 1 g was extracted. Saponification to hydrolyze carotenoid esters was achieved by adding 2 ml methanolic KOH (10 N) to the 30 ml of extraction solvent. Extraction without saponification permitted separation and determination of esters. Purification of Extracted Carotenoids. Hexane (30 ml) was added to the total extract which then was diluted to 100 ml with 10% aqueous Na2 S 0 4 , mixed, and let stand for 1 hr before taking 10 ml of the hexane layer and evaporating it under N 2 . The dried material was
dissolved in 1.5 ml hexane and transferred quantitatively to a Sep-Pak silica gel cartridge (Millipore-Waters, Milford, MA), prewet with 5 ml hexane, and the carotenoids were eluted with 15 ml hexane: acetone (79:21). The eluate was evaporated in a vial under N 2 and stored in the dark until analyzed. High Pressure Liquid Chromatography Conditions. Analysis was by the method of Tyczkowski and Hamilton (1984). The dried eluates were dissolved in 400-fil hexane, and a 10-^tl aliquot was injected with UK-6 injector into a silica gel (5-jJ.m particle size) cartridge column held in a Z-module, eluted with isooctane: acetone: methanol (80:15:5) witha flow-rate at 1.5 ml for 6 min from a Model M-45 pump. The Model 440 absorbance detector (MilliporeWaters, Milford, MA) had a 436-nm filter, and the peaks were recorded on a Recordall 6000 (Fisher Scientific Co., Raleigh, NC). Quantitation was by the peak height method, using as standards lutein diesters (Xantho-Glow) and free lutein prepared by saponification of the diesters and crystallizing from hexane. Lutein monoesters were identified by alkaline hydrolysis to lutein.
RESULTS
In the initial analyses, total lutein of the contents of the jejunum and large intestine and of serum, liver, and toe web were measured by saponification of the extracts prior to HPLC. The facile, hydrolytic conversion of lutein diesters, under alkaline conditions to free lutein via lutein monoester, is illustrated in Figure 1. The concentration of total lutein (Fig. 2) in each of the tissues was a linear function of the dietary concentration of lutein except for a single point at the highest dietary concentration (80 //g/g) in the large intestine, where a greater amount of lutein was found than expected from the other linear relationships. When tissues from birds fed free lutein in the foregoing experiment were analyzed by HPLC without saponification so that the degree of esterification of lutein in the tissues could be ascertained, an unexpected pattern was revealed (Fig. 3). The major form of lutein in the intestinal contents (except in the large intestine at the highest concentration of dietary lutein, 80 Mg/g diet) was lutein monoester. Generally, the concentration of lutein monoester was about twice that of free lutein and about four times that of lutein diester. In the serum, where
1143
LUTEIN AND PIGMENTATION
carotenoids are t r a n s p o r t e d from t h e gastrointestinal tract t o d e p o t sites, a b o u t 96% of t h e t o t a l lutein occurred as t h e free alcohol. T h e remainder was in t h e form of t h e m o n o e s t e r , since n o diester peak was d e t e c t e d at t h e i n s t r u m e n t a l a t t e n u a t i o n used. Lutein occurred in t h e liver primarily as t h e free alcohol with a b o u t 2 0 % as t h e m o n o e s t e r and only trace quantities as t h e lutein diester. T h e p a t t e r n of cartenoids in t h e t o e w e b (Fig. 4) was different from those in t h e liver and especially from those in t h e serum. A b o u t half of t h e t o t a l lutein in t h e toe web occurred as t h e diester. T h e remainder occurred as a b o u t equal a m o u n t s of free lutein and lutein diester. DISCUSSION
-Jj UNSAPONIFED
PARTIALLY SAPONIFIED
SAPONIFIED
FIG. 1. High pressure liquid chromatography analysis of the saponification of lutein diester. The short vertical line on the left of each tracing is the event marker for time of injection. The order of elution was lutein diester (A), lutein monoester (B), and free lutein (C).
