Frank A. Csonka

The Demethoxylation of Lignin in the Animal Body.

We recently reported 1 that lignin when fed to a dog or cow causes a substantial increase in the quantity of benzoic acid eliminated in the urine. Since this fact is at variance with results published by others, we undertook to obtain more direct evidence bearing on the metabolism of lignin. The OCH3, or methoxyl group, has been definitely shown to be present in lignin. The Kirpal and Bühn 2 modification of the Zeisel method was employed by us for the quantitative estimation of the methoxyl group. Knowing the methoxyl content of the lignin and also that of the feces before and after the feeding of lignin, it was possible to follow up the demethoxylation in the animal organism. Experiments conducted with a dog showed that over 15% of the methoxyl initially present in the lignin was liberated and, in the case of the cow, 37% loss of methoxyl was observed. We believe that in these experiments we have direct evidence that lignin is metabolized in the animal organism. We have also conducted experiments in vitro in which known quantities of lignin and fresh material taken from the 4 compartments of the cows stomach were incubated at 38° C. for 8 days. It was found that demethoxylation took place in all our experiments, irrespective of whether or not toluene had been added to the medium. Experiments conducted in a similar manner, using fresh material taken from the large and small intestine of a cow gave entirely negative results. It would appear then that the demethoxylation of the lignin takes place in the stomach of the animal and that this is not brought about by bacteria, but rather by some other agent, possibly in the nature of an enzyme which is present in the gastric juice of the animal. In connection with our animal experiments, we observed that when lignin was fed in larger doses than 2.0 gm. per kilo weight, toxic symptoms were developed. In 2 instances we found increased amounts of non-protein nitrogen in the blood, pointing to an impaired renal function.

Function of egg white in embryonic development.

In an attempt to decide whether or not the yolk or the egg white is the carrier of a growth factor transmitted through the egg from the hens feed to the chick, the experimental technic described here was devised. In the past it has been assumed that egg white has a purely nutritional function in the growth and development of the chick embryo, but no experimental evidence exists to support this supposition. The yolk also furnishes food, but in addition the yolk-sac serves as an organ for the digestion and assimilation of food by the chick embryo. This function of the yolk-sac indicates a structural relationship between the embryo and the yolk. In this experiment fertile hens eggs from crosses of Rhode Island Reds and White Leghorns were used. A circular window approximately 1/4 inch in diameter was bored in the small end of each egg and the egg-white was removed into a glass ground-joint wash bottle by suction. To combat bacterial infection 0.1 cc of an aqueous solution containing 3000 I.U. of penicillin and 3.3 mg of streptomycin was injected into each of the eggs so treated. The egg white from another fertile egg was then transferred by air pressure from the wash bottle into the egg from which the egg white had previously been removed. When one egg white was not sufficient to completely refill the shell, that from another fertile egg was added. Thus, at times egg white from more than 1 egg was used for replacement in a single egg. After the egg was completely filled with egg white, the window in the shell was closed with a strip of scotch tape and the egg placed into an incubator for 3 weeks. At intervals of 7 days, the condition and development of the chick embryo was observed by candling the eggs. In the first successful series, 12 such eggs were prepared; of these 3 chicks hatched which were normal in every respect. Of the rest of the embryos, two lived 2 days, one 4, two 6, one 7, one 13, one 15 and one 21 days. These are probably the first live chicks produced after such drastic treatment. This experiment demonstrates that the chief function of the white of the hen egg is mainly to furnish food for the developing embryo. It has been previously held that the two chalazae serve to orient the embryo in a proper position for hatching by keeping the yolk more or less fixed. In these experiments, however, the two chalazae were removed with the egg white and thus the position of the developing embryo depended upon gravity. Further work is in progress on the nutrition of the chick embryo employing this technic.

Varieties of protein as factors in hippuric acid production.

Parker and Lusk were the first to point out the importance of the total N and hippuric acid ratio. Wiechowsky and Magnus-Levy observed a higher ratio than 4.7, which exists when the glycine in the body protein is utilized in the formation of hippuric acid, and they interpreted this increased ratio to a synthetic formation of glycine. There is no doubt that glycine is synthetized in the animal body. The new-born baby, for instance, lives on practically a glycine-free diet and yet increases its weight, and the newly developed tissue contