The experiments summarized in Table 1 were designed to compare the effect of leucine isomers on the reversal of norleucine toxicity. Weanling albino rats of the Yale strain which were previously fed on a nitrogen-free diet for a 2-week period were used in this study. Following this period the animals were fed an amino-acid diet containing various levels of L- and D-leucine in the conjunction of 2 per cent DL-norleucine, as outlined in Table 1. The composition of the basal diet and the amino-acid mixture have been described elsewhere2. It can be seen from Table 1 that both enantiomorphs of leucine are antagonized by norleucine, but it is also apparent from the results that the antagonism is greater in the case of D-leucine. Although the exact quantitative relationship was not determined, it appears that at least twice as much of D-leucine is required to reverse norleucine toxicity when compared with the L-isomer. This is in good agreement with the previous observation that L-leucine is approximately twice as effective as the D-leucine for growth of young rats2. Note the close correlation between water consumption and growth. Although norleucine does not seem to be a natural constituent of proteins3 it is readily metabolized by mammals4-7. When incubated with rat liver homo-genate it gives rise to oc-ketocaproic acid, valeric acid and p-hydroxyvaleric acid. On the basis of such results, Kinnory et al.8 concluded that norleucine is catabolized by conversion to the corresponding oc-keto-acid, followed by oxidative decarboxylation and the subsequent oxidation to the resulting mono-carboxylic acid by (3-oxidation.In order to learn more about the growth-depressing effect of norleucine an additional study was undertaken in which two of the above-mentioned intermediates of norleucine catabolism (a-ketocaproic and valeric acids) and two other compounds closely related to norleucine (caproic and e-aminocaproic acids) were compared with norleucine for their effect on growth of rats. Since the growth-retarding effect of norleucine was previously demonstrated with the diet containing L-leucine at a level corresponding to 50 per cent of the requirement, that is, 0 -43 per cent of the diet, it was decided to use this amount of L-leucine in our control diet. The compounds under test were added to the diet at molar equivalent levels corresponding to 1 per cent of norleucine. Normal weanling male albino rats of the Sprague-Dawley strain were used for assaying the above compounds. Each experiment covered 2 weeks. The experimental results which are shown in Table 2 demonstrate that of all the substances tested only norleucine and a-ketocaproic acid retarded the growth of rats. The slower rate of growth of animals was accompanied by lower consumption of food and decreased food efficiency. The inhibitory effect of the keto-acid is not unexpected since the first major step in the catabolism of most amino-acids, that is, formation of keto-acid, is readily reversible9. Since the keto-acid was less inhibitory than norleucine, while valeric acid was without any demonstrable effect, it can be concluded that the deleterious effect manifested by norleucine is due to the intact molecule of the latter rather than to some of its catabolic products. Once the amino-acid undergoes oxidative decarboxylation it loses its growth-depressing effect. Since neither caproic nor s-aminocaproic acids affected the growth of animals it is apparent that the amino- or oxo-group must be present at the a-carbon position in order to produce a growth depression in rats. Table 1. AVERAGE GROWTH RESPONSE OF FOUR PROTEIN-DEPLETED WEANLING RATS TO L- AND D-LETJCINE IN THE PRESENCE OF DL-NORLEUCINESupplement to leucine-free diet Days Initial weight (gm.) Final weight (gni.) Change in weight (gm./day) ]Water intake j (ml. /day) I0 -85 per cent D-leucine +2-0 per cent DL-norleucine 1 -70 per cent D-leucine +2-0 per cent DL-norleucine 0 -85 per cent D-leucine +2-0 per cent DL-norleucine 2 -20 per cent D-leucine +2-0 per cent DL-norleucine 0 -43 per cent L-leucine +2-0 per cent DL-norleucine 0 -43 per cent L-leucine 0-43 per cent L-leucine + 2-0 per cent DL-norleucine 0 -85 per cent L-leucine + 2-0 per cent DL-norleucine 0-43 per cent L-leucine +2-0 per cent DL-norleucine 12 5 6 43 6 3 9 40 39 44 42 50 51 62 58 69 39 44 42 50 51 62 58 69 67 -0-08 0-95 -0-30 1-90 0-15 3-55 -0-60 3-65 -0-20 5-6 ! 7-8 ! 6-3 9-2 8-7 i 12-3 ! 10 -5 14-3 10-7 j I Table 2. EFFECT OF NORLEUCINE AND RELATED COMPOUNDS ON THE GROWTH OF WEANLING RATS FED DIETS Low IN LEUCINESupplement to leucine-free diet No. of rats Initial weight (gm.) Average weight gain (W) (gm.) Average food intake (JF)(gm.) WIF 0 -43 per cent L-leucine 0 -43 per cent L-leucine + 1-0 per cent DL-norleucine 0 -43 per cent L-leucine + 2-0 per cent DL-norleucine 0 -43 per cent L-leucine + 0 -99 per cent a-ketocaproic acid 0 -43 per cent L-leucine + 0 -78 per cent valeric acid 0 -43 per cent L-leucine + 0 -89 per cent caproic acid 0 -43 per cent L-leucine + 1 -00 per cent e-amino caproic acid 4 4 4 4 4 4 4 57 57 57 57 57 57 57 16-0 5-3 2-0 8-5 16-0 19-3 15-3 71-5 57-5 62-0 56-0 61-5 73-5 69-0 0-22 0-09 0-03 0-15 0-26 0-26 0-22