FAT-SOLUBLE VITAMINS.

XXVII. THE QUANTITATIVE DETERMINATION OF VITAMIN A.*

BY H. STEENBOCK AND KATHARINE H. COWARD.

(From the Department of Agricultural Chemistry, University of Wisconsin, Madison.)

(Received for publication, February 3, 1927.)

In April, 1923, Steenbock and Nelson (l), experimentally veri- fying observations of Hume (2) and of Goldblatt and Soames (3), advanced the theory that under certain conditions the anti- rachitic vitamin is essential for normal growth. They arrived at this conclusion because rats which had become stationary in weight on a diet deficient in vitamin A were made to resume growth either by exposing them to the radiations of a quartz mercury vapor lamp or by feeding them cod liver oil in which vitamin A had been destroyed by aeration. They suggested that when growth first ceased, it resulted from a deficiency of the antira- chitic vitamin and not of vitamin A because ophthalmia and symptoms of the respiratory tract did not make their appearance until much later and were not influenced as to time of incidence by irradiation. With the exhaustion of this reserve of vitamin A neither radiations nor aerated cod liver oil could effect mainte- nance or induce growth.

They emphasized these correlations because it had been uni- versally accepted that failure of growth on rations otherwise com- plete but low in fat-soluble vitamins was due to a lack of vitamin A. These conclusions attracted a great deal of attention because obviously, if correct, most of the observations on vitamin A as recorded in the literature would be in error except in those cases where by chance the animal had been furnished a sufficiency of the antirachitic factor and the very isolated cases where ophthal- mia instead of growth had been used as a criterion.

* Published with the permission of the Director of the Wisconsin Agri- cultural Experiment Station.

765

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766 Fat-Soluble Vitamins. XXVII

Goldblatt and Soames (4) in a second paper published by them extended the scope of their experiments and demonstrated that livers from irradiated rats were growth-promoting. Steenbock and Black (5) verified these facts and by further experimentation showed that this growth promoting property was not related to vital activity of the material exposed nor due to translocation of vitamin A. Steenbock, Black, and Nelson (6) found that it could be induced with antirachitic activation in macerated excised animal tissues; in the basal synthetic ration usually used by them for fat-soluble vitamin studies; in crude fats, and in the unsaponifiable fraction of crude fats as well. Steenbock, Black, Nelson, Nelson, and Hoppert (7) later showed that besides a considerable array of naturally occurring foodstuffs, of known compounds, only choles- terol and probably phytosterol were capable of acting in a similar capacity.

In the meantime Hess and coworkers among their other notable experiments, demonstrated the antirachitic activation of oils (8) and cholesterol (9) directly without correlating this phenomenon with the process of growth. Their experiments, in fact, closely followed in time of execution those of Steenbock and Black (5), and their announcement (9) in regard to the antirachitic activa- tion of cholesterol was, in fact, made simultaneously with that of the latter workers (7). In Europe, entirely independently Drum- mond, Rosenheim, and Coward (10) showed that irradiated cho- lesterol could serve as a growth-promoting factor; and later Drum- mond, Coward, and Handy (11); accepting the demonstration from this laboratory that irradiated cholesterol promoted growth by providing the antirachitic factor, made use of irradiated cho- lesterol in connection with a new technique for vitamin A deter- mination. In this technique they provided their rats with 1 mg. of irradiated cholesterol daily in addition to their fat-soluble vitamin-free diet. This allowed them to dispense with irradiation of the rats as Steenbock, Nelson, and Black (12) had advised.

As a matter of fact we had been in doubt for some time as to the permissibility of irradiating our rats, not so much when we used growth as the determining reaction but when we used change in eye condition instead. Ophthalmia as produced by vitamin A deficiency is a complex pathological reaction, and it is conceivable that the short ultra-violet rays emitted by the quartz mercury

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vapor lamp may act destructively or constructively according to the sensitivity of the affected tissues and the invading micro- organisms. At any rate by exposure to ultra-violet radiations there always exists the possibility that the course of the ophthal- mic reaction may be changed independently of vitamin A addi- tions. The appreciation of this possibility led to our adoption, soon after the discovery of antirachitic activation, of irradiation of the basal synthetic ration in place of irradiation of the animals.