50
-
40
JEJUNUM LARGE INTESTINE SERUM LIVER TOE WEB
T h e foregoing d a t a s u p p o r t t h e usefulness of models in investigating pigmentation in p o u l t r y . In every tissue studied ( c o n t e n t s of j e j u n u m and large intestine, serum, liver, and t o e w e b ) , t h e c o n c e n t r a t i o n s of t o t a l lutein and t h e esterification f o r m s of lutein b o r e an essentially linear relationship t o t h e c o n c e n t r a t i o n of free lutein a d d e d t o t h e diet. These observations agree with earlier r e p o r t s that t h e t o t a l carot e n o i d c o n t e n t of t h e i n t e g u m e n t ( K u z m i c k y et
• LUTEIN DIESTER
0 LUTEIN MONOESTER c LUTEIN
30
ui
20
10
0
40 FREE LUTEIN
80
( (19/9 d i e t )
FIG. 2. Proportionality between total lutein in various tissues and concentration of free lutein in the diet. Each data point is the mean of four groups of 10 birds, and the vertical bars on the points are the standard errors of the means. Absence of bars on a point indicate the standard error was smaller than the space occupied by the data point. Concentrations of tissue carotenoids are expressed on an as is basis.
FREE LUTEIN ADDED ( p . g / 9
diet)
FIG. 3. Concentrations of the free alcohol, monoester, and diester of lutein in tissues as a function of concentration of dietary free lutein. Concentrations of tissue caroteinoids are expressed on an as is basis.
1144
«qz.5
TYCZKOWSKI AND HAMILTON A LUTEIN o LUTEIN MONOESTER • LUTEIN DIESTER
40 FREE LUTEIN ADDED ( |tg/g diet )
80
FIG. 4. Concentrations of different forms of lutein deposited in toe web as a function of concentration of dietary free lutein. Each data point is the mean of four groups of 10 birds, and the vertical bars on the points are the standard errors of the means. Absence of bars on a point indicate the standard error was smaller than the space occupied by the point.
al, 1968; Dua et al, 1967) and the serum (Dua et al, 1967; Twining et al, 1971) were directly proportional to concentration of carotenoids in natural dietary ingredients such as alfalfa meal. Such linearity should be very helpful in studying carotenoid metabolism and its abnormalities during infections and toxicoses. Departures from linearity would indicate loci of action of different factors and agents. The finding that lutein in the intestinal contents occurred as three forms with the concentrations being lutein monoester > free lutein > lutein diester was contrary to expectations based on an earlier observation that lutein diester in the diet of chickens is deacylated to free lutein prior to deposition in egg yolks (Philip et al, 1976) and that this deacylation occurs in the intestinal contents of young chickens (Tyczkowski and Hamilton, unpublished data). These findings appear paradoxical with lutein diester in diet being hydrolyzed and free lutein in the diet being acylated, but similar interconversions occur when free and esterified cholesterol and free and esterified vitamin A are fed chickens (Ganguly et al, 1957). Regardless of the form of dietary lutein, the intestinal mucosa are bathed in a mixture of free alcohol, monoester, and diester. The role, if
any, of these interconversions in the absorption of lutein is unknown, but speculation on a beneficial role is tempting. The data do not define the form of lutein when it is absorbed, but the finding of mainly free lutein (96%) in the serum suggests that the free alcohol is primarily the form that is absorbed. This suggestion is buttressed by the direct proportionality between dietary lutein and serum lutein (Fig. 3), which implies that absorption is an unsaturable process and occurs passively down a concentration gradient. A passive diffusion mechanism for the absorption of many nonpolar compounds, like carotenoids, has been demonstrated (Bensadoun and Rothfeld, 1972). Such a mechanism would also account for finding small concentrations of serum lutein monoester that were directly proportional to the dietary concentration. Regardless of the status of lutein when it is absorbed, the finding of an overwhelming percentage (96%) of free lutein in the serum (Fig. 3) implies that the transport form of lutein is the free alcohol. The occurrence of 20% of the total lutein in the liver as monoester suggests there may be local enzymatic activity that esterifies free lutein, the primary form in which it is transported in the serum to the liver. The role of storage and esterification of lutein in liver is not known, but perhaps the liver acts as an auxiliary depot site for carotenoids in chickens that dampens violent swings in the serum concentration of carotenoids available through the diet (i.e., the liver acts much as a surge tank does in regulating the flow of liquids in closed systems). This hypothesis would require that liver