THE NUTRITIVE PROPERTIES OF KAFIRIN.

THE NUTRITIVE PROPERTIES OF KAFIRIN. BY ALISXT G. I1C)GAN. (From the Llepartmd of Chemistry, Kansas Stnte Ag~ic~cltuml E xperiment Station: Jlanhattan.) (Reeeivcd for publication, November 22, 1917.) Agriculturists have known for years t,hnt the sorghum grains are peculiarly adapted to regions of light or insufficient rainfall, and for that reason they have urged that larger nrcas in the semiarid regions be devoted to the culture of kafir, fcterita, and mile. In view of the drouth-resist,ing properties of t,hese grains, and their increasing economic importance, it seemed desirable to investightc more closely their nutritive wtlue. Such knowledge as we have of the individual proteins of kafir is Iargcly due t,o Johns and Brewster (1916). These authors found that most of the nitrogenous matter of knfir, about 67 per cent of the total protein, was soluble in alcohol, ltnd they coined the term X-~~firz% for this particular protein. They made a careful chemical examination of the material, and for convenience, some of their results are included below. Analysis of an average of thirteen samples of k&in gave: C, 55.19 per cent; II, 7.36; N, 16.44; S, 0.60; 0, 20.41. The authors also determined the percentage of diamino-acids in knfirin by the Van Slyltc method, with the following results: arginine, 1.58 per cent; lysine, 0.90; histidine, 1.00; and tryptophane, present. Because kafirin forms such a large proportion of the protein of kafir, it,s nutritive properties were studied in some detail. A b:~~l rat.ion was prepared in which, for all practical purposes, 1 12 per c:cnt 15 per cent kafirin. katirin. g,n elm. Protein...._... 126 157 Phein-free milk.. 257 257 Butter.. 300 300 Starch... 1:. I ::....:::::: ::.::.:.: 295 265 Agnr... 20 20 151

152 Kafirin kafirin formed the sole source of protein supply. In all cases this diet resulted in nutritive failure (Chart l), and accordingly kafirin was supplemented with other deficient proteins, gliadin and gelatin, in the hope t.hat some clue might be obtained to the amino-acid deficiencies of kafirin. These proteins were added t,o the basal rat.ion, in the proportion of 100 gm. supplement to each 1,000 gm. of the ration. The addition of gliadin, for a short time at least, barely sufficed to maintain the live weight of the animals; but the addition of gelatin enabled them to grow slowly. Bot,h proteins were then added to the basal ration (Chart 2) in the proportion of 50 gm. of each to 1,000 gm. of t.he rat,ion, with the result that apparently the animals grew still more rapidly than they did when gelatin alone was added. The data are illustrated graphically in Charts 1 and 2. It is evident, taking the period as a whole, that the rats receiving both protein supplements grew more rapidly than the rat,s receiving only gelatin. Isolated periods could be selected in which the animals of this latter lot grew as rapidly as the others, but probably no especial significance should be attached to that point,. Two or three obvious explanations for the behavior of t.hese rats could be advanced, but the theory chosen tentatively was that there are at least, two limiting factors in kafirin as a source of protein. Gelatin supplies one, and gliadin the other. The most conspicuous advantage of gelatin over gliadin as a source of a,mino-acid supply is its much larger percentage of lysine. In other respects, however, gelatin is decidedly deficient, as it lacks tyrosine, cystine, and 6ryptophane. Gliadin, on the other hand, is deficient in lysine, but contains the other three amino-acids in fairly satisfactory proportions. It seemed logical therefore to assume that lysine is the first limiting factor in kafirin, and that the second is one of the three other amino-acids mentioned, tyrosine, cystmine, or tryptophane. Some of the earlier work concerning the rble of tryptophane in nutrition has been summarized in an earlier paper (1917) from this laboratory. The important role that cystine plays in nutrition has been demonstrated by Osborne and Mendel (1915). Their data show that when casein is the only protein in the ration cystine under certain conditions becomes a limiting factor. According to-mathews (1915) casein contains 0.8 per cent of sulfur,

