FAT-SOLUBLE VITAMIN. XXVI. THE ANTIRACHITIC PROPERTY OF MILK AND ITS IN- CREASE BY DIRECT IRRADIATION AND BY IRRADIATION OF THE ANIMAL.* BY H. STEENBOCK, E. B. HART, C. A. HOPPERT, AND ARCHIE BLACK. (From the Department of Agricultural Chemistry, University of Wisconsin, Madison.) (Received for publication, September 22, 1925.) Some time ago we (1) made the statement that cow's milk, well known to be an antirachitic agent of questionable value, can have its antirachitic potency materially increased by direct exposure to the radiations of a quartz mercury vapor lamp. Hess (2) has made a similar statement and Cowell (3) has used irradiated milk successfully in stimulating calcium deposition in children. It appeared desirable that the data of our experiments which brought out this fact should be published in detail because they indicate again to what extremes the animal organism is dependent upon ultra-violet radiations for the endowment of its food with the requisite antirachitic properties; and because they also suggest the possibility of a practical utilization of the principle in the preparation of infant foods. This latter is not, however, without its practical difficulties as will be pointed out later, and therefore when considered from the standpoint of increasing the antirachitic properties of milk alone, we believe that direct irradiation of the animal has much to offer. We shall discuss this later. EXPERIMENTAL. Our first experiments were carried out in the month of February, 1925, with cow's milk obtained from stall-fed cows. These cows received a mixture of grains, silage, and alfalfa as their regular * Published with the permission of the Director of the Wisconsin Agricultural Experiment Station. 441
442 Fat-Soluble Vitamin. XXVI ration and were allowed to exercise in a paddock exposed to the sun for a few hours daily when the weather was not too cold or disagreeable. As a test for antirachitic properties the milk from these cows was fed in quantities of.5 to 12 cc. daily to rachitic rats. For irradiation 5 cc. of milk were poured into a galvanized iron pan measuring 12 by 24 inches, which was then tipped from side to side so as to distribute the milk as evenly as possible. It was exposed to ultra-violet light generated by a quartz mercury vapor burner of the Cooper-Hewitt BY type at a distance of 2 feet for 3 minutes. The burner was run at a burner voltage of 4 to 6 and 4 amperes. The rats used for the tests were animals of our own breeding started at approximately 6 gm. in weight on our rickets-producing ration No. 2965. This, as we have already published (4), consists of 76 parts of yellow corn, 2 of wheat gluten, 3 of calcium carbonate, and 1 of sodium chloride. It always produces rickets of uniform severity in our rats in a few weeks. After 29 days on this ration, by which time moderate rickets had developed in all our rats, their diets were supplemented for days by the addition of milk measured out individually into small glass dishes. The rats were then killed and the distal ends of their radii and ulnse split with a scalpel and stained with silver nitrate. In most cases the bones were not stained immediately after dissection because it was found that fixing in per cent formalin, not only had no harmful effects, but gave much clearer preparations. The results of these experiments tabulated in Table I show that it took 12 cc. of non-irradiated milk to produce a response in our animals, but only 1 cc. of the irradiated. It was a question in our minds whether or not we had irradiated our milk sufficiently long to secure maximum results. Purposely we had used as thin a film of milk as we could obtain by pouring it into a large pan, but we were apprehensive lest a prolonged exposure might result in an inactivation such as we had experienced with oils (4). Arbitrarily we selected 3 minutes as the period of time because that had given us optimum results in previous trials with other foods (4). Table II gives the results of feeding milk exposed to our quartz mercury vapor light for periods varying from to 12 minutes.
Steenbock, Hart, Hoppert, and Black 443 This time 5 cc. of milk were exposed in a smaller pan (a circular' pie tin 7 inches in diameter) because the pans used before were TABLE I. The Increase of the Antirachitic Properties of Cow's Milk by Irradiation. Lot No. Rat No. Milk added Weight when weight Final ~umption. Average depo Calcium dailyt. changed. consumption. Non-irradiated. cc. pm. gm. gm. 3143 2217.5 98 92 6-2218 1. 4 8 5-6 - 2219 2. 88 1 4-222 4. 5 118 6-2221 8. 115 137 6? 2222 12. 2 3-4 + 3144 2223.5 88 9 5-6 - 2224 1. 95 2 6-7 - 2225 2. 112 123 8-2226 4. 96 4 6-2227 8. 96 9 5 + 2228 12. 7 122 5 + Irradiated. 3145 2229.5 97 9 8? 223 1. 85 94 5 + 2231 2. 94 7 + 2232 4. 95 92 6 + 2233 8. 89 88 4-5 + 2234 12. 96 5 4 + 3146 2235.5 85 85 6 2236 1. 94 98 6 + 2237 2. 98 2 5-6 + 2238 4. 88 85 5 + 2239 8. 8 75 5 + 224 12. 9 5-6 + too large to allow simultaneous exposure of a number of samples. The rats were given 1 cc. of the milks daily for days after they had been on our Ration 2965 for 3 weeks. The table shows that 3 minutes gave us optimum results in activation.