lutein be depleted early when birds are placed on diets limited in lutein. The finding that the diester form of lutein predominates in the integument (Fig. 4) and that free lutein is only about 25% of the total has several implications. The esterified forms, and particularly the diester, appear to be the integumentary depot forms of lutein. The finding of appreciable quantities of lutein monoester suggest that the formation of lutein diester from free lutein has two discrete enzymatic steps (free lutein -> lutein monoester -»• lutein diester) and the equilibria for the reactions are not far removed from unity. The occurrence of free lutein as the transport form suggests that the free lutein in the integument would be more labile than the diester when carotenoid deprivation occurs. The eventual
LUTEIN AND PIGMENTATION paling of birds placed o n diets low in carotenoids (Herrick et al, 1 9 7 1 ) implies t h e existence of deacylation e n z y m e s (esterases), if free lutein c o n t i n u e s t o be t h e t r a n s p o r t form u n d e r these conditions. T h e existence in t h e i n t e g u m e n t of e n z y m a t i c steps for t h e acylation a n d deacylation of lutein implies t h a t t h e r e is some benefit t o t h e bird in having stable forms of d i h y d r o x y c a r o t e n o i d s in their skin. T h e findings of this s t u d y can be summarized b y saying t h a t at least part of t h e ingested free lutein is esterified t o t h e m o n o e s t e r a n d diester during its passage d o w n t h e intestinal tract. Regardless of its status w h e n absorbed, it is t r a n s p o r t e d primarily in t h e b o d y as t h e free alcohol. When lutein enters d e p o t sites such as liver and i n t e g u m e n t , it is esterified, at least in p a r t , p r e s u m a b l y as t h e result of local enzymatic activity. T h e primary d e p o t form in t h e i n t e g u m e n t is t h e diester. These findings raise m a n y questions a b o u t c a r o t e n o i d m e t a bolism and carcass p i g m e n t a t i o n in chickens. ACKNOWLEDGMENTS We t h a n k Scott J o h n s o n , N a n c y Bailey, and G o r u m Whitaker for technical assistance. T h e d o n a t i o n of X a n t h o - G l o w b y A. L. Laboratories and Oro-Glo b y Kemin Industries, Inc. is appreciated. This w o r k was c o n d u c t e d in p a r t with t h e s u p p o r t of a c o n t r a c t (USDA-NERA R S 58-32U4-2-398) from t h e US D e p a r t m e n t of Agriculture.
REFERENCES Bensadoun, A., and A. Rothfeld, 1972. The form of absorption of lipids in the chicken, Gallus domesticus. Proc. Soc. Exp. Biol. Med. 141:814-818. Borenstein, B., and R. Burnell, 1967. Carotenoids: Properties, Occurrence, and Utilization in Foods. Adv. Food Res. 15:195-276.
1145
Dua, P. N., E. J. Day, J. E. Hill, and C. O. Grogan, 1967. Utilization of xanthophylls from natural sources by the chick. J. Agric. Food Chem. 15:324-328. Fritz, J. C , F. D. Wharton, and L. J. Classen, 1957. Influence of feed on broiler pigmentation. Feedstuffs29(43):18-24. Ganguly, J., S. Krishnamurthy, and S. Mahadevan, 1957. The transport of carotenoids, vitamin A and cholesterol across the intestines of rats and chickens. Biochem. J. 71:756-762. Grogan, C. O., and C. W. Blessin, 1968. Characterization of major carotenoids in yellow maize lines of differing pigment concentration. Crop Sci. 8:730732. Herrick, G. M„ J. L. Fry, and R. H. Harms, 1971. Repletion and depletion of pigmentation in broiler skin and shanks. Poultry Sci. 50:14671475. Kuzmicky, D. D., G. O. Kohler, A. L. Livingston, R. E. Knowles, and J. W. Nelson, 1968. Pigmentation potency of xanthophyll sources. Poultry Sci. 47:389-397. Marusich, W. L., and J. C. Bauernfeind, 1981. Oxycarotenoids in poultry feeds. Pages 319-462 in Carotenoids as Colorants and Vitamin A Precursors. J. C. Bauernfeind, ed. Academic Press, Inc., New York, NY. Philip, T , C. W. Weber, and J. W. Berry, 1976. Utilization of lutein and lutein-fatty acid esters by laying hens. J. Food Sci. 41:23-25. Quackenbush, F. W., 1970. Analysis for carotenes and xanthophylls in dried plant materials. J. Assoc. Off. Anal. Chem. 53:181-185. Quackenbush, F. W., J. G. Firch, W. J. Rabourn, M. McQuistan, E. W. Petzold, and T. E. Kargl, 1961. Analysis of carotenoids in corn grain. J. Agric. Food Chem. 9:132-135. Twining, P. V., E. H. Bossard, P. G. Lund, and O. P. Thomas, 1971. Relative availability of xanthophylls from ingredients based on plasma levels and skin measurements. Proc. MD Nutr. Conf. 1971:90-95. Tyczkowski, J. K., and P. B. Hamilton, 1984. HPLC method for analysis of carotenoids in poultry feed and tissues. Page 42 in 98th Annu. Mtg. Assoc. Off. Anal. Chem. (Abstr.) Williams, W. P., R. E. Davies, and J. R. Couch, 1963. The utilization of carotenoids by the hen and chick. Poultry Sci. 42:691-699.
1 Paper Number 10028 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC. 2 The use of trade names in this publication does not imply endorsement by the North Carolina Agricultural Research Service, nor criticism of similar ones not mentioned. 3 To whom correspondence should be addressed.
Blessin, 1968; Quackenbush et al, 1961) and a very effective pigmenter of chickens and their products is lutein, a dihydroxycarotenoid (Fritz et al, 1957; Williams et al, 1963; Kuzmicky et al, 1968; Marusich and Bauernfeind, 1981). As a class, monohydroxycarotenoids, such as cryptoxanthin, which occurs in corn (8 to 10% of total carotenoids), are less effective pigmenters than dihydroxycarotenoids. Carotenes, the other main class of carotenoids in corn, scarcely pigment at all (Borenstein and Bunnell, 1967; Quackenbush, 1970; Marusich and Bauernfeind, 1981). It appears that lutein, a dihydroxycartenoid, has the essential characteristics of a model for the study of pigmentation in chickens. Briefly, lutein is readily absorbed from the gastrointestinal tract and transported to depot sites, where it is deposited in tissues. Also, there are reliable techniques for identifying and quantitating low levels of lutein in diets and tissues; it is the major carotenoid in corn and poultry tissues and is available in a purified form. The present communication describes the development and characterization of a model, which utilizes a white corn-soy based diet supplemented with known amounts of purified lutein available commercially or by laboratory procedures, for the study of carotenoid metabolism in chickens.
1141
1142
TYCZKOWSKI AND HAMILTON MATERIALS AND METHODS
Poultry Husbandry. Day-old male broiler chickens (Arbor Acre X Arbor Acre) were obtained from the university farm and housed in electrically heated batteries under continuous illumination with feed and water available ad libitum. The diet was a commercial type based on corn and soy bean meal with white corn substituted for the customary yellow corn ; it did not contain corn gluten meal or alfalfa meal. The basal diet contained 1.35 Mg total carotenoids/g diet. Four groups of 10 birds/level of lutein (0, 5, 10, 20, 40, and 80 £(g/g diet) were fed for 3 weeks, at which time the experiment was terminated. In an initial experiment, lutein (91% free alcohol, 8% monoester, and 1% diester) was prepared by saponification of lutein diester (Xantho-Glow, A. L. Laboratories, Englewood Cliffs, NJ), extraction with hexane, and stabilization with butylated hydroxytoluene (Marusich and Bauemfeind, 1981). Thereafter, lutein was added to the diet as Oro-Glo (Kemin Industries, Inc., Des Moines, I A), zeaxanthin (3%), lutein monoester (3%), and lutein diester (<1%). Both Xantho-Glow and Oro-Glo are from marigold (Tagetes erecta) concentrates. The birds were bled for serum collection and killed by cervical dislocation prior to removing contents of the jejunum and large intestine, liver, and feet which were combined on a group basis and stored at —20 C until analyzed for carotenoid content. Extraction of Samples. The samples were thawed and extracted with 30 ml hexane: acetone: toluene: ethanol (10:7:7:6) overnight at room temperature. The amounts of tissues extracted were 2 ml of serum, 2 ml of liver homogenate (1:5 ratio of liver to deionized water), 20 punches of toe web (obtained with a paper punch of 6-mm diameter from which the area was calculated), or 1-g intestinal contents. When the diet or ingredients were analyzed, 1 g was extracted. Saponification to hydrolyze carotenoid esters was achieved by adding 2 ml methanolic KOH (10 N) to the 30 ml of extraction solvent. Extraction without saponification permitted separation and determination of esters. Purification of Extracted Carotenoids. Hexane (30 ml) was added to the total extract which then was diluted to 100 ml with 10% aqueous Na2 S 0 4 , mixed, and let stand for 1 hr before taking 10 ml of the hexane layer and evaporating it under N 2 . The dried material was
dissolved in 1.5 ml hexane and transferred quantitatively to a Sep-Pak silica gel cartridge (Millipore-Waters, Milford, MA), prewet with 5 ml hexane, and the carotenoids were eluted with 15 ml hexane: acetone (79:21). The eluate was evaporated in a vial under N 2 and stored in the dark until analyzed. High Pressure Liquid Chromatography Conditions. Analysis was by the method of Tyczkowski and Hamilton (1984). The dried eluates were dissolved in 400-fil hexane, and a 10-^tl aliquot was injected with UK-6 injector into a silica gel (5-jJ.m particle size) cartridge column held in a Z-module, eluted with isooctane: acetone: methanol (80:15:5) witha flow-rate at 1.5 ml for 6 min from a Model M-45 pump. The Model 440 absorbance detector (MilliporeWaters, Milford, MA) had a 436-nm filter, and the peaks were recorded on a Recordall 6000 (Fisher Scientific Co., Raleigh, NC). Quantitation was by the peak height method, using as standards lutein diesters (Xantho-Glow) and free lutein prepared by saponification of the diesters and crystallizing from hexane. Lutein monoesters were identified by alkaline hydrolysis to lutein.
RESULTS
In the initial analyses, total lutein of the contents of the jejunum and large intestine and of serum, liver, and toe web were measured by saponification of the extracts prior to HPLC. The facile, hydrolytic conversion of lutein diesters, under alkaline conditions to free lutein via lutein monoester, is illustrated in Figure 1. The concentration of total lutein (Fig. 2) in each of the tissues was a linear function of the dietary concentration of lutein except for a single point at the highest dietary concentration (80 //g/g) in the large intestine, where a greater amount of lutein was found than expected from the other linear relationships. When tissues from birds fed free lutein in the foregoing experiment were analyzed by HPLC without saponification so that the degree of esterification of lutein in the tissues could be ascertained, an unexpected pattern was revealed (Fig. 3). The major form of lutein in the intestinal contents (except in the large intestine at the highest concentration of dietary lutein, 80 Mg/g diet) was lutein monoester. Generally, the concentration of lutein monoester was about twice that of free lutein and about four times that of lutein diester. In the serum, where
1143
LUTEIN AND PIGMENTATION
carotenoids are t r a n s p o r t e d from t h e gastrointestinal tract t o d e p o t sites, a b o u t 96% of t h e t o t a l lutein occurred as t h e free alcohol. T h e remainder was in t h e form of t h e m o n o e s t e r , since n o diester peak was d e t e c t e d at t h e i n s t r u m e n t a l a t t e n u a t i o n used. Lutein occurred in t h e liver primarily as t h e free alcohol with a b o u t 2 0 % as t h e m o n o e s t e r and only trace quantities as t h e lutein diester. T h e p a t t e r n of cartenoids in t h e t o e w e b (Fig. 4) was different from those in t h e liver and especially from those in t h e serum. A b o u t half of t h e t o t a l lutein in t h e toe web occurred as t h e diester. T h e remainder occurred as a b o u t equal a m o u n t s of free lutein and lutein diester. DISCUSSION
-Jj UNSAPONIFED
PARTIALLY SAPONIFIED
SAPONIFIED
FIG. 1. High pressure liquid chromatography analysis of the saponification of lutein diester. The short vertical line on the left of each tracing is the event marker for time of injection. The order of elution was lutein diester (A), lutein monoester (B), and free lutein (C).
50
-
40
JEJUNUM LARGE INTESTINE SERUM LIVER TOE WEB
T h e foregoing d a t a s u p p o r t t h e usefulness of models in investigating pigmentation in p o u l t r y . In every tissue studied ( c o n t e n t s of j e j u n u m and large intestine, serum, liver, and t o e w e b ) , t h e c o n c e n t r a t i o n s of t o t a l lutein and t h e esterification f o r m s of lutein b o r e an essentially linear relationship t o t h e c o n c e n t r a t i o n of free lutein a d d e d t o t h e diet. These observations agree with earlier r e p o r t s that t h e t o t a l carot e n o i d c o n t e n t of t h e i n t e g u m e n t ( K u z m i c k y et
• LUTEIN DIESTER
0 LUTEIN MONOESTER c LUTEIN
30
ui
20
10
0
40 FREE LUTEIN
80
( (19/9 d i e t )
FIG. 2. Proportionality between total lutein in various tissues and concentration of free lutein in the diet. Each data point is the mean of four groups of 10 birds, and the vertical bars on the points are the standard errors of the means. Absence of bars on a point indicate the standard error was smaller than the space occupied by the data point. Concentrations of tissue carotenoids are expressed on an as is basis.