With this modification of our vitamin A technique, Dr. E. M. Nelson and Professor Lois K. Stewart obtained very satisfactory results in the spring and summer of 1925 (13). The rats continued to grow practically up to the time of incidence of ophthalmia and sometimes even longer; and later with the addition of vitamin A they responded with growth much more promptly.

Irradiation of the basal synthetic ration accomplished the object that we were after, but a number of objections can be raised against it. In the first place with the growing tendency to feed more highly purified diets-which practice is most certainly to be encouraged-there obtains the possibility that the diet may be completely freed from activatible compounds. The probability of this at the present time appears rather remote because we have experienced no difficulty in activating alcohol-extracted and heated casein, corn-starch, or the dextrin which is prepared from corn-starch. In fact, we have found that corn-starch may be extracted with alcohol for weeks without losing its ability to be- come activated. Furthermore, we have always obtained very decided antirachitic response when only 5 per cent of the diet was composed of irradiated starch or dextrin. Nevertheless, we must admit that certain preparations of potato and rice starch, sugars, salts, and some pure proteins cannot be activated. Thus there always obtains the possibility that the experimenter compounding his ration from different sources may secure a diet which will not respond as expected. In the second place, it may sometimes be desirable to avoid the possibility of destruction of vitamins pres- ent in the basal diet. In our experience both vitamin A and vita- min B have shown remarkable stability to ultra-violet light. But here again we cannot be absolutely certain that these compounds are stable to ultra-violet radiat,ions under all conditions, and there- fore the use of ultra-violet light on the ration itself may be contraindicated.

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768 Fat-Soluble Vitamins. XXVII

Taking these objections into consideration, with a view to the needs of the future rather than the immediate present, we believe that the use of irradiated cholesterol with proper precautions is to be highly recommended for the rat. As to its use for other animals, we are not so convinced because it is not established that cholesterol is absorbed so well as the activatible compounds found in plant materials. Witness, for instance, the results obtained by Hart, Steenbock, Kletzien, and Scott (14) who found that the antirachitic unsaponifiable constituents of cod liver oil did not establish a positive calcium balance in a goat unless fed in solution in a liquid fat such as corn oil. Not only does this present. con- crete evidence of the variation in the assimilability of the antira- chitic constituents with the diet’, but it should make us very skeptical of the equivalency of antirachitic agents from different sources. It is probably not unwarranted to surmise that the anti- rachitic compounds which exist in the finely divided condition, often even in chemical combination, in plant tissues are much more easily assimilated than activated cholesterol administered en masse. In recognition of this factor we have always taken the precaution when administering cholesterol of feeding it by evapo- rating its ether solution directly on the basal ration. Further- more, it is absolutely necessary to standardize each preparation of cholesterol on rachitic animals. Different preparations vary tremendously in activity, and an activity once induced is far from being a permanent property.

Whatever method of supplying the antirachitic factor is selected, we are convinced that there should be used a more specific reac- tion than growth to indicate whether or not vitamin A is present. As to what this should be, we are not absolutely positive because it is very evident that the absence of vitamin A from the diet leads to degenerative processes in many different organs with corre- sponding variation in symptoms. Tentatively, we have accepted the ophthalmic reaction and have found it to serve our purpose admirably. In the first place, in the course of the last few years, we have had ophthalmia incident in our animals on a vitamin A- free diet almost without fail. In the second place, ophthalmia is incident in the very early stages of vitamin A depletion and is readily detectable as such; and, in the third place, it responds very readily to treatment with vitamin A.