444 Fat-Soluble Vitamin. XXVI Dur next experiments were carried out with goat's milk because we wanted to determine not only the extent to which its antirachitic properties could be increased by exposing it to ultraviolet light, but also the effect of direct irradiation of the animal. For such an experiment a goat is obviously a more convenient animal than a cow. Goat I, a young goat which had been confined in a dimly illuminated basement all winter, was used for the first trials beginning in the month of April, 1925. She weighed 5 pounds and was producing an average of 213 cc. of milk daily. Previous to the experiment and during the experiment she received all she would consume of a ration of 2 parts of a grain mixture and 1 part of chopped alfalfa hay. The grain mixture was composed of 35 parts of corn-meal, 3 parts of bran, 3 parts of ground oats, and 5 parts of linseed oil meal. The goat was exposed to the quartz mercury vapor are for 3 minutes daily when irradiation was started. Exposure was made at a distance varying from 12 to 3 inches from the lamp depending upon whether the animal was standing up or lying down. As her coat was exceedingly heavy, the long hair was clipped from her back before irradiation. This did not, however, expose her skin because her coat was almost felt-like in thickness. Milk was fed to rachitic rats before irradiation and after, beginning respectively 4, 9, and 17 days after irradiation was started. In addition samples of milk irradiated directly were fed as controls to determine the degree of activation possible. In all cases the rachitic rats were prepared as before, being fed our ricketsproducing ration No. 2965 for 21 to 28 days before milk was added to the diet. Milk was fed for a period of days. It was measured out individually for each rat. The results of these trials are presented in Table III in abbreviated form to conserve space. The table is self-explanatory. It shows that while it took 12 cc. of control milk to bring about incipient calcium deposition, after irradiation of the milk it took only.5 cc. and after irradiation of the animal 2 cc. Evidently irradiation of the animal was not so effective as direct irradiation of the milk itself, but nevertheless it is noteworthy that the increase in potency was sixfold. This appears the more remarkable when it is taken into consideration that the milk was taken
Steenbock, Hart, Hoppert, and Black 445 beginning 4 days after irradiation was started and ending after 14 days irradiation, by which time the animal had received a total of only 7 hours of exposure. By longer treatment with ultra-violet radiations no further increase in activity occurred, but it was a noticeable fact that the animal consumed its ration with greater avidity after such treatment. It became more active and increased in weight in 4 weeks TABLE II. Time of Exposure Necessary to Activate Cow's Milk Completely. to Each Rat Daily. z 3374 3375 d 3 3377 3378 3379 338 3381 3382 3383 3384 3385 3386 3387 3388.2 2965 2965 2965 2965 a 21 21 21 21 21 - no calcium deposition. + narrow line of calcium. average line of calcium. moin. 5 3 6 12 5 3 6 12 D P gm. 85 88 91 81 92 73 87 8 79 7 7 W gm. 118 1 8 98 89 3 86 78 91 87 78 8 a E. gm. 7-8 7-8 7-8 6 6-7 7 6 6-7 6 6 5-6 6 I Cc. Fed a. from 5 pounds to 56 pounds. Evidently the resultant changes in the physiology of the animal were more deep seated than mere increase in the antirachitic potency of its milk. We have already pointed out how animal tissues (5) become endowed with antirachitic properties by irradiation and how an animal can also metabolize calcium salts with greater efficiency after such treatment (6).
446 Fat-Soluble Vitamin. XXVI The rapidity with which the milk is affected in antirachitic properties as a result of irradiation of the animal is shown in Table IV. This presents results obtained with Goat II. Goat II weighed 93 pounds. She had dropped her kid about 4 weeks before she was used for the experiment and had been kept for months on a ration of grain and alfalfa in the dimly lighted basement of our barn. She produced from 7 to cc. of milk daily. Goat I. Amount of milk fed daily. cc..2.5 1. 2. 4. 6. 8. 12. TABLE III. Calcium Deposition in Rachitic Rats after Feeding Milk from an Irradiated Goat. Milk not irradiated. -? + -? $: Milk irradiated 3 min. +? - no calcium deposition. + narrow line of calcium. average line of calcium. + wide line of calcium. * union with diaphysis. Milk from goat irradiated 3 min. daily. Feeding of milk begun after irradiation of: 4 days. 9 days. - I- + + * It-t +? + 17 days. +? + + The experimental procedure with this animal was about the same as before, the important exceptions being that the animal was irradiated 1 hour daily instead of hour and that samples of milk for the 1, 2, and 4 day periods were sterilized daily for hour at 15 pounds steam pressure and then kept in the refrigerator during the day feeding interval. For the 8th day period the milk was, however, again taken daily and fed directly. The rachitic rats used for the test were prepared in the same way as before on Ration 2965 on which they were kept from 21