A. G. Hogan 153 while according to Johns and Brewster (1916) kafirin contains only 0.6 per cent of that element. It is an assumption to calculate that all the sulfur of these proteins is cystine sulfur, but the data make it seem possible that the amount of oystine in kafirin is well below the optimum. The statement is frequently encountered in the literature that tyrosine is an essential amino-acid. Totani (1916) believes that tyrosine is not necessarily essential, and apparently the facts in the case have not been finally established. In an effort to investigate the deficiencies of kafirin more definitely, the amino-acids2 mentioned were added directly to the basal rations. These additions were made singly or collectively as follows: lysine, cystine, lysine plus cystine, lysine plus tyrosine, lysine plus tryptophane. The lot receiving the basal ration plus lysine grew slowly, in accordance with our expectations. Evidently lysine (Chart 2) is the first limiting factor in kafirin. Accordingly, in our other feeding trials (Chart 3), lysine was always made a part of the basal ration. The further addition of tryptophane to the diet did not increase the rate of growth, and a similar addition of tyrosine (Chart 4) was equally ineffective. When cystine was superimposed on this ration, however, the animals grew somewhat more rapidly. Small variations might seem insufficient to establish this point, but the unanimity of the results seems conclusive. Furthermore, the marked acceleration in growth that follows the addition of cystine after it has been withheld for some time seems even more conclusive. Accordingly, our interpretation of the data is that cystine is the second limiting factor in kafirin. Charts 3 and 4 give the data in greater detail. 2 In some cases the tyrosine used was a Kahlbaum preparation, in others it was prepared by the author. The cystine, trypt,ophane, and lysine were prepared in this laborat,ory. The amount of lysine added varied from 3 to 6 per cent of the kafirin in the ration, but the larger amount seemed no more effective than the smaller. The cystine formed 5 per cent of the kafirin, and enough tyrosine was added to equal 4 per cent of the protein.

Kafirin The Lysirle Requirement of Immature Animals. The data bring out one fact of general application; namely, that lysine is indispensable, even for the maintenance of young animals. This point has been in doubt for some time because of the difficulty in proving that a diet contains no lysine. Gliadin was formerly thought to lack that particular amino-acid, and because of that belief, earlier experimental work on the nutritive properties of gliadin was assumed to have special significance. Osborne and Mendel (1914) stated that rats maintained their live weight unchanged, but failed to grow when gliadin formed the sole source of protein in the ration. Their interpretation was that lysine is necessary for growth, but is not necessary for maintenance. When somewhat later the presence of lysine in gliadin was established, they (1916) recognized the element of doubt attached to their earlier pronouncement. The behavior of animals receiving their protein in the form of zein has also been interpreted as an indication that lysine is not required for the maintenance of nutritive equilibrium. Willcock and Hopkins (1906) found that mice receiving zein as the sole source of protein died in less than 2 weeks. Those receiving zein and tryptophane were active after 16 days of zein feeding, but had lost weight. Wheeler (1913) obtained similar results, but even after fortifying the zein with tryptophane the animals constantly lost weight. Similar results are recorded by Osborne and Mendel (1914) in their series of experiments. Their animals receiving a zein plus tryptophane diet were at a nutritive standstill or lost weight, though they grew rather rapidly on the further tum.itian of lysim. The precise status of lysine in nutrition remained uncertain, however, because of the difficulty in estimating or even detecting small quantities of that amino-acid. Recently Osborne, Van Slyke, and collaborators (1915) published data indicating no inconsiderable quantity of lysine in gliadin. The method of Van Slyke yielded an average result of 1.33 per cent, and the direct method of Kossel and Kutscher resulted in the recovery of 0.64 per cent of the gliadin as lysine. There is no doubt, therefore, that gliadin contains approximately 1 per cent of lysine, while kafirin, according to Johns and Brewster (1916),

A. G. Hogan 155 contains 0.90 per cent. On the other hand, such evidence as is available points strongly to the complete absence of lysine from zein. Osborne and Leavenworth (1913) found no trace. Furthermore, according to a theory advanced by Van Slyke and B&hard (1914), proteins containing lysine possess free amino groups that react with nitrous acid; but zein gives no evidence of such a reaction. Gliadin, on the contrary, according to their data yields 1.10 per cent of the total nitrogen as free amino nitrogen. Although this evidence may be questioned, yet such weight as it has is all against the presence of lysine in the zein complex. If the facts deduced from zein feeding contradict our findings while feeding kafirin, we have no explanation for the discrepancy. SUMMARY. Lysine is the first limiting factor in kafirin and cystine is the second. Lysine is indispensable for the maintenance of young animals. BIBLIOGRAPHY. Hogan, A. G., J. Biol. Chem., 1917, xxix, 485. Johns, C. O., and Brewster, J. F., J. Biol. Chtm., 1916-17, xxviii, 59. Mathews, A. P., Physiological Chemistry, New York, 1915, 110. Osborne, T. B., and Leavenworth, C. S., J. Biol. Chem., 1913, xiv, 481. Osborne, T. B., and Mendel, L. B., J. Biol. Chern., 1914, xvii, 325. Osborne and Mendel, J. Biol. Chem., 1915, xx, 351. Osborne and &Iendel, J. Biol. Chem., 191G, xxv, 1. Osborne, T. B., Van Slyke, D. D., Leavenworth, C. S., and Vinograd, M., J. Biol. Chem., 1915, xxii, 259. Totani, G., Biochem. J., 1916, x, 382. Van Slyke, D. D., and Birchard, F. J., J. Biol. Chew, 1913-14, xvi, 539. Wheeler, R., J. Exp. Zool., 1913, xv, 209. Willcock, E. G., and Hopkins, F. G., J. Physiol., 1906-07, xxxv, 88.

30 1 30 i3 CHART 1. One lot of animals received 12 per cent, anot.her 15 per cent kafirin. The inadequacy of this protein is indicated by the behavior of these rats. They lost weight and manifested the usual symptoms of malnutrition, such as diseased eyes, roughened coat, and muscular weakness. Rat 234 died at the point indicated by X. The lot receiving cystine as a supplement is included here inasmuch as these animals had obviously not been affected in any way by that addition to the ration. $4

CHART 2. All animals received the same basal ration, 15 per cent, kafirin. (10 per cent, of the entire ration) permitted fairly rapid growth. The addition of gelatin as a supplement Gliadin in equal quantity was much less eflective. One rat receiving this adjuvant grew very slowly, the other died at the point, indicated by X. When the gelatin and gliadiu diets were combined in equal proportions and fed to anot,her group of rats, growth occurred more rapidly than on the larger quantity of gelat in given alone. These farts see&d t,o indicate that lysine is the first limiting factor in kafirin, and when this ammo-acid (5 per cent of the protein) was added to,the basal ration, that inadequate diet was converted into one permitting a slow rate of growth. II,,,,,,., 8, 8,,,,,,,

Kafirin CHART 3. All these animals received a ration containing 15 per cent kafirin, and lysine. Three of them, Rats 259, 260, and 261, rcccived in addition tryptophane and cystine at the point shown by a. At point b, the cystine feeding was discontinued and thereafter these rats received as amino-acid supplements only lysine and tryptophane. The other three animals, Rats 219, 233, and 265, received cystine in addition to the lysine at all times. The effects of cystine feeding are not marked, but the lot receiving this aminoacid made a distinctly greater gain than the other. The males normally grow faster than the females, yet Rat 260 on the tryptophane ration, a male, grew somewhat more slowly than cit.her of the females receiving the cystine diet. For a short time, the tryptophnne lot received cystine, and it seems significant that in this brief period they should exhibit such a marked increase in the rate of growth. If the cystine feeding had been omitted, it seems probable that these rats would have made a still more unfavorable showing as compared with the cystine lot.

A. G. Hogan 159 CHART 4. The data shown in this chart are not complete, as the investigation was unavoidably interrupted. In spite of the fact that the period of these feeding trials was short, however, we believe that it was sufficiently long, at least when considered in relation to Chart 3, to admit of a final conclusion. Rats 259 and 261 had been receiving 15 per cent kafirin and lysine? plus tryptophane,? and Rat 265 had been on a diet of 15 per cent kafirin plus lysine plus cystine.2 At the point indicated by a all were changed to a ration of 15 per cent kafirin plus lysine plus tyrosine.* The rate of growth of the animals was apparently not affected, certainly it was not increased. At point b tyrosine was replaced by cystine, and the rate of gain was increased in all ewes. In two instances it was marked.

THE NUTRITIVE PROPERTIES OF KAFIRIN Albert G. Hogan J. Biol. Chem. 1918, 33:151-159. Access the most updated version of this article at http://www.jbc.org/content/33/1/151.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 0 references, 0 of which can be accessed free at http://www.jbc.org/content/33/1/151.citation.full.ht ml#ref-list-1