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H. Steenbock and K. H. Coward

In comparison with the ophthalmic reaction, growth is so much more complex and dependent upon so many more factors, that it obviously cannot be considered in the same category. On ad- ministering a very small dose of vitamin A, we have often noted the cure of ophthalmia without resumption of growth in the fol- lowing few weeks. A somewhat larger dose of vitamin A will cure ophthalmia in 10 to 14 days during which time growth will be at a standstill but will be resumed when the trouble has cleared up. A really large dose of vitamin A will result in the curing of the disease and continuation of growth at the same time. The use of growth only, as a criterion of vitamin A depletion, may have dis- astrous results. While waiting to be certain that growth has really ceased and that the cessation is not merely temporary, the rat may decline in weight so rapidly that recovery cannot be established even with the addition of large amounts of vitamin A. In these latter cases we have observed that failure is usually due to infections of the respiratory tract or congestion of the mesen- teric vessels of the upper part of the small intestine which by its severe nature tends to terminate the animal’s existence rapidIy.

It is also to be noted that where only small doses of vitamin A have been administered, recovery may be only temporary, a second attack of ophthalmia developing and growth again ceasing. On the other hand, where large doses of vitamin A have been ad- ministered, the animal grows to maturity. It is evidently of the greatest importance in making comparative studies of the vitamin content of different materials, to carry on the test for long periods of time, 10 to 12 weeks generally being necessary where the amount of vitamin A administered is small.

The experimental work reported in this paper deals exclusively with the determination of vitamin A in various grains while the antirachit.ic factor was supplied by irradiation of the ration. We have obtained such clean cut results in this manner that we believe them worthy of presentation at this time.

EXPERIMENTAL.

At an age of a little more than 3 weeks when weighing from 40 to 45 gm., black and white piebald rats were given a basal ration consisting of purified casein 18 gm., agar 2, salts 40 (1) 4, dried brewer’s yeast 8, and partially dextrinized starch 68.

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770 Fat-Soluble Vitamins. XXVII

The constituents were mixed dry-no water was added. To make it antirachitic, 100 gm. of the ration were spread over an area of 4 sq. ft. in a metal tray, and then irradiated for half an hour at a distance of 2 ft. with a BY Cooper Hewitt quartz mercury vapor lamp run at 4 amperes, 40 volts. It was stirred after 15 minutes. The irradiation was not carried out every day; a week’s or even a month’s supply was prepared at a time because the activation induced is a very stable property.

The members of one litter were kept together in a large cage (2’ X 2’ X 1:‘) on wire screens during the preparatory period, but in separate cages during the test period. They were weighed individually once a week for the first 3 weeks, then 2 or 3 times per week. Careful examination for the onset of ophthalmia or re- spiratory disturbances was made daily and it was found that such trouble was definitely established generally within 5 weeks of the beginning of the experiment and within 3 or 4 days of the appear- ance of the first signs. It is significant that all the members of any one litter generally developed ophthalmia within a very few days of each other.

Vitamin A Content of Grains.

In illustration of this technique may be quoted some experi- ments carried out with various grains as the source of vitamin A. Recent experiments on the vitamin A content of yellow corn seeds and seedlings had suggested that yellow corn contained more vitamin A than had previously been suspected. In order to con- firm this, one rat from a litter showing definite signs of depletion of vitamin A was given one yellow corn seed per day, two others were given two yellow corn seeds, while two others were given four yellow corn seeds. The rat on one seed recovered completely and grew for a time but eventually declined in weight and died (Chart I). The rats on two seeds also recovered and grew for a longer period of time but eventually died also; while those on four seeds grew to maturity though eventually they too developed respiratory trouble. The experiment was terminated before they died. Reproduction was not attempted. Consumption records showing what percentage of the total food intake the corn seeds constituted in the later stages of the experiment are shown in Table I.

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H. Steenbock and K. H. Coward 771

A further experiment was carried out a little later than the above in order to determine what percentage of the total food intake the corn formed during the early weeks of the experiment. Three rats from one litter were given one yellow corn seed each per day

Gms.

200

1.60

I_ l2C

8c 1.

