THE AVAILABILITY OF DL-LANTHIONINE FOR THE PROMOTION OF GROWTH IN YOUNG RATS

In an earlier communication (1) it WBS reported that the internally compensated thio ether amino acid, mesolanthionine, is not utilized for the growth of young rats subsisting on a cystineand methionine-deficient diet. Two alternative explanations were given for this failure of the animals to utilize mesolanthionine, either that the animals were unable to split the thio ether or that cleavage did occur in such a way that the unutilizable D-cysteine (2) was formed instead of n-cysteine. In order to develop information on this question, feeding experiments have now been conducted with nn-lanthionine. By feeding the racemic form under the same conditions by which the mesolanthionine was fed, it was believed that evidence could be obtained to show which of the above alternative explanations for the failure of mesolanthionine to support growth is correct. I f the L component of the nn-lanthionine were split, one-fourth of the nn-lanthionine would supply n-cysteine irrespective of what side of the sulfur in the molecule cleavage occurred. The results of the experiments here reported show that DL-lanthionine supports growth to the extent that the L component is cleaved to yield 1 molecule of L-cysteine. nn-Lanthionine usually accompanies mesolanthionine in acid hydrolysates of alkali-treated proteins (3). The DL-lanthionine used in these experiments was obtained from acid hydrolysates of human hair that had been previously boiled for 1 hour with 2 per cent Na&03 solution.1

(Received for publication, May 12, 1948) In an earlier communication (1) it WBS reported that the internally compensated thio ether amino acid, mesolanthionine, is not utilized for the growth of young rats subsisting on a cystine-and methionine-deficient diet. Two alternative explanations were given for this failure of the animals to utilize mesolanthionine, either that the animals were unable to split the thio ether or that cleavage did occur in such a way that the unutilizable D-cysteine (2) was formed instead of n-cysteine. In order to develop information on this question, feeding experiments have now been conducted with nn-lanthionine. By feeding the racemic form under the same conditions by which the mesolanthionine was fed, it was believed that evidence could be obtained to show which of the above alternative explanations for the failure of mesolanthionine to support growth is correct. If the L component of the nn-lanthionine were split, one-fourth of the nn-lanthionine would supply n-cysteine irrespective of what side of the sulfur in the molecule cleavage occurred. The results of the experiments here reported show that DL-lanthionine supports growth to the extent that the L component is cleaved to yield 1 molecule of L-cysteine.
nn-Lanthionine usually accompanies mesolanthionine in acid hydrolysates of alkali-treated proteins (3). The DL-lanthionine used in these experiments was obtained from acid hydrolysates of human hair that had been previously boiled for 1 hour with 2 per cent Na&03 solution.1 EXPERIMENTAL In the previous investigation (1) on the nutritional availability of mesolanthionine, to supply the B vitamins a cystine and methionine basal diet having the following percentage composition was satisfactorily used: casein 6.0, dextrin 37.0, sucrose 15, salt mixture 4 (4), agar 2.0, lard 19, cod liver oil 5.0, and a commercial milk-vitamin concentrate 12. When fed this diet the animals invariably lost weight.
Since the milk-vitamin concentrate previously used was no longer available, a simpler basal diet containing synthetic vitamins and choline was first employed.
It was found, however, that with 6 per cent casein in the diet, as previously used with the milk-vitamin concentrate, the animals grew too fast to make it possible to detect the effect of adding the amino acid supplements.
By omitt,ing choline and reducing the level of casein to 4.21 per cent, a satisfactory basal diet was obtained. When fed t.his diet, the animals declined rapidly in weight.
Addition of the sulfur arnino acids promptly arrested the decline in weight, and growth proceeded at a satisfactory rate. Salmon (5) has pointed out that the primary deficiency of casein at low levels is in labile methyl groups. This is in accord with the decline in weight of our rats when choline was omitt,ed from the diet. However, this diet with a low content of fat, devoid of choline, and containing 4.21 per cent of casein, supported growth at a satisfactory rate after it had been supplemented with cystine as well as with choline, methionine, or nL-lanthionine.
Unless the cod liver oil or corn oil used in the diet contained an appreciable amount of choline, the methionine in the casein may have supplied enough methyl groups to be adequate for growth when cystine was added.
The data presented represent the results obtained with eleven lots of albino rats.
In addition to these, several lots were used in preliminary work on developing a satisfactory basal diet,. Each lot consisted of four or six animals from the same litter, equally divided with respect to sex and having initial weights of 50 to 60 gm. They were housed in individual cages having wide mesh screen bottoms and kept in a room maintained at about 24.4". The animals were weighed twice weekly.
After considerable experimentation a basal diet having the following percentage composition was found satisfactory: casein (Labco) 4.21, dextrirized corn-starch 90.79, salt mixture 2.00 (4), cod liver oil 2.00, snd corn oil 1.00. R vitamin mixture was incorporated in the dextrinized corn-starch which provided the following constituents in each 100 gm. of diet: thiamine hydrochloride 0.2 mg., pyridoxine hydrochloride 0.2 mg., riboflavin 0.3 mg., calcium pantothenate 0.3 mg., and niacin 1 mg. These vitamins, in an aqueousalcoholic solution, were added to the dextrinized starch and the mixture was dried at 50" to the original weight of the starch.
The diet was fed ad libitum and a record of the food intake is given in Table I. It is of interest to note that in general the food consumption was definitely lower during the periods when the animals subsisted on the basal diet alone than when the amino acid supplements were incorporated.
The animals fed the basal diet alone invariably lost weight rapidly, and when the feeding was prolonged they died in about 45 days. For comparison with the behavior of the rats given nn-lanthionine, L-cystine and DL-methionine were also used. The addition of 0.3 gm. of cystine or 0.37 gm. of methionine to 100 gm. of the basal diet made it effective in promptly arresting the decline in weight, of the animals, and in enabling them to grow at a satisfactory rate. Likewise, equally prompt and effect,ive responses followed the addition of 1.04 gm. of DLJanthionine to the basal diet. The above-stated amounts of the three supplements added to the diet represent biologically available sulfur equivalents.
In six of the lots all of the animals were fed the basal diet from the start for periods of 15 to 25 days. Following the fore periods on the basal diet the animals were fed one of the supplements.
In most cases, one rat, of a lot was allowed to continue on the basal diet throughout the remainder of the feeding period to serve as a control.
In several lots the effect of the supplements was studied throughout successive periods on the same animal. Without exception, DL-lanthionine proved to be as effective as cystine or methionine in its capacity to promote growth at, the start, or to restore it after a period of decline on the basal diet, alone. The growth curves for representative animals from the various lots are given in Figs. 1 and 2.
These are typical of the others which are omitted to save space.

SUMMARY
Feeding experiments have shown that racemic lanthionine can replace cystine and methionine in the diet. Young albino rats fed a low protein (casein) basal diet deficient in cystine, but adequately suppIied with the non-protein dietary essential factors, declined in weight rapidly.
For comparison with the behavior of rats given racemic lanthionine, L-cystine and DL-methionine were also fed. The addition of 0.3 gm. of L-cystine or 0.37 gm. of DL-methionine per 100 gm. of basal diet caused immediate resumption of growth, The addition of m-lanthionine likewise resulted in corresponding growth resumption.
An immediate decline occurred when the lanthionine was omitted from the basal diet, and growth again was resumed when the lanthionine was supplied.
Previous experiments similarly conducted with mesolanthionine showed that this isomer is not utilized for growth. BIBLIOGRAPHY