CAROTIN-THE PRINCIPAL NATURAL YELLOW PIGMENT OF MILK FAT: ITS RELATIONS TO PLANT CAROTIN AND THE CAROTIN OF THE BODY FAT, CORPUS LUTEUM AND BLOOD SERUM.l

II. THE PIGMENTS OF THE BODY FAT CORPUS LUTEUM AND SKIN SECRETIONS OF THE COW.

BY LEROY S. PALR4ER AND C. H. ECKLES.

(From the Dairy Chemistry Laboratory, University of Missouri, in colipera- tion with the U. S. Department of Agriculture.)

(Received for publication, January 23, 1914.)

In the previous study2 we have shown that the commonly ob- served yellow lipochrome of butter fat from cows’ milk is corn. posed principally of a pigment identical with carotin, the unsat- urated hydrocarbon pigment widely distributed in plants. The carotin of the butter fat, like the carotin of plants, has associated with it one or more xanthophyll pigments, but in much smaller proportion than is usually found in green plants.

We have furthermore shown that these pigments are present in the milk fat as a result of feeding a ration containing an abun- dant amount of these pigments. The presence of carotin and xanthophyll in milk fat therefore is not due to any synthetic powers which the cow possesses, but merely to the fact that the organism absorbs the pigments along with the digestion products of the food and subsequently secretes them dissolved in the milk fat. We were accordingly able to vary the amount of pigment in the milk fat by choosing the proper feeds, i.e., either deficient in carotin and xanthophylls or very rich in these pigments. The above relations between the carotin and xanthophylls of milk fat and the carotin and xanthophylls of the feed were found to hold good for all breeds of dairy cows.

The above results at once opened the question of a similar re- lation of the food pigments to the yellow pigment which is so

1 Published by permission of the Secretary of Agriculture. * This JournaZ, xvii, p. 191, 1914.

211

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212 Pigments of Cows’ Body Fat

often observed in the body fat of cows, especially those of cer- tain breeds, such as the Jersey and Guernsey. In addition the questions were raised, whether the carotin of the corpus luteum has associated with it the xanthophyll constituents found in milk fat; and whether the yellow pigment of the skin secretions of some breeds of cows is the same that characterizes the butter fat.

The investigation here reported was undertaken for the pur- pose of studying these questions.

EXPERIMENTAL.

The methods of studying and identifying the pigments were the macroscopic ones used in the preceding study of the lipo- chrome of milk fat, and may be found in detail in the paper deal- ing with that pigment. These methods were: 1. A careful differentiation of the pigment into its carotin and xanthophyll constituents by using their property of relative greater solubility in low-boiling-point petroleum ether and in 80 per cent alcohol respect,ively. 2. A further demonstration of the character of each constituent by studying its adsorption properties toward calcium carbonate, when in anhydrous carbon bisulphide. 3. A careful comparison of the spectroscopic absorption bands of each constituent obtained in (2) and (3) with the bands of caro- tin and xanthophylls of plants obtained in a similar manner.

The pigments of body fat.

The pigment of the body fat of cows has never been subjected to critical examination. Newbigins reports the only attempt to identify it. He compared the pigment with a yellow non-lipo- chrome pigment which he isolated from the salmon, and also with the yellow pigment of maize. Newbigin concluded from his study that the body fat pigment, like the yellow salmon pigment was not a lipochrome. The maize pigment was similar in all re- spects except that it gave the lipochrome color reactions. On account of this result, we paid particular attention to the lipo- chrome propert,ies of the body fat pigment.

The same method was used for isolating the body fat pigment as was used for the milk fat pigment. It consisted in careful

3 Cf. D. Kiiel Paton, Report of Investigations on the Life History of the Salmon, 1898, Article XV.

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I,. S. Palmer and C. H. Eckles 213

saponification of the fat with alcoholic potash (2 cc. of 20 per cent solution per gram of fat) and subsequent extraction of the unsaponifiable matter from the diluted soap (three volumes of water to one of soap) with ether. The ether extract was some- times purified by re-saponification and was sometimes freed from cholesterol with digitonin. Twenty-five to thirty grams of fat were used for isolating the pigment for study.

