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Broiler Pigmentation Potency of Neoxanthin and Violaxanthin Relative to Lutein
Broiler Pigmentation Potency of Neoxanthin and Violaxanthin Relative to Lutein D. D. KUZMICKY, G. O. KOHLER, A. L. LIVINGSTON, R. E. KNOWLES AND J. W. NELSON Western Regional Research Laboratory, Agricultural Research Service, V. S. Department of Agriculture, Albany, California 94710 (Received for publication August 13, 1968)
T
TABLE 1.—Basal ration Ingredients Soybean meal Corn starch Saffloweroil Dried fish solubles Vitamin mix W U-24G2 Mineral mix MK-5 2 CaC0 3 DL-Methionine
% of ration 44.64 41.84 7.00 1.00 1.00 3.00 1.50 0.20
1 Contains 22% protein and .0125% ethoxyquin added to the safflower oil. 2 For composition see Kuzmicky et al. (1968).
GENERAL PROCEDURE 1
For the two experiments conducted in this study, day-old Cornish-White Rock type cross-bred cockerels (Kimber) were depleted of pigment on a 22% protein soystarch ration (Table 1) for 13 days. After this period all chicks with greenish colored feet (less than 5%) were discarded and the chicks used were distributed into replicate 1 Reference to a company or product name does not imply approval or recommendation of the product by the U. S. Department of Agriculture to the exclusion of others that may be suitable.
groups equalized in weight. The chicks were housed in electrically heated brooders in a laboratory with continuous fluorescent lighting. The laboratory temperature was maintained at 23.5-26.5°C. (75-80°F.). The lutein, neoxanthin and violaxanthin used in this study were isolated from an extract of fresh alfalfa by a procedure similar to that of Bickoff et al. (1954). Briefly, the procedure consisted of the preparation of an acetone extract, transfer of the pigments to hexane, and chromatography of the hexane extract on MgO-Celite (1-1). Washing the column with hexane-acetone-methanol (89-10-1) gave four fractions. These were neoxanthin, a mixture of violaxanthin and zeaxanthin, lutein and cryptoxanthin. Final purification of neoxanthin and lutein was accomplished by recrystallization in the dark under an inert atmosphere, first from methanol-hexane followed by acetonehexane. It was necessary to rechromato-
326
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HE xanthophylls from dehydrated alfalfa meal as measured by the new Western Regional Research Laboratory (W.R.R.L.) analytical procedure include lutein, zeazanthin, cryptoxanthin, neoxanthin, violaxanthin, carotenoid oxidation products, and various cis isomers of these (Kohler et al., 1967). Lutein, zeaxanthin, and cryptoxanthin have been reported to be good skin pigmenters while indications are the polyoxycarotenoids are relatively poor skin pigmenters (Quackenbush et al., 1965; Kuzmicky et al., 1968). Violaxanthin has been reported to be ineffective as a yolk pigmenter (Marusich et al., 1960). However, the effectiveness of violaxanthin and neoxanthin as skin pigmenters has not been established. This study was undertaken to determine the relative skin pigmentation potency (R.S.P.P.) of pure all-trans neoxanthin and all-trans violaxanthin relative to pure all-trans lutein.
327
PIGMENTATION FROM NEOXANTHIN AND VIOLAXANTHIN TABLE 2.—Broiler pigmentation results from Experiment 1 Dose ' Vkn»12 *«• Xanthophyll Supplements mg./chick/ v l 5 ~ " per 100 cm.'
Control
0
Lutein
.25 .50 .75
Neoxanthin
3.0
RSPP''
0.8
21.9
1.9 2.2 3.0
42.2 51.4 65.1
100
1.5
32.6
8
1
Three chicks were used for the control and each lutein dose and 2 chicks for the neoxanthin. The pigments were administered for 5 days. 2 Average value of 6 people using 1965 model Roche yolk color fan. 3 All-trans lutein equivalents. 4 Estimated relative skin pigmentation potency.
E X P E R I M E N T A L AND RESULTS
Experiment 1. This was a preliminary experiment to determine the approximate RSPP of pure neoxanthin. One zero level control, 3 doses of lutein at .25, .50 and .75 mg. per chick per day, and one dose of neoxanthin at 3 mg. per chick per day The pigments were dissolved in corn oil were used. The lutein doses were disor safflower oil and administered to the solved in 0.5 ml. corn oil and the neochicks orally at 9:00-10:00 a.m. each morning for five days after the 13-day xanthin in 1 ml. corn oil. One group of 3 depletion period. A syringe with a 2-inch chicks was used for the control and each 18 gauge needle was used. A smooth round lutein level. One group of 2 chicks was ball of solder was attached to the tip of the used for the neoxanthin. For the toe web needle. The needle was inserted into the analysis 2 toe web plugs (3/16-inch diachick's esophagus about 2 inches past the meter) were taken from both the left and point of the beak and then the xantho- right foot. The xanthophyll (all-trans phyll solution was slowly injected into the lutein equivalents) per unit toe web area crop. Ad libitum feeding of the soy-starch was determined using the absorptivity of ration continued during this 5-day experi- 236 and the absorbance at 475 mju. The results are shown in Table 2. mental period. From the toe web analysis (plotting the The toe web analyses and visual scoring (using 1965 model Roche yolk color fan) xanthophyll dose vs. xanthophyll per unit were done as described earlier (Kuzmicky toe web area) a significant (P < .05) linear el al., 1968). From the toe web analysis the regression was obtained for the lutein relative skin pigmentation potency standard and control (Y= 24.3+55.5 X (RSPP) was determined. In Experiment 1 (r=.99)). A regression line for the neothe slope of the dose-response curve of the xanthin was determined by extending a unknown was determined and compared line from the zero intercept of the lutein to the slope of the standard. In Experi- curve to the neoxanthin point. A ratio of ment 2 from which more data was avail- the two slopes yielded an estimated RSPP able the procedure as described by Bliss of 8% for neoxanthin. The visual scoring of the foot pads of both the right and left (1946) was used.
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graph the violaxanthin and zeaxanthin mixture on micro-eel C (developed with hexane-methanol (99-1)) to separate the two pigments. Violaxanthin crystals were collected upon concentration of the eluting solvent and then purified by recrystallization from methanol-hexane and acetone-hexane. Thin-layer chromatographic data, absorption spectra and m.p. determinations were obtained on the isolated compounds. These physical data were compared with those reported by Strain (1938) to ascertain identity and purity. An abundant supply of lutein was prepared, however only 300 mg. of neoxanthin and 175 mg. of violaxanthin were obtained. Consequently, the two experiments which were conducted were restricted in size because of the limited amounts of pigments available.
328
KuZMICKY, KOHLEE, LIVINGSTON, KNOWLES AND NELSON TABLE 3.—Broiler pigmentation results from Experiment 2
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(Bliss, 1946) for the neoxanthin and lutein yields an RSPP of 8% for the neoxanthin Dose 1 (relative to lutein). The visual scoring was 1 ng. Xanthophyll per 100 cm. 2 Supplements mg./chick/ Visual RSPP< score toe web area 3 consistent with these results. day Following the toe web analysis and (%) 20.1 Control 0 0.8 visual scoring, the feet from the control 40.2 Lutein .25 1.9 52.6 .50 2.3 100 chicks and the chicks receiving lutein, 52.8 .75 2.3 neoxanthin and violaxanthin were cut into Neoxanthin 1.5 0.7 22.2 very small pieces, placed in 250 ml. amber 37.0 3.0 1.7 8 33.6 1.5 4.5 colored Erlenmeyer flasks and extracted Violaxanthin 1.5 0.9 29.8 at room temperature for 5 days with three 3.0 0.8 29.8 4.5 1.3 28.3 100 ml. portions of hexane-acetone (1-1). 