4( );

CHART I. All the rats in this experiment were from the same litter and all gave a response to yellow corn seed additions. In Rat 34, receiving one yellow corn seed daily, the eye symptoms improved temporarily but became worse later and the rat was never cured. In Rats 31 and 33, each receiving two yellow corn seeds daily, the eye symptoms were cleared up quickly, and growth was resumed for a considerable period of time. The rats did not reach maturity, however, and eventually died of some form of respiratory trouble. In Rats 32 and 35, each receiving four yellow corn seeds daily, the eye symptoms cleared up quickly, growth was more nearly normal than in the rats on two corn seeds, and duration of life was longer.

and consumption records were taken daily (Chart II). For a week’s ration for each rat, seven seeds were chosen weighing al- together 2.5 gm., and any one of the seven seeds was given to the rat daily. This constituted some 5 per cent of the total food in- take at the beginning of the experiment but a smaller percentage

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172 Fat-Soluble Vitamins. XXVII

as the intake increased with the growth of the rat (Table II). The effectiveness of the low dosage of yellow corn in curing ophthalmia and, in the rather higher dosage, promoting normal growth when added to a basal ration made definitely antirachitic by irradiation, indicates that yellow corn is comparatively rich in vitamin A; and the fact that previously 85 per cent had been

TABLE I.

Food Intake of Rats A 31, SS, 32, 35 (Chart I) during the i&h to 18th Weeks of the Test Period.

Rat No. Average body

weight for period.

Yellow corn seeds

gm. gm. gm.

A 31 196 232 25 7.5 I‘ 33 155 258 25 9.7 “ 32 213 375 50 13.2 “ 35 216 352 50 13.0

Gms.

160

40

&ART II. All the rats in this experiment were from the same litter. Ophthalmia was cured and growth was promoted temporarily in each case. In Rat 37, ophthalmia and the first attack of respiratory trouble were cured about the same time.

found necessary in a diet which was almost certainly not antirachi- tic (15), shows that yellow corn is remarkably poor in the antira- chitic factor and that its previous failure to promote growth was due to a deficiency of the latter factor rather than of vitamin A. A similar vitamin content of hog millet has been recorded by Steenbock, Nelson, and Black (12).

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H. Steenbock and K. H. Coward 773

It was considered desirable at this point to make a comparison between yellow and white corn on the new basal diet; that is, a definitely antirachitic one. One test was made using one yellow corn seed to four white ones; another was made using four yellow corn seeds to four white ones. The dosage of four white corn seeds was in each case totally inadequate to indicate even traces of vitamin A, while the results obtained with one or four yellow corn seeds confirmed the previous ones (Chart III). The yellow and white seeds were of approximately equal size and weight.

TABLE II.

Food Intake of Rats A 37, 58, 36 (Chart II) during Each Mod of ftk Test Period, Compiled fl .om Daily Consumption Records.

Rat No.

A 37

A 38

A 36

-

-.

-_

-.

-

Average body Total food weight. intake.

Qm.

99 145 161 140

95 135 160 130

103 112

om.

220 250 203 233

5 wks.

190 239 200

89 2 wks.

177 125

3 wks.

-7

c

-1-

--

Total weight of torn seeds eaten.

Qm.

10 10 10 12.5

10 5.3 10 4.2 10 5.0

5 5.6

10 7.5

Yellow corn seeds per cent of total

food intake.

4.5 4.0 5.0 5.4

5.6 6.0

An attempt was then made to discover whether the vitamin A was concentrated in the endosperm or in the embryo of the yellow corn seed. The embryo of one seed gave no indication of the pres- ence of vitamin A, nor did a daily dosage of sixteen embryos, a weight equal to that of the endosperm of an average seed. The endosperm of one seed, however, indicated the presence of vitamin A. Hence it may be concluded that the vitamin is largely, if not entirely, concentrated in the endosperm of the yellow cornseed (Chart IV). This confirms previous unpublished experiments.