FREE LUTEIN ADDED ( p . g / 9
diet)
FIG. 3. Concentrations of the free alcohol, monoester, and diester of lutein in tissues as a function of concentration of dietary free lutein. Concentrations of tissue caroteinoids are expressed on an as is basis.
1144
«qz.5
TYCZKOWSKI AND HAMILTON A LUTEIN o LUTEIN MONOESTER • LUTEIN DIESTER
40 FREE LUTEIN ADDED ( |tg/g diet )
80
FIG. 4. Concentrations of different forms of lutein deposited in toe web as a function of concentration of dietary free lutein. Each data point is the mean of four groups of 10 birds, and the vertical bars on the points are the standard errors of the means. Absence of bars on a point indicate the standard error was smaller than the space occupied by the point.
al, 1968; Dua et al, 1967) and the serum (Dua et al, 1967; Twining et al, 1971) were directly proportional to concentration of carotenoids in natural dietary ingredients such as alfalfa meal. Such linearity should be very helpful in studying carotenoid metabolism and its abnormalities during infections and toxicoses. Departures from linearity would indicate loci of action of different factors and agents. The finding that lutein in the intestinal contents occurred as three forms with the concentrations being lutein monoester > free lutein > lutein diester was contrary to expectations based on an earlier observation that lutein diester in the diet of chickens is deacylated to free lutein prior to deposition in egg yolks (Philip et al, 1976) and that this deacylation occurs in the intestinal contents of young chickens (Tyczkowski and Hamilton, unpublished data). These findings appear paradoxical with lutein diester in diet being hydrolyzed and free lutein in the diet being acylated, but similar interconversions occur when free and esterified cholesterol and free and esterified vitamin A are fed chickens (Ganguly et al, 1957). Regardless of the form of dietary lutein, the intestinal mucosa are bathed in a mixture of free alcohol, monoester, and diester. The role, if
any, of these interconversions in the absorption of lutein is unknown, but speculation on a beneficial role is tempting. The data do not define the form of lutein when it is absorbed, but the finding of mainly free lutein (96%) in the serum suggests that the free alcohol is primarily the form that is absorbed. This suggestion is buttressed by the direct proportionality between dietary lutein and serum lutein (Fig. 3), which implies that absorption is an unsaturable process and occurs passively down a concentration gradient. A passive diffusion mechanism for the absorption of many nonpolar compounds, like carotenoids, has been demonstrated (Bensadoun and Rothfeld, 1972). Such a mechanism would also account for finding small concentrations of serum lutein monoester that were directly proportional to the dietary concentration. Regardless of the status of lutein when it is absorbed, the finding of an overwhelming percentage (96%) of free lutein in the serum (Fig. 3) implies that the transport form of lutein is the free alcohol. The occurrence of 20% of the total lutein in the liver as monoester suggests there may be local enzymatic activity that esterifies free lutein, the primary form in which it is transported in the serum to the liver. The role of storage and esterification of lutein in liver is not known, but perhaps the liver acts as an auxiliary depot site for carotenoids in chickens that dampens violent swings in the serum concentration of carotenoids available through the diet (i.e., the liver acts much as a surge tank does in regulating the flow of liquids in closed systems). This hypothesis would require that liver lutein be depleted early when birds are placed on diets limited in lutein. The finding that the diester form of lutein predominates in the integument (Fig. 4) and that free lutein is only about 25% of the total has several implications. The esterified forms, and particularly the diester, appear to be the integumentary depot forms of lutein. The finding of appreciable quantities of lutein monoester suggest that the formation of lutein diester from free lutein has two discrete enzymatic steps (free lutein -> lutein monoester -»• lutein diester) and the equilibria for the reactions are not far removed from unity. The occurrence of free lutein as the transport form suggests that the free lutein in the integument would be more labile than the diester when carotenoid deprivation occurs. The eventual