The results of the study of the pigment from several samples of high-colored body fat were uniform in showing that the prin- cipal pigment is, as in the case of milk fat, carotin. It is readily ext,racted from the 80-90 per cent alcohol by petroleum ether (b.p. 30”-50°C.) and cannot be extracted from the latter solvent by 80-90 per cent alcohol. The pigment gives a blood-red car- bon bisulphide solution and in this solvent is unadsorbed by cal- cium carbonate. In carbon bisulphide as well as in alcohol, the pigment shows the characteristic absorption bands of carotin. The above properties were true only of the freshly prepared pig- ment, and in this state only showed the characteristic lipochrome color reactions with concentrated H&SO4 and HNOB.

Like the carotin of milk fat, the body fat carotin has associated with it one or more constituents which show the properties of the xanthophylls. They are readily extracted from low-boiling- point petroleum ether by 80 per cent alcohol and cannot be reex- tracted from the latter solvent by the petroleum ether. In car- bon bisulphide they are partially adsorbed by calcium carbonate and in the same solvent as well as in alcohol show the absorption bands of the xanthophylls, which are noticeably shifted toward the more refrangible part of the spectrum from the carotin bands. The measurements of the bands of both carotin and xanthophylls from body fat, made according to the arbitrary standard scale explained in the previous paper dealing with the milk fat pig- ment, are given in the following Table 1.

TABLE 1.

Absorption bands of carotin and xanthophylk of body fat. --____.

BAND NO. CAROTIN BAND NO. XANTHOPHYLLS

I. ; 224-242 I. 235-252 II. 262-288 II. 278302

III. 302-322 III. 315335

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214 Pigments of Cows’ Body Fat

The relation between the color of the body fat and the food of the cow.

The discovery that the pigment of the body fat of cows is composed of the same pigments found in the milk fat naturally leads to the conclusion that these pigments, i.e., carotin and xanthophylls, arise from the same source as the milk-fat pig- ments. This conclusion is strikingly substantiated by the fol- lowing experiment which was carried out for that purpose.

Two barren, dry, pure bred Jersey cows, in moderate flesh were fed wheat straw alone for sixty days, or until they had lost as much fat as was considered necessary for the second part of the experiment. The daily ration of Cow 25 .was then changed to 9 pounds of yellow corn and 20 pounds of green alfalfa hay, rich in carotin and xanthophylls. Cow 21 was given a ration averaging 11.4 pounds of white corn and 14 pounds of bleached clover hay, very deficient in carotin and xanthophylls, per day. Cow 25 was slaughtered at the end of eighty-one days. Her gain in weight was 160 pounds. Cow 21 was slaughtered at the end of ninety-five days. She had gained materially in condition during her “fattening” period, although the scales showed little gain in weight, due to a much greater “fill” when receiving wheat straw. Samples of fat from various parts of the body were taken from each animal at slaughtering and used for color readings. The results are given in Table 2, p. 215.

The results of this experiment are even more striking when the amount of fat on the various parts of the bodies of the two cows is taken into consideration. Aside from the kidney fat and pelvic cavity fat, which were probably not disturbed to any ex- tent during the starvation period, and which furthermore were of equal color in the two animals, the largest proportion of the en- tire fat of the two cows was represented by the caul fat. It is here that the great difference in the color is noticeable. In fact, if all the fat on the body of No. 21 had been rendered and its color compared with the same from No. 25, the difference in color would have been very marked.

The data resulting from this experiment have some significance aside from the relation of the color of the body fat to the food of the cow. The fact that the outside fats of the two cows were of equal color and the inside fats, especially the caul and rib plate

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L. S. Palmer and C. H. Eckles

TABLE 2.

215

The relation of .feed to the color of body fat.4

Rib plate ......................... Gaul. ............................. Thoracic cavity ................... Around ovaries and uterus ........ Attached to fourth stomach ....... In pelvic cavity. ................. Kidney. .......................... Crops ............................ Over last rib ...................... Over outside chuck ...............

cow NO. 25.

Red Light

2.3 1

2.1 1 1.3 1 2.3 1 1.6 1 2.3 1 1.6 1 2.3 1 2.3 1 2.0 1

cow NO. 21.

I Yellow ’ Red

1.4 3.6 8.0 2.5

24.0 47.0 50.0 47.0 ijo .o 47.0

!.

