W. W. Cravens

Crotalaria spectabilis toxicity in chickens.

Summary Concentrations of 0.05% to 5% Crotalaria spectabilis seed were fed to chicks. All concentrations of the legume were shown to be toxic. Concentrations in excess of 0.3% produced death in all birds within 18 days. When less than 0.2% was fed there was a marked reduction in weight gain. In birds which died, hemorrhage was frequently observed in liver, lungs and the pericardium. Atrophy of liver and ascites were constantly encountered in test birds after death.

Reversal of aminopterin inhibition in the chick embryo with desoxyribosides.

Summary Aminopterin, injected prior to, or early in, the incubation period, is highly toxic to the chick embryo. Ten micrograms or more of this compound produce 100% mortality; 100 μg or more per egg completely inhibit embryonic development. The inhibitor becomes relatively less toxic as embryonic development progresses; at nine days or more, twenty micrograms is non-toxic. The same amount injected at three days results in almost immediate embryonic death. Thymidine partially counteracted the inhibitory effects of aminopterin. A combination of hypoxanthine desoxyriboside and thymidine was more effective than thymidine alone. In the absence of thymidine, hypoxanthine desoxyriboside was without effect. An enzymatic digest of desoxyribonucleic acid also counteracted aminopterin inhibition partially. Thymine, hypoxanthine, folic acid, vitamin B12, and concentrates of the Leuconostoc citrovorum factor, alone and in various combinations, were ineffective in counteracting aminopterin inhibition. These experiments indicate that aminopterin acts in animal tissues in part by preventing synthesis of thymidine and one or more of the purine desoxyribosides, and that these compounds are immediately essential for embryonic growth. Since aminopterin is thought to act by creating a deficiency in folic acid, the necessity for folic acid in synthesis of these compounds in animal tissues is implied.

Activity of Vitamin B12 in the Growth of Chicks.

Summary Vitamin B12 administered orally or parenterally completely counteracted a thyrotoxic condition in chicks produced by feeding a basal ration containing 0.05% iodinated casein. A level of 0.75 γ vitamin B12 per 100 g of ration resulted in a half maximal growth response. At a level of 1.5 γ vitamin B12 per 100 g of ration the growth response after a two week test period was 88 g compared to 86 g for a group supplemented with 3% condensed fish solubles. A response of 72 g resulted from the intramuscular injection of 0.1 γ vitamin B12 per bird per day. Pure vitamin B12 can replace the animal protein factor activity of condensed fish Solubles and injectable liver preparations.

Reversal of Aminopterin Inhibition in the Chick Embryo with the Leuconostoc citrovorum Factor.

Summary Concentrates of the Leuconostoc citrovorum factor (CF) and of “folinic acid” partially counteract the inhibitory action of aminopterin for the chick embryo. Folic acid and formylfolic acid were ineffective or only slightly effective under the same conditions. The magnitude of the effects obtained with CF were similar to those obtained previously with mixtures of thymidine and hypoxanthine desoxyriboside 1). The viewpoint is developed that conversion of folic acid to CF is a necessary preliminary to the catalytic action of the former compound, that synthesis of thymidine and one or more of the purine desoxyribosides are among the synthetic reactions for which CF or folic acid) is required, and that these synthetic reactions are those inhibited in the chick embryo by small amounts of aminopterin. The great variation that exists in the ability of CF to counteract aminopterin inhibition in various organisms is pointed out.

Growth-Promotion by Lyxoflavin. II. Relationship to Riboflavin in Bacteria and Chicks.

Summary 1. L-lyxoflavin, D-galactoflavin and isoriboflavin markedly increase the growth response of Lactobacillus casei to suboptimal amounts of riboflavin, but do not promote growth in the absence of this vitamin. Appropriate differential assays indicated that lyxoflavin was not deposited in cells of L. casei under conditions where it stimulated growth, and actually decreased the concentration of riboflavin deposited in such cells. Thus it does not serve as a partial substitute for riboflavin in L. casei, but appears to enhance the efficiency with which limited supplies of riboflavin are utilized. When riboflavin is present in excess, lyxoflavin does not increase the cell yield or the growth-rate. At higher concentrations, lyxoflavin and galactoflavin inhibit growth by acting as competitive antagonists of riboflavin. Dichlororiboflavin and isoriboflavin are ineffective as inhibitors of this organism. 2. L-lyxoflavin promotes maximum growth of Lactobacillus lactis in the absence of riboflavin, and such growth continues indefinitely upon subculture. Differential assay with L. casei and L. lactis showed that cells of the latter organism grown with lyxoflavin contain no riboflavin, but do contain lyxoflavin; i.e., in this organism lyxoflavin can fill the essential metabolic roles normally played by riboflavin. Similar assays of tissues from rats grown on natural rations indicate that lyxoflavin does not occur naturally in significant quantities. 3. In chicks, lyxoflavin stimulated growth when fed at levels 2.5 to 10 times the amount of riboflavin in the diet. At ratios of lyxoflavin to riboflavin higher than 40 to 1. lyxoflavin inhibited growth. Amounts of lyxoflavin that inhibited growth on diets of low riboflavin content were non-toxic when the amount of riboflavin was increased. 4. Although the growth-responses observed in chicks when L-lyxoflavin is added to diets low in riboflavin may result in part from a sparing action similar to that found with L. casei, such a sparing action does not explain the growth effects of the analog observed in rations of high riboflavin content.