1 Two replicates of 3 chicks each were used for the control The extracts were filtered and saponified and each lutein dose and one group of 3 chicks for each neoxanthin and violaxanthin dose. The pigments were administered with methanolic KOH. Following removal for 5 days. 2 Average value of 7 people using 1965 model Roche yolk of the alkali and alcohol by washing with color fan. 3 All-trans lutein equivalents. water and drying over sodium sulfate, the 4 Relative skin pigmentation potency calculated by the slope ratio assay using data from the toe web analysis. respective extracts were chromatographed on MgO-Celite (1-1). The columns were feet of all chicks by a panel of six persons washed with increasing quantities of acetone in hexane or acetone-methanol in was consistent with these results. Experiment 2. The purpose of this ex- hexane. The purified xanthophyll bands periment was to determine the RSPP of were identified by thin-layer chromatoneoxanthin and violaxanthin with lutein graphic comparison (Nelson and Livingas the standard. A zero level control and ston, 1967) with known xanthophylls three levels of each pigment were used. isolated from alfalfa and by preparation of The levels of lutein were .25, .50, and .75 their visible absorption spectra including mg. per chick per day. The levels of the addition of small amounts of acid to neoxanthin and violaxanthin were 1.5, determine if epoxide derivatives were 3.0, and 4.5 mg. per chick per day. In each present (Curl, 1961). The spectra thus case the pigment was dissolved in 1 ml. of prepared were compared with those of safflower oil. Two replicates of 3 chicks purified alfalfa xanthophylls as well as each were used for the control and each those reported in the literature (Strain, lutein level. One group of 3 chicks was 1938). The visible spectra of the xanused for each neoxanthin and violaxanthin thophylls isolated from chick skin are prelevel. The toe web analysis was done the sented in Table 4. same as in Experiment 1. A panel of seven Lutein was detected in the skin of all people visually scored both the right and the chicks. Neoxanthin and neochrome left feet of all chicks. The results are were detected in the skin of the chicks shown in Table 3. receiving neoxanthin. Violaxanthin, luteoSignificant (P<.05) linear dose-re- xanthin and auroxanthin were detected sponse regressions were obtained for the in the skin of the chicks receiving violalutein and neoxanthin but not for the xanthin. violaxanthin. Both the visual scoring and DISCUSSION toe web analysis indicate that violaxanthin is deposited in only trace amounts in the The results from this study show that skin. Employing the slope-ratio assay compared to lutein, neoxanthin is a weak
PIGMENTATION FROM NEOXANTHIN AND VIOLAXANTHIN
TABLE 4.—Xanthophylls present in chick skin {Experiment 2) Chick supplement
Band
Absorption maxima, nut.
Apparent xanthophyll
None (control)
1
472,
443,
415
Lutein
Lutein
1
473,
444,
419
Lutein
Neoxanthin
1 2 3
469, 465, 447,
442, 437, 420,
4161 4202 396
Lutein Neoxanthin Neochrome
Violaxanthin
1 2 3 4
474, 471, 449, 427,
445, 445, 427, 401,
4181 420» 4003 381
Lutein Violaxanthin Luteoxanthin Auroxanthin
1 No spectra shift upon addition of acid, indicating no 5,6epoxide present. 2 Upon addition of acid spectra shifted to 448, 421, 399 nut. indicating a 5,6-epoxide forming a 5,8-epoxide. 3 Upon addition of acid spectra shifted to 427, 401, 380 nut. indicating 5,6,5',6'-diepoxide or 5,6,5',8'-diepoxide forming a 5,8,5',8'-diepoxide.
falfa meal as the pigmenting source. Consequently, from this type of a ration, the contribution towards skin pigmentation from the neoxanthin and violaxanthin present would be negligible. The results reported here coupled with our earlier results (Kuzmicky et al., 1968) and those of Quackenbush et al. (1965) further clarify the picture of broiler pigmentation from dehydrated alfalfa meal. It seems clear that lutein plus its cis isomers or derivatives are primarily responsible for skin pigmentation from alfalfa meal. Zeaxanthin and cryptoxanthin are present in alfalfa meal in such small quantities that their contribution would be small even though they may be efficiently utilized. A recent extensive xanthophyll analysis of fresh and dehydrated alfalfa meal by Livingston et al. (1968) has shown that lutein plus its cis isomers or derivatives comprise a considerably greater portion of the total xanthophylls than reported earlier by Bickoff et al. (1954). In the more recent work lutein plus its cis isomers or derivatives comprised 82% of the xanthophylls measured while neoxanthin and violaxanthin amounted to only 18%. The authors also reported that during the dehydration of alfalfa up to 94% of the neoxanthin is destroyed and up to 87% of the violaxanthin is lost. Thus, use of the new WRRL xanthophyll analytical procedure for fresh or sun-cured alfalfa could result in lower pigmentation results than expected since both these products contain appreciably greater proportions of violaxanthin and neoxanthin than dehydrated alfalfa meal. Possible modifications could be incorporated into the analytical procedure for such products high in epoxyxanthophylls. SUMMARY Day-old cockerels were depleted of pig-