0.1 0.5 1.0 0.3 1.7 2.1 2.1 1.9 2.1 1.8

Light

fat, were of such widely different colors, shows in a striking manner what fats are drawn upon first in starvation in this class of ani- mals, and what fats are laid on first during fattening.

The relation between the color of the body fat and the breed of the cow.

It is not uncommon to find the body fat of cows, especially of the Jersey and Guernsey breeds, characterized by a very high yellow color. This has led to the belief that this phenomenon is characteristic of only those breeds. As a matter of fact, butch- ers and the consumers also look with disfavor upon beef from these animals on account of the high color of the fat. Although it has been clearly shown that the color of the body fat of Jersey cows is as much dependent upon the food as the color of the milk fat, it was necessary to study the normal coloration of the body fat of different breeds which had accumulated under the ordi- nary conditions, in order to determine the normal breed relation.

For this purpose we have compared the color of the fat on the various parts of the body of one Jersey cow, other than the two

4 The color readings were taken with a l-inch layer of rendered, melted fat, using the Lovibond tintometer.

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216 Pigments of Cows’ Body Fat

given in Table 2, and three Holstein cows. The data are given in Table 3.

TABLE 3.

The relation 0.f breed to the color ?f body fat. -___

cow NO. 22G (Holstein)

I -

cow NO. 2oi (Holstein)

cow NO. 221 (Holstein)

54 63 17 50 47 63

54 47

L

I- Red Red Yellon Red

Rib plate.. crops......,......... Thoracic cavity.. _. Caul . . Pelvic cavity. Over last rib.. Ovaries, uterus.. Chuck. Kidney. On fourth stomach..

47 14 36 54 61 17 62 14 64

1 .7 1.2 1.5 1.7 1.8 1.3 1.8 1 .o 1.8

15 21

6 12 10 23

22 20 11

1.2 1.2 1.2 1.2 1.2 1.2

1.3 1.2 1.2 24

The significant points brought out by the data in the above table are as follows: First, that it is possible to find pure bred Holstein cows with body fat of as high a color as is apparently normal to Jersey cows. Second, that the body fat of Holstein cows is often characterized by a much lower color than is appar- ently normal to Jersey cows. An interesting feature of this dif- ference is that Holstein cow 207 was ‘known to give high colored milk fat, while Holstein cow 221 never gave milk fat of a color which exceeded the maximum color found in her body fat even under the most favorable conditions for the production of a high color. Both of these cows were dry and barren at the time of slaughtering and had been fattened largely on pasture grass and grain, a ration rich in carotin. This result points to an individ- ual variation, as well as a breed variation, in regard to the color of the body fat.

The relation between the maximum color of the body fat and milk fat which an individual cow will produce under a given feeding condition is an important one. It will be shown in a later paper that this maximum color, especially of the milk fat, is independent of the amount of carotin in the blood. This is also true in regard to the color of the body fat. One would ex-

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L. S. Palmer and C. H. Eckles 217

pect this by analogy and it was found to be true from an ex- perimental standpoint. Without entering into details here in regard to the method of analysis, which will be given in a later paper, it may be stated that the blood of Cow 221 was found to be as rich in carotin as is normal to a Jersey under the same con- ditions. These results point to the fact that whatever differences occur among individuals or breeds in regard to maximum color- ation of milk fat or body fat, arise at the point of formation of the fat, i.e., at the milk glands and body cells.

The pigments of the corpus luteum.

Escher5 has recently shown by chemical analysis that the pig- ment which can be crystallized from the high-colored corpus lu- teum of the cow is identical with the carotin of plants. In view of the fact that our investigations have shown that the lipochromes of the milk fat and body fat of the same animal are the same pigment and especially that they have associated with them one or more minor xanthophyll constituents, it became important to study the corpus luteum pigment in this connection also.

Only a few corpora lutea were available for this study, they being obtained at various times at the slaughtering of experi- mental cows at the experiment station. In some cases the cows had well developed corpora lutea and at other times only the remains of former corpora lutea were obtained. In each case, however, the pigmented tissue was carefully cut away and mac- erated with sand and the pigment extracted with ether. In some cases the extract was saponified before examining the pigment.