Steenbock, Hart, Hoppert, and Black 447 to 22 days before the milk additions were made. For comparative purposes one series of rats was given milk drawn before irradiation and another the same after it had been irradiated as in the previous experiment. The results are presented in Table IV. They suggest that the milk was somewhat improved in antirachitic properties by the 2nd day of irradiation and by the 7th day maximum activity had apparently been imparted. The 8 to 18 day samples gave no better results. With this animal the effect of irradiation was apparently not so pronounced as with Goat I because it took 4 cc. Goat II. TABLE IV. Calcium Deposition in Rachitic Rats after Feeding Milk from an Irradiated Goat. Milk from goat irradiated 1 hr. daily. Feeding Amount Milk Dot Milk of milk begun after irradiation of: of milk irradiated fed daily. irradiated. 3 min. I day. 2 days. 4 days. 8 days. CC..2.5 + 1. --... 2. -.. 4. - - +? + + 6. - - + + * 8. - - + : 12. 4 * rather than 2 cc. of milk to produce an antirachitic response. Whether this is due to variation in responsiveness of the two animals, we are not in position to say. The animals were treated exactly alike except that Goat II was irradiated twice as long. DISCUSSION. The data presented in this paper supplement our previously published data which showed that exposure of the animal to ultra-violet radiations effected an economy in its metabolism of calcium salts. These data make us realize that the offspring of irradiated animals may benefit as well and that such benefit may be secured indirectly through the medium of the milk. In 1924
448 Fat-Soluble Vitamin. XXVI Luce (7) studied the antirachitic properties of milk from the standpoint of its relation to sunlight and feed and came to the conclusion that the feed was the more important factor. We have come to realize that this may or may not be the case--depending entirely upon whether or not the feeds have been exposed to ultraviolet light. It does, however, appear to be true that sunlight acting upon the animal itself is not such an extremely active agent.as one might be led to assume. With the broadening of our conceptions of the r6le of calcium in the body and the realization that, at least in comparison to the animal's needs, calcium usually occurs in foods and feeds in quantities bordering on a deficiency, the part that the antirachitic factor plays becomes more and more impressive. We (8) have already emphasized the fact that to obtain a hay of first quality, it should be cured exposed to sunlight. In the light of data presented in this paper, it now appears justifiable to emphasize the fact that the producer of so called high grade milk has undoubtedly gone to extremes in protecting his cows from sunlight. This he has done not entirely by intent because his concern primarily has been to keep his herd away from the heat, flies, and dust. To effect this he has stabled his animals sometimes the year around. It is true he has seen the necessity of having his barns well provided with windows but unfortunately sunlight passing through glass is no longer antirachitic. The question immediately arises if some of the difficulties experienced in high producing herds, such as failure to breed, non-infectious abortion, ahd physical breakdown, may not have as a contributing cause, if not often their origin, a low calcium ration coupled with low antirachitic content. Outside of this it appears advisable that cows should be exposed liberally to direct sunlight in order that they may be able to produce a first class product for human consumption as well as for the production of good calves. In lieu of exposure to sunlight and for the sake of cleanliness and convenience, if not efficiency, the time is probably not far distant when every producer of high grade milk will find it necessary to irradiate his cows artificially. The lessons to be drawn by the hygienist, the pediatrican, and the general practitioner of medicine from these studies are obvious.
Steenbock, Hart, Hoppert, and Black 449 SUMMARY. By exposure to the radiations of a quartz mercury vapor lamp, the antirachitic properties of cow's milk were increased eight or more times. Under the same conditions a sample of goat's milk increased in activity about twenty-four times. This increase in activity can also be induced rather promptly, though to a lesser degree, by direct irradiation of the animal. Attention is called to the beneficial results to be expected by the dairyman. For man in general the indirect benefits are obvious. BIBLIOGRAPHY. 1. Steenbock, H., and Daniels, A. L., J. Am. Med. Assn., 1925, lxxxiv, 93. 2. Hess, A. F., J. Am. Med. Assn., 1925, lxxxiv, 19. 3. Cowell, S. J., Brit. Med. J., 1925, i, 594. 4. Steenbock, H., and Black, A., J. Biol. Chem., 1925, xiv, 263. 5. Steenbock, H., and Black, A., J. Biol. Chem., 1924, lxi, 45. 6. Hart, E. B., Steenbock, H., and Elvehjem, C. A., J. Biol. Chem., 1924-25, lxii, 117. 7. Luce, E. M., Biochem. J., 1924, xviii, 716, 1279. 8. Steenbock, H., Hart, E. B., Elvehjem, C. A., and Kletzien, S. W. F., J. Biol. Chem., 1925. xvi, 425.
FAT-SOLUBLE VITAMIN: XXVI. THE ANTIRACHITIC PROPERTY OF MILK AND ITS INCREASE BY DIRECT IRRADIATION AND BY IRRADIATION OF THE ANIMAL H. Steenbock, E. B. Hart, C. A. Hoppert and Archie Black J. Biol. Chem. 1925, 66:441-449. Access the most updated version of this article at http://www.jbc.org/content/66/2/441.citation Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's e-mail alerts This article cites references, of which can be accessed free at http://www.jbc.org/content/66/2/441.citation.full.html#ref-lis t-1