LUTEIN AND PIGMENTATION paling of birds placed o n diets low in carotenoids (Herrick et al, 1 9 7 1 ) implies t h e existence of deacylation e n z y m e s (esterases), if free lutein c o n t i n u e s t o be t h e t r a n s p o r t form u n d e r these conditions. T h e existence in t h e i n t e g u m e n t of e n z y m a t i c steps for t h e acylation a n d deacylation of lutein implies t h a t t h e r e is some benefit t o t h e bird in having stable forms of d i h y d r o x y c a r o t e n o i d s in their skin. T h e findings of this s t u d y can be summarized b y saying t h a t at least part of t h e ingested free lutein is esterified t o t h e m o n o e s t e r a n d diester during its passage d o w n t h e intestinal tract. Regardless of its status w h e n absorbed, it is t r a n s p o r t e d primarily in t h e b o d y as t h e free alcohol. When lutein enters d e p o t sites such as liver and i n t e g u m e n t , it is esterified, at least in p a r t , p r e s u m a b l y as t h e result of local enzymatic activity. T h e primary d e p o t form in t h e i n t e g u m e n t is t h e diester. These findings raise m a n y questions a b o u t c a r o t e n o i d m e t a bolism and carcass p i g m e n t a t i o n in chickens. ACKNOWLEDGMENTS We t h a n k Scott J o h n s o n , N a n c y Bailey, and G o r u m Whitaker for technical assistance. T h e d o n a t i o n of X a n t h o - G l o w b y A. L. Laboratories and Oro-Glo b y Kemin Industries, Inc. is appreciated. This w o r k was c o n d u c t e d in p a r t with t h e s u p p o r t of a c o n t r a c t (USDA-NERA R S 58-32U4-2-398) from t h e US D e p a r t m e n t of Agriculture.
REFERENCES Bensadoun, A., and A. Rothfeld, 1972. The form of absorption of lipids in the chicken, Gallus domesticus. Proc. Soc. Exp. Biol. Med. 141:814-818. Borenstein, B., and R. Burnell, 1967. Carotenoids: Properties, Occurrence, and Utilization in Foods. Adv. Food Res. 15:195-276.
1145
Dua, P. N., E. J. Day, J. E. Hill, and C. O. Grogan, 1967. Utilization of xanthophylls from natural sources by the chick. J. Agric. Food Chem. 15:324-328. Fritz, J. C , F. D. Wharton, and L. J. Classen, 1957. Influence of feed on broiler pigmentation. Feedstuffs29(43):18-24. Ganguly, J., S. Krishnamurthy, and S. Mahadevan, 1957. The transport of carotenoids, vitamin A and cholesterol across the intestines of rats and chickens. Biochem. J. 71:756-762. Grogan, C. O., and C. W. Blessin, 1968. Characterization of major carotenoids in yellow maize lines of differing pigment concentration. Crop Sci. 8:730732. Herrick, G. M„ J. L. Fry, and R. H. Harms, 1971. Repletion and depletion of pigmentation in broiler skin and shanks. Poultry Sci. 50:14671475. Kuzmicky, D. D., G. O. Kohler, A. L. Livingston, R. E. Knowles, and J. W. Nelson, 1968. Pigmentation potency of xanthophyll sources. Poultry Sci. 47:389-397. Marusich, W. L., and J. C. Bauernfeind, 1981. Oxycarotenoids in poultry feeds. Pages 319-462 in Carotenoids as Colorants and Vitamin A Precursors. J. C. Bauernfeind, ed. Academic Press, Inc., New York, NY. Philip, T , C. W. Weber, and J. W. Berry, 1976. Utilization of lutein and lutein-fatty acid esters by laying hens. J. Food Sci. 41:23-25. Quackenbush, F. W., 1970. Analysis for carotenes and xanthophylls in dried plant materials. J. Assoc. Off. Anal. Chem. 53:181-185. Quackenbush, F. W., J. G. Firch, W. J. Rabourn, M. McQuistan, E. W. Petzold, and T. E. Kargl, 1961. Analysis of carotenoids in corn grain. J. Agric. Food Chem. 9:132-135. Twining, P. V., E. H. Bossard, P. G. Lund, and O. P. Thomas, 1971. Relative availability of xanthophylls from ingredients based on plasma levels and skin measurements. Proc. MD Nutr. Conf. 1971:90-95. Tyczkowski, J. K., and P. B. Hamilton, 1984. HPLC method for analysis of carotenoids in poultry feed and tissues. Page 42 in 98th Annu. Mtg. Assoc. Off. Anal. Chem. (Abstr.) Williams, W. P., R. E. Davies, and J. R. Couch, 1963. The utilization of carotenoids by the hen and chick. Poultry Sci. 42:691-699.