The result of several such studies completely confirmed the carotin properties of the principal pigment. In addition in two cases it was found that after saponification a minor portion could be extracted by 80 per cent alcohol from a petroleum ether solu- tion of the entire pigment, which could not be reextracted by fresh petroleum ether. In another case where a complete pe- troleum ether extract of two ovaries, one of which had a very large corpus luteum, was studied, no xanthophyll pigments were obtained after saponification. We were unfortunate in not hav- ing sufficient corpora lutea to carry this study to completion.

5 Zeitschr. f. physiol. Chem., lxxxiii, p. 198, 1913.

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218 Pigments of Cows’ Body Fat

Undoubtedly the normal corpus-luteum carotin sometimes has associated with it some xanthophyll, which is probably present in the fat which may be extracted along with the corpus-luteum carotin.

The pigments of the waxy secretions in the ears and on the skin of Jersey cows.

The skin secretion of Jersey (and Guernsey) cows is often characterized by a more or less yellow color. This is considered by the breeders of these cattle to indicate their ability to secrete yellow milk fat. It was thought that a brief investigation of this pigment might be of interest and possibly of some scientific value.

The yellow skin pigment is sometimes very abundant in the ears of these animals. A few grams of the yellow wax was scraped from the ears of several pure bred Jersey cows. The wax was macerated with ether, which readily dissolved the pigment. Af- ter saponification and extraction with ether, the pigment was sub- mitted to a macroscopic examination with respect to its carotin and xanthophyll properties.

The result was that the pigment was readily divided into a major constituent which showed all the properties of carotin, and a very minor constituent which was a xanthophyll. It is thus seen that the pigments which characterize the skin secretions of the Jersey breed are the same pigments that are found in the milk fat and body fat.

The body fat and blood serum pigments of the new-born calf.

Carotin and xanthophylls having been found to be normal con- stituents of the body fat of cows which had received an abun- dance of these pigments in the food, an interesting question was raised whether these pigments are present in the body of the new-born calf. Should these pigments be absent from the new- born calf, additional proof would be obtained that their presence in the mature animal is the result of feeding. Should they be present in the new-born calf, it would merely indicate that they were able to traverse the placental barrier from the mother whose body is normally rich in carotin and xanthophylls. In this con- nection the question would be especially interesting in view of

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L. S. Palmer and C. H. Eckles 219

the fact that Mendel and Daniel@ have found that fat-soluble dyes, such as Sudan III, do not traverse the placental barrier of small animals such as cats and rats, whose milk fat and body fat, however, is readily tinted as the result of feeding the dyes.

In order to study this question, a new-born Jersey calf was not allowed to suckle its mother but was slaughtered a few hours after birth. The blood was caught in a cylinder and allowed to clot. The serum was carefully investigated for the presence of carotin. No pigment was found which could be precipitated from the serum with the proteins and extracted therefrom with alcohol. The filtrate also yielded no lipochrome-like pigments.

A very small amount of fat could be obtained from the body of the calf, in all weighing about 40 grams. The rendered fat had a very faint yellow color. On saponification, it yielded a small amount of unsaponifiable pigment which could be differentiated into two constituents which showed properties identical with carotin and xanthophyll respectively.

It appears that carotin and xanthophylls do traverse the pla- cental barrier of the cow to a very slight extent. It is possible that their presence in the body fat was due to a direct transfer of pigmented fat from the mother to the foetus. On this ground the absence of the pigments in the blood could be explained.

Discussion of results.

Viewing the results of the above studies from a physiological standpoint, it is readily seen that the establishment of the source of the yellow pigments of the body of the cow, and the ease with which they are therefore increased and decreased,7 throws great doubt upon any physiological significance which they have been supposed to exert in the animal body. In the case of the corpus luteum for instance, the accumulation of carotin during the formation of this body is merely a phenomenon incidental to the rupture of the Graafian follicle and the subsequent formation of the cellular tissue around the central blood clot, and to the fact that the blood serum is normally very rich in carotin as will be shown in a subsequent paper.

6 This Journal, xiii, p. 72, 1912. 7 This is especially true of the milk fat and, as will be shown in a sub-

sequent paper, the blood serum.