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774 Fat-Soluble Vitamins. XXVII

Wheat embryo has been reported almost from the time of begin- ning of vitamin research (16) as containing small though definite amounts of vitamin A, and it now appeared conceivable that in this case also, a lack of the antirachitic vitamin rather than a lack of vitamin A might be responsible for this conclusion. Wheat embryo (a representative commercial sample obtained from and selected by one of our largest milling companies as being of excel- lent quality) was tested by adding it to the irradiated basal diet to

CHART III. Rats 4555 to 4560 were from one litter, Rats 4750 to 4755 from another. There was no improvement in the condition of the rats on four white corn seeds. The rats on one yellow corn seed (except Rat 4755) recovered quickly and even resumed growth. Those on four yellow corn seeds recovered quickly also and grew more steadily and quickly than those on only one.

t,he extent of (a) 10 per cent, and (6) 20 per cent of the ration. 10 per cent furnished some indication of the presence of vitamin A, while 20 per cent gave much more definite evidence, one rat grow- ing on this amount remarkably well (Chart V). But it must be remembered when evaluating the nutritive properties of the entire wheat kernel that embryo constitutes about 1.5 per cent of the dry weight of a whole wheat grain (17) and that, therefore, 20 per cent of this sample (which admittedly was far from being pure embryo)

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H. Steenbock and K. H. Coward 775

would represent an amount of whole grain equivalent to more than 13 times the entire ration. Actually the amount was far less than this because the sample of germ was not pure.

A further comparison was made between whole grains of wheat, oats, and white corn using our basal synthetic ration as a control.

CHART IV. Rats 53 to 58 were from one litter, Rats 47 to 52 from another There was definite though uneven improvement in the general condition of the rats on the whole yellow corn seed and of those on the corn endosperm, except in Rat 50 which was probably in too bad a condition for the experi- ment when the change in diet was made. There was no improvement at all in the rats on the embryo only, whether one embryo was fed or sixteen embryos. In Rat 52, the ophthalmia was never cured, though the rat’s duration of life was much prolonged.

Each of the grains was ground up in a mill, made up into a diet similar to the basal synthetic ration, substituting 68 per cent of the ground grain for the 68 per cent dextrin. The diet was ir- radiated as needed. To eliminate differences due to variations in the appetite of the rats, the possible consumption was limited to 5 gm. per rat per day during the 1st week. As even this small

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776 Fat-Soluble Vitamins. XXVII

amount was not eaten by all the rats, ih was later decreased to 4 gm. during the 2nd and succeeding weeks. By the end of the 3rd week, only 2 rats had not eaten in toto as much as the others, and these were on the basal synthetic control ration. To elimi- nate individual differences as much as possible there were used four rats, one from each of four litters divided into four comparable groups (Chart VI). They were 23 to 27 days old and weighed 41 to 49 gm.

All four rats on the basal synthetic ration developed ophthalmia permanently in 6 to 63 weeks; those on the wheat diet showed some

CHART V. All the rats were from the same litter. Rats 4577 and 4573, receiving 10 per cent wheat embryo, show some evidence of the presence of vitamin A; Rats 4576 and 4578, receiving 20 per cent wheat embryo, gave more definite evidence of t,he presence of vitamin A.

signs of it in 6 weeks, but Rats 195 and 196 recovered during the 7th and 8th weeks to succumb again shortly afterwards. Those on the oats diet developed ophthalmia in 54 to 6+ weeks-two of these dying within the next few days of some respiratory trouble and a third in 10 days with slight hemorrhage of the stomach. On the white corn diet, 3 developed ophthalmia in 6 to 6$ weeks (one of these dying in the next 10 days) ; while one rat had not developed ophthalmia at the end of 8 weeks.

Apparently all these grains are very poor in vitamin A, but of the three, wheat appears to Fe the richest; however, for con- clusive evaluation further experiments need to be performed.

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H. Steenbock and K. H. Coward 777

The data as at present available are presented primarily to indi- cate the applicability of the technique to the study of these relations.