Effects of Desoxypyridoxine and Vitamin B6 on Development of the Chick Embryo.

Summary Desoxypyridoxine (1000 fig) injected into eggs prior to the start of the incubation period resulted in 100% mortality of the embryos; this inhibition was prevented by simultaneous injection of any of the three forms of vitamin B6. The ratio of vitamin to inhibitor which permitted approximately 50% of the embryos to produce live chicks was 1/20 for pyridoxal, 1/50 for pyridoxamine and 1/100 for pyridoxine. When injected after 4 or more days of incubation, 1000 μg of desoxypyridoxine proved non-toxic. Higher levels injected at 6 days of incubation proved toxic but this toxicity could not be prevented by any of the 3 forms of vitamin B6. A possible explanation for this observed variation in toxicity of desoxypyridoxine at different stages of incubation is offered. Pyridoxal hydrochloride and pyridoxine hydrochloride also proved toxic at high levels while pyridoxamine dihydro-chloride was not toxic at the highest level tested. Neither nicotinic acid nor its amide affected the inhibitory action of desoxypyridoxine; negative results were also obtained when DL-alanine was injected alone or with nicotinic acid. These experiments demonstrate that vitamin B6 becomes essential for embryonic growth very early in the incubation period.

The effect of folic acid on glycine toxicity in the chick

Abstract Studies were conducted to determine the effect of folic acid and vitamin B 12 on glycine toxicity. Folic acid was found effective in reversing the toxic effects of glycine. Folic acid determinations on the livers of chicks receiving glycine indicate an increased storage of the vitamin. The total creatinine content of blood, muscle, and liver is higher in folic acid-deficient birds. The possible significance of these results is discussed.

Effect of Nucleic Acid Derivatives in the Reversal of Aminopterin Inhibition in the Chick Embryo.

Summary Several purine bases and related nucleosides were tested for their effectiveness in potentiating the reversal by thymidine of aminopterin inhibition of the chick embryo. Of the free purine bases, hypoxan-thine was most active; adenine and guanine were also active, while xanthine was inactive. Of the nucleosides tested, only inosine was active. Inosine was more active than hypo-xanthine; together with thymidine it completely overcame the effects of the low level of aminopterin employed. The results are interpreted to mean that aminopterin prevents embryonic growth by preventing synthesis of compounds essential for formation of nucleic acids; by supplying these substances preformed, growth occurs even though amino-pterin is present. It follows that thymidine, and the active purine bases and derivatives listed above, are utilizable for nucleic acid synthesis by the chick embryo.

Growth-promoting activity of L-lyxoflavin.

Summary L-lyxoflavin stimulates growth of chicks under these conditions. Its mode of action remains obscure. Since it has no riboflavin activity, and its growth-promoting action occurs on a ration rich in riboflavin, it is quite possible that it acts as a true vitaminlike entity. The possibility that it acts in the same manner as the known antibiotics is unlikely, since the ration contained aureomycin.

Relation Between Induced Hyperthyroidism and an Unidentified Chick Growth Factor.

Summary A thyrotoxic condition in the chick, induced by feeding desiccated thyroid or iodinated casein was effectively counteracted either by supplementing the diet with condensed fish solubles or by the injection of reticulogen. An increased growth response was obtained upon the addition of either desiccated thyroid or iodinated casein to a ration containing adequate amounts of the known and unidentified chick growth factors. The addition of either 0.125% desiccated thyroid or 0.02 to .03% iodinated casein to the basal ration resulted in an improved assay range for the unidentified chick growth factor (s) in condensed fish solubles and reticu-logen.