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skin pigmenter (RSPP = 8%) and violaxanthin is essentially ineffective. However, the chromatographic analysis of the chick skin showed neoxanthin and traces of violaxanthin to be deposited in the skin. Neochrome, a 5,8-epoxide isomer of neoxanthin, and luteoxanthin and auroxanthin, 5,8-epoxide isomers of violaxanthin, were also detected in the chick skin in trace amounts. Apparently, after ingestion by the chick, the neoxanthin and violaxanthin undergo partial acid isomerization to form these compounds. In measuring the total xanthophylls (all-trans lutein equivalents) per unit toe web area, by using the absorptivity of 236 andtheabsorbanceat 475 m/x., neoxanthin, neochrome, luteoxanthin, and auroxanthin are partially discounted due to the fact that their absorption peaks are farther toward the violet end of the spectrum. The error involved here is relatively small. Compensating for this error would only change the RSPP of neoxanthin from 8% to about 10% and essentially have no effect on the estimation of potency of violaxanthin. The levels of neoxanthin and violaxanthin used in this study are very high compared to the amounts that would be present in a broiler ration containing al-
329
330
KUZMICKY, KOHLER, LIVINGSTON, KNOWLES AND NELSON
ACKNOWLEDGMENTS T h e authors gratefully acknowledge Ann R. Gramps and Robert L. Patterson for assisting in carrying out chick assays. Grateful appreciation is also extended to Dr. Clyde D . Mueller, Kimber Farms, Inc., Fremont, California, for arranging a special supply of Kimber cockerels for our purchase.
REFERENCES Bickoff, E. M., A. L. Livingston, G. E. Bailey and C. R. Thompson, 1954. Alfalfa carotenoids—• xanthophylls in fresh and dehydrated alfalfa. J. Agr. Food Chem. 2: S63-567. Bliss, C. I., 1946. An experimental design for sloperatio assays. Ann. Math. Stat. 17: 232-237. Curl, A. L., and G. F. Bailey, 1961. An improved test for carotenoid epoxides. J. Agr. Food Chem. 9: 403-405. Kohler, G. 0., R. E. Knowles and A. L. Livingston, 1967. An improved analytical procedure for the determination of xanthophyll. J. Assoc. Offic. Analyt. Chemists, 50: 707-711. 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. Livingston, A. L., R. E. Knowles, J. W. Nelson and G. O. Kohler, 1968. Xanthophyll and carotene loss during pilot and industrial scale alfalfa processing. J. Agr. Food Chem. 16: 84-87. Marusich, W., E. DeRitter and J. C. Bauernfeind, 1960. Evaluation of carotenoid pigments for coloring egg yolks. Poultry Sci. 39: 1338-1345. Nelson, J. W., and A. L. Livingston, 1967. Stabilization of xanthophyll and carotene by ethoxyquin during thin-layer chromatography. J. Chromatog. 28: 465-467. Quackenbush, F. W., S. Kvakovszky, T. Hoover and J. C. Rogler, 1965. Deposition of individual carotenoids in avian skin. J. Assoc. Offic. Agr. Chemists, 48: 1241-1244. Strain, H. H., 1938. Leaf xanthophylls. Carnegie Institute of Washington, Pub. 490, Washington, D.C.
NEWS AND NOTES (Continued from page 306)
signatures normally necessary for a contract. This will be done after the contracts are awarded. Research projects for the consideration of the Committee are to be presented in accordance with the following format and are not to exceed two typewritten pages. Title. Project leader and other investigators (brief biography). Objectives. Plan of work (a review of leader's present work related to this project, if any).
Starting date and duration of project. Facilities available for project. Itemized budget requested. Project leader signature. Proposals, in compliance with the protocol format, are to be sent to Dr. D. E. Davis, Caroline Foods, Federalsburg, Maryland 21632, the Committee Chairman. Assistance, counsel or guidance can be obtained from any Committee Member listed below. In an attempt to cover all possible areas where research might be conducted, the information re-
(Continued on page 337)
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ment for 13 days on a soy-starch ration. Then once each day for 5 days the chicks received oral doses of either lutein, neoxanthin or violaxanthin. T h e skin pigmentation was measured by a toe web analysis and visual scoring. Compared to lutein, neoxanthin was found to be a weak (Relative Skin Pigmentation Potency = 8%) skin pigmenter and violaxanthin was essentially ineffective. Chromatographic analysis of the chick skin showed neoxanthin and traces of violaxanthin were deposited in the skin. Traces of neochrome, an isomer of neoxanthin, and luteoxanthin and auroxanthin, isomers of violaxanthin, were also detected in the chick skin. Apparently, after ingestion by the chick, the neoxanthin and violaxanthin undergo partial acid isomerization to form these compounds.