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220 Pigments of Cows’ Body Fat

The popular opinion that the body fat of Jersey cows is nor- mally characterized by a higher yellow color than Holstein cows has been at least partially confirmed by experimental study, al- though it was found that Holstein cows may also possess high- colored body fat. At least there seems to be more breed charac- teristic in this respect than in the case of the pigmentation of the milk fat. There is no foundation, however, for the belief that beef has a lower value because its fat has a high color. If this pigment is the same as is demanded by the consumer for butter, why should not beef with high-colored fat also be more desirable? It is recognized, of course, that some of the unfavor- able attitude toward beef with highly colored fat arises partially from the fact that it indicates that the beef probably came from a dairy cow. The two ideas are neverthelessvery closely associated.

The normally high color of the body fat of Jersey cows, and also of Guernsey cows explains why cows of these breeds often appear to be producing well colored butter on a ration deficient in carotin and xanthophylls. When cows whose body fat has a high yellow color are put upon a ration deficient in carotin and xanthophylls and also, as is usually the case with such rations, deficient in food value, the body fat is called upon to furnish energy for the animal and also in many cases to supplement the food digestion products in the production of milk fat. It is readily seen that in such cases an important source is opened up for pigments for the milk fat. The importance of this source would depend upon the amount of highly colored body fat avail- able for the needs of the body, and upon the rapidity with which it would be used up. If our experimental data are correct showing that the inside fats, such as the caul fat and rib plate fat, are first drawn upon in starvation of this class of animals, then the amount of available highly colored fat would be rather large. Dairy cows usually have a fairly abundant amount of these fats, especially the caul fat. It is thus readily seen that a contin- uous drawing upon these inside fats for a long period of time would result in a very slow and gradual reduction of the color of the milk fat, especially since, as will be shown in a subsequent paper, the blood serum furnishes the principal, normal source of carotin for the milk fat. The deduction that the animal was actually producing colored milk fat on a carotin-xanthophyll-free ration would therefore be quite natural but nevertheless entirely false.

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L. S. Palmer and C. H. Eckles 221

In a similar manner, it is readily seen why the breeders of Jersey and Guernsey cattle have been led to believe that the yellow skin secretions of these breeds are indicative of their ability to produce yellow milk fat. It is interesting to find that the yellow pigments of these secretions are carotin and xan- thophylls. It should be clearly borne in mind, however, that the only indication that a cow will secrete yellow milk fat is that the food contains an abundance of carotin and xanthophylls.

CONCLUSIONS.

1. The yellow lipochrome of the body fat, corpus luteum, and skin secretions of the cow, like the lipochrome of butter fat, is composed principally of carotin, which sometimes has associated with it one or more minor xanthophyll constituents.

2. The carotin and xanthophyll pigments of the body fat are derived from the food of the cow. The body fat of Jersey cows formed on a ration deficient in carotin and xanthophylls is devoid of color.

3. The body fat of Jersey and Guernsey cows is usually char- acterized by a higher yellow color than the body fat of other breeds. This is of great importance in explaining why Jersey and Guernsey cows sometimes show a much slower elimination of pigment from the milk fat on a non-pigmented ration, as during the winter months. In these cases the body fat furnishes a sup- plementary source of carotin and xanthophylls for the milk fat.

4. The yellow body fat of Jersey and Guernsey cows should not be a point against the use of these animals for beef. The pigments here are the same as those for which the consumer will pay a higher price when present in butter.

5. The breeders of Jersey and Guernsey cattle are no doubt correct in their belief that the yellow skin and skin secretions of these animals are characteristic of these breeds. It is not cor- rect, however, that this characteristic is indicative of the ability of the breeds to secrete yellow milk fat under all conditions. The only indication of this is whether the food contains an abun- dance of carotin and xanthophylls.

6. The blood serum of the new-born Jersey calf is free from carotin and xanthophylls. The small amount of fat on the body is tinted faintly with these pigments.

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Leroy S. Palmer and C. H. EcklesSKIN SECRETIONS OF THE COWBODY FAT CORPUS LUTEUM AND

SERUM: II. THE PIGMENTS OF THE CORPUS LUTEUM AND BLOOD

THE CAROTIN OF THE BODY FAT, RELATIONS TO PLANT CAROTIN ANDYELLOW PIGMENT OF MILK FAT: ITS

THE PRINCIPAL NATURAL−−CAROTIN

1914, 17:211-221.J. Biol. Chem. 

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