80

80

80

CHART VI. Rats 190, 194, 198, 202 were from one litter, Rats 191, 195, 199, 203 from another, Rats 192, 196, 200, 204 from another, and Rats 193, 197, 201, 205 from still another. ,411 the rats on the basal synthetic diet developed ophthalmia permanently in 6 to 6% weeks; those on the wheat diet developed faint signs of it at the same time but a remission occurred in the case of Rats 195 and 196 in the 7th and 8th weeks for about a week in each case; those on the oats diet developed ophthalmia in 6 weeks and three died in 6 to 7 weeks; all those except one on the white corn diet de- veloped ophthalmia in 6 to 7 weeks, two of them dying in 8 weeks.

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778 Fat-Soluble Vitamins. XXVII

SUMMARY.

A method of testing substances for vitamin A is described in which the antirachitic factor is supplied by irradiation of all or part of the basal diet.

Excellent results were obtained by this method but in view of the desirability of feeding synthetic diets of highly purified known constituents the advantages of feeding irradiated cholesterol in place of irradiating the ration have been pointed out.

The use of the incidence of ophthalmia is advocated as a sign of exhaustion of the animal’s store of vitamin A in preference to cessation of growth. The two are often simultaneous, but the use of the former criterion prevents loss of animals through the very sudden and rapid decline that may ensue while one is waiting to become certain that growth has really ceased.

Growth ceases during the worst stages of ophthalmia and is only resumed when definite improvement in the animal’s condition is observable.

Yellow corn seeds were used to demonstrate the technique described and it is to be noted that they appear to be richer in vitamin A than was previously supposed, but presumably they are poor in the antirachitic factor. Vitamin A is located in the endosperm rat,her than in the embryo. Commercial wheat germ when fed as 20 per cent of the diet furnishes definite evidence of the presence of vitamin A.

Whole wheat, white corn, and oats were all found poor in vita- min A, but of the three wheat appeared to contain the most. Whether these relations hold true generally remains to be established.

BIBLIOGRAPHY.

1. Steenbock, H., and Nelson, E. M., J. Biol. Chem., 1923, lvi, 355. 2. Hume, E. M., Lancet, 1922, ii, 1318. 3. Goldblatt, H., and Soames, K. M., Lancet, 1922, ii, 1321. 4. Goldblatt, H., and Soames, K. M., Biochem. J., 1923, xvii, 446. 5. Steenbock, H., and Black, A., J. Biol. Chem., 1924, lxi, 405. 6. Steenbock, H., Black, A., and Nelson, M. T., Science, 1924, lx, 530. 7. Steenbock, H., Black, A., Nelson, E. M., Nelson, M. T., and Hoppert,

C. A., J. Biol. Chem., 1925, lxiii, p. xxv. 8. Hess, A. F., and Weinstock, M., Am. J. Dis. Child., 1924, xxviii, 517.

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H. Steenbock and K. H. Coward 779

9. Hess, A. F., and Weinstock, M., J. BioZ. Chem., 1925, lxiii, p. xxv. 10. Drummond, J. C., Rosenheim, O., and Coward, K. H., J. Sot. Chem.

Id., 1925, xliv, 123. 11. Drummond, J. C., Coward, K. H., and Handy, J., Biochem.J., 1925, xix,

1068. 12. Steenbock, H., Nelson, M. T., and Black, A., J. Biol. Chem., 1924-25,

Ixii, 275. 13. Unpublished data. 14. Hart, E. B., Steenbock, H., Kletzien, S. TV., and Scott, H., J. Biol.

Chem., 1927, lxxi, 271. 15. Steenbock, H., and Boutwell, P. W., J. Biol. Chem., 1920, xli, 81. 16. McCollum, E. V., and Davis, M., J. Biol. Chem., 1915, xxi, 179. 17. Osborne, T. B., and Mendel, L. B., J. BioZ. Chem., 1919, xxxvii, 557.

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H. Steenbock and Katharine H. CowardVITAMIN A

QUANTITATIVE DETERMINATION OF FAT-SOLUBLE VITAMINS: XXVII. THE

1927, 72:765-779.J. Biol. Chem. 

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