T
TABLE 1.—Basal ration Ingredients Soybean meal Corn starch Saffloweroil Dried fish solubles Vitamin mix W U-24G2 Mineral mix MK-5 2 CaC0 3 DL-Methionine
% of ration 44.64 41.84 7.00 1.00 1.00 3.00 1.50 0.20
1 Contains 22% protein and .0125% ethoxyquin added to the safflower oil. 2 For composition see Kuzmicky et al. (1968).
GENERAL PROCEDURE 1
For the two experiments conducted in this study, day-old Cornish-White Rock type cross-bred cockerels (Kimber) were depleted of pigment on a 22% protein soystarch ration (Table 1) for 13 days. After this period all chicks with greenish colored feet (less than 5%) were discarded and the chicks used were distributed into replicate 1 Reference to a company or product name does not imply approval or recommendation of the product by the U. S. Department of Agriculture to the exclusion of others that may be suitable.
groups equalized in weight. The chicks were housed in electrically heated brooders in a laboratory with continuous fluorescent lighting. The laboratory temperature was maintained at 23.5-26.5°C. (75-80°F.). The lutein, neoxanthin and violaxanthin used in this study were isolated from an extract of fresh alfalfa by a procedure similar to that of Bickoff et al. (1954). Briefly, the procedure consisted of the preparation of an acetone extract, transfer of the pigments to hexane, and chromatography of the hexane extract on MgO-Celite (1-1). Washing the column with hexane-acetone-methanol (89-10-1) gave four fractions. These were neoxanthin, a mixture of violaxanthin and zeaxanthin, lutein and cryptoxanthin. Final purification of neoxanthin and lutein was accomplished by recrystallization in the dark under an inert atmosphere, first from methanol-hexane followed by acetonehexane. It was necessary to rechromato-
326
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HE xanthophylls from dehydrated alfalfa meal as measured by the new Western Regional Research Laboratory (W.R.R.L.) analytical procedure include lutein, zeazanthin, cryptoxanthin, neoxanthin, violaxanthin, carotenoid oxidation products, and various cis isomers of these (Kohler et al., 1967). Lutein, zeaxanthin, and cryptoxanthin have been reported to be good skin pigmenters while indications are the polyoxycarotenoids are relatively poor skin pigmenters (Quackenbush et al., 1965; Kuzmicky et al., 1968). Violaxanthin has been reported to be ineffective as a yolk pigmenter (Marusich et al., 1960). However, the effectiveness of violaxanthin and neoxanthin as skin pigmenters has not been established. This study was undertaken to determine the relative skin pigmentation potency (R.S.P.P.) of pure all-trans neoxanthin and all-trans violaxanthin relative to pure all-trans lutein.
327
PIGMENTATION FROM NEOXANTHIN AND VIOLAXANTHIN TABLE 2.—Broiler pigmentation results from Experiment 1 Dose ' Vkn»12 *«• Xanthophyll Supplements mg./chick/ v l 5 ~ " per 100 cm.'
Control
0
Lutein
.25 .50 .75
Neoxanthin
3.0
RSPP''
0.8
21.9
1.9 2.2 3.0
42.2 51.4 65.1
100
1.5
32.6
8
1
Three chicks were used for the control and each lutein dose and 2 chicks for the neoxanthin. The pigments were administered for 5 days. 2 Average value of 6 people using 1965 model Roche yolk color fan. 3 All-trans lutein equivalents. 4 Estimated relative skin pigmentation potency.
E X P E R I M E N T A L AND RESULTS
Experiment 1. This was a preliminary experiment to determine the approximate RSPP of pure neoxanthin. One zero level control, 3 doses of lutein at .25, .50 and .75 mg. per chick per day, and one dose of neoxanthin at 3 mg. per chick per day The pigments were dissolved in corn oil were used. The lutein doses were disor safflower oil and administered to the solved in 0.5 ml. corn oil and the neochicks orally at 9:00-10:00 a.m. each morning for five days after the 13-day xanthin in 1 ml. corn oil. One group of 3 depletion period. A syringe with a 2-inch chicks was used for the control and each 18 gauge needle was used. A smooth round lutein level. One group of 2 chicks was ball of solder was attached to the tip of the used for the neoxanthin. For the toe web needle. The needle was inserted into the analysis 2 toe web plugs (3/16-inch diachick's esophagus about 2 inches past the meter) were taken from both the left and point of the beak and then the xantho- right foot. The xanthophyll (all-trans phyll solution was slowly injected into the lutein equivalents) per unit toe web area crop. Ad libitum feeding of the soy-starch was determined using the absorptivity of ration continued during this 5-day experi- 236 and the absorbance at 475 mju. The results are shown in Table 2. mental period. From the toe web analysis (plotting the The toe web analyses and visual scoring (using 1965 model Roche yolk color fan) xanthophyll dose vs. xanthophyll per unit were done as described earlier (Kuzmicky toe web area) a significant (P < .05) linear el al., 1968). From the toe web analysis the regression was obtained for the lutein relative skin pigmentation potency standard and control (Y= 24.3+55.5 X (RSPP) was determined. In Experiment 1 (r=.99)). A regression line for the neothe slope of the dose-response curve of the xanthin was determined by extending a unknown was determined and compared line from the zero intercept of the lutein to the slope of the standard. In Experi- curve to the neoxanthin point. A ratio of ment 2 from which more data was avail- the two slopes yielded an estimated RSPP able the procedure as described by Bliss of 8% for neoxanthin. The visual scoring of the foot pads of both the right and left (1946) was used.
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graph the violaxanthin and zeaxanthin mixture on micro-eel C (developed with hexane-methanol (99-1)) to separate the two pigments. Violaxanthin crystals were collected upon concentration of the eluting solvent and then purified by recrystallization from methanol-hexane and acetone-hexane. Thin-layer chromatographic data, absorption spectra and m.p. determinations were obtained on the isolated compounds. These physical data were compared with those reported by Strain (1938) to ascertain identity and purity. An abundant supply of lutein was prepared, however only 300 mg. of neoxanthin and 175 mg. of violaxanthin were obtained. Consequently, the two experiments which were conducted were restricted in size because of the limited amounts of pigments available.
328
KuZMICKY, KOHLEE, LIVINGSTON, KNOWLES AND NELSON TABLE 3.—Broiler pigmentation results from Experiment 2
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(Bliss, 1946) for the neoxanthin and lutein yields an RSPP of 8% for the neoxanthin Dose 1 (relative to lutein). The visual scoring was 1 ng. Xanthophyll per 100 cm. 2 Supplements mg./chick/ Visual RSPP< score toe web area 3 consistent with these results. day Following the toe web analysis and (%) 20.1 Control 0 0.8 visual scoring, the feet from the control 40.2 Lutein .25 1.9 52.6 .50 2.3 100 chicks and the chicks receiving lutein, 52.8 .75 2.3 neoxanthin and violaxanthin were cut into Neoxanthin 1.5 0.7 22.2 very small pieces, placed in 250 ml. amber 37.0 3.0 1.7 8 33.6 1.5 4.5 colored Erlenmeyer flasks and extracted Violaxanthin 1.5 0.9 29.8 at room temperature for 5 days with three 3.0 0.8 29.8 4.5 1.3 28.3 100 ml. portions of hexane-acetone (1-1). 1 Two replicates of 3 chicks each were used for the control The extracts were filtered and saponified and each lutein dose and one group of 3 chicks for each neoxanthin and violaxanthin dose. The pigments were administered with methanolic KOH. Following removal for 5 days. 2 Average value of 7 people using 1965 model Roche yolk of the alkali and alcohol by washing with color fan. 3 All-trans lutein equivalents. water and drying over sodium sulfate, the 4 Relative skin pigmentation potency calculated by the slope ratio assay using data from the toe web analysis. respective extracts were chromatographed on MgO-Celite (1-1). The columns were feet of all chicks by a panel of six persons washed with increasing quantities of acetone in hexane or acetone-methanol in was consistent with these results. Experiment 2. The purpose of this ex- hexane. The purified xanthophyll bands periment was to determine the RSPP of were identified by thin-layer chromatoneoxanthin and violaxanthin with lutein graphic comparison (Nelson and Livingas the standard. A zero level control and ston, 1967) with known xanthophylls three levels of each pigment were used. isolated from alfalfa and by preparation of The levels of lutein were .25, .50, and .75 their visible absorption spectra including mg. per chick per day. The levels of the addition of small amounts of acid to neoxanthin and violaxanthin were 1.5, determine if epoxide derivatives were 3.0, and 4.5 mg. per chick per day. In each present (Curl, 1961). The spectra thus case the pigment was dissolved in 1 ml. of prepared were compared with those of safflower oil. Two replicates of 3 chicks purified alfalfa xanthophylls as well as each were used for the control and each those reported in the literature (Strain, lutein level. One group of 3 chicks was 1938). The visible spectra of the xanused for each neoxanthin and violaxanthin thophylls isolated from chick skin are prelevel. The toe web analysis was done the sented in Table 4. same as in Experiment 1. A panel of seven Lutein was detected in the skin of all people visually scored both the right and the chicks. Neoxanthin and neochrome left feet of all chicks. The results are were detected in the skin of the chicks shown in Table 3. receiving neoxanthin. Violaxanthin, luteoSignificant (P<.05) linear dose-re- xanthin and auroxanthin were detected sponse regressions were obtained for the in the skin of the chicks receiving violalutein and neoxanthin but not for the xanthin. violaxanthin. Both the visual scoring and DISCUSSION toe web analysis indicate that violaxanthin is deposited in only trace amounts in the The results from this study show that skin. Employing the slope-ratio assay compared to lutein, neoxanthin is a weak
PIGMENTATION FROM NEOXANTHIN AND VIOLAXANTHIN
TABLE 4.—Xanthophylls present in chick skin {Experiment 2) Chick supplement
Band
Absorption maxima, nut.
Apparent xanthophyll
None (control)
1
472,
443,
415
Lutein
Lutein
1
473,
444,
419
Lutein
Neoxanthin
1 2 3
469, 465, 447,
442, 437, 420,
4161 4202 396
Lutein Neoxanthin Neochrome
Violaxanthin
1 2 3 4
474, 471, 449, 427,
445, 445, 427, 401,
4181 420» 4003 381
Lutein Violaxanthin Luteoxanthin Auroxanthin
1 No spectra shift upon addition of acid, indicating no 5,6epoxide present. 2 Upon addition of acid spectra shifted to 448, 421, 399 nut. indicating a 5,6-epoxide forming a 5,8-epoxide. 3 Upon addition of acid spectra shifted to 427, 401, 380 nut. indicating 5,6,5',6'-diepoxide or 5,6,5',8'-diepoxide forming a 5,8,5',8'-diepoxide.
falfa meal as the pigmenting source. Consequently, from this type of a ration, the contribution towards skin pigmentation from the neoxanthin and violaxanthin present would be negligible. The results reported here coupled with our earlier results (Kuzmicky et al., 1968) and those of Quackenbush et al. (1965) further clarify the picture of broiler pigmentation from dehydrated alfalfa meal. It seems clear that lutein plus its cis isomers or derivatives are primarily responsible for skin pigmentation from alfalfa meal. Zeaxanthin and cryptoxanthin are present in alfalfa meal in such small quantities that their contribution would be small even though they may be efficiently utilized. A recent extensive xanthophyll analysis of fresh and dehydrated alfalfa meal by Livingston et al. (1968) has shown that lutein plus its cis isomers or derivatives comprise a considerably greater portion of the total xanthophylls than reported earlier by Bickoff et al. (1954). In the more recent work lutein plus its cis isomers or derivatives comprised 82% of the xanthophylls measured while neoxanthin and violaxanthin amounted to only 18%. The authors also reported that during the dehydration of alfalfa up to 94% of the neoxanthin is destroyed and up to 87% of the violaxanthin is lost. Thus, use of the new WRRL xanthophyll analytical procedure for fresh or sun-cured alfalfa could result in lower pigmentation results than expected since both these products contain appreciably greater proportions of violaxanthin and neoxanthin than dehydrated alfalfa meal. Possible modifications could be incorporated into the analytical procedure for such products high in epoxyxanthophylls. SUMMARY Day-old cockerels were depleted of pig-
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skin pigmenter (RSPP = 8%) and violaxanthin is essentially ineffective. However, the chromatographic analysis of the chick skin showed neoxanthin and traces of violaxanthin to be deposited in the skin. Neochrome, a 5,8-epoxide isomer of neoxanthin, and luteoxanthin and auroxanthin, 5,8-epoxide isomers of violaxanthin, were also detected in the chick skin in trace amounts. Apparently, after ingestion by the chick, the neoxanthin and violaxanthin undergo partial acid isomerization to form these compounds. In measuring the total xanthophylls (all-trans lutein equivalents) per unit toe web area, by using the absorptivity of 236 andtheabsorbanceat 475 m/x., neoxanthin, neochrome, luteoxanthin, and auroxanthin are partially discounted due to the fact that their absorption peaks are farther toward the violet end of the spectrum. The error involved here is relatively small. Compensating for this error would only change the RSPP of neoxanthin from 8% to about 10% and essentially have no effect on the estimation of potency of violaxanthin. The levels of neoxanthin and violaxanthin used in this study are very high compared to the amounts that would be present in a broiler ration containing al-
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ACKNOWLEDGMENTS T h e authors gratefully acknowledge Ann R. Gramps and Robert L. Patterson for assisting in carrying out chick assays. Grateful appreciation is also extended to Dr. Clyde D . Mueller, Kimber Farms, Inc., Fremont, California, for arranging a special supply of Kimber cockerels for our purchase.
REFERENCES Bickoff, E. M., A. L. Livingston, G. E. Bailey and C. R. Thompson, 1954. Alfalfa carotenoids—• xanthophylls in fresh and dehydrated alfalfa. J. Agr. Food Chem. 2: S63-567. Bliss, C. I., 1946. An experimental design for sloperatio assays. Ann. Math. Stat. 17: 232-237. Curl, A. L., and G. F. Bailey, 1961. An improved test for carotenoid epoxides. J. Agr. Food Chem. 9: 403-405. Kohler, G. 0., R. E. Knowles and A. L. Livingston, 1967. An improved analytical procedure for the determination of xanthophyll. J. Assoc. Offic. Analyt. Chemists, 50: 707-711. 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. Livingston, A. L., R. E. Knowles, J. W. Nelson and G. O. Kohler, 1968. Xanthophyll and carotene loss during pilot and industrial scale alfalfa processing. J. Agr. Food Chem. 16: 84-87. Marusich, W., E. DeRitter and J. C. Bauernfeind, 1960. Evaluation of carotenoid pigments for coloring egg yolks. Poultry Sci. 39: 1338-1345. Nelson, J. W., and A. L. Livingston, 1967. Stabilization of xanthophyll and carotene by ethoxyquin during thin-layer chromatography. J. Chromatog. 28: 465-467. Quackenbush, F. W., S. Kvakovszky, T. Hoover and J. C. Rogler, 1965. Deposition of individual carotenoids in avian skin. J. Assoc. Offic. Agr. Chemists, 48: 1241-1244. Strain, H. H., 1938. Leaf xanthophylls. Carnegie Institute of Washington, Pub. 490, Washington, D.C.
NEWS AND NOTES (Continued from page 306)
signatures normally necessary for a contract. This will be done after the contracts are awarded. Research projects for the consideration of the Committee are to be presented in accordance with the following format and are not to exceed two typewritten pages. Title. Project leader and other investigators (brief biography). Objectives. Plan of work (a review of leader's present work related to this project, if any).
Starting date and duration of project. Facilities available for project. Itemized budget requested. Project leader signature. Proposals, in compliance with the protocol format, are to be sent to Dr. D. E. Davis, Caroline Foods, Federalsburg, Maryland 21632, the Committee Chairman. Assistance, counsel or guidance can be obtained from any Committee Member listed below. In an attempt to cover all possible areas where research might be conducted, the information re-
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ment for 13 days on a soy-starch ration. Then once each day for 5 days the chicks received oral doses of either lutein, neoxanthin or violaxanthin. T h e skin pigmentation was measured by a toe web analysis and visual scoring. Compared to lutein, neoxanthin was found to be a weak (Relative Skin Pigmentation Potency = 8%) skin pigmenter and violaxanthin was essentially ineffective. Chromatographic analysis of the chick skin showed neoxanthin and traces of violaxanthin were deposited in the skin. Traces of neochrome, an isomer of neoxanthin, and luteoxanthin and auroxanthin, isomers of violaxanthin, were also detected in the chick skin. Apparently, after ingestion by the chick, the neoxanthin and violaxanthin undergo partial acid isomerization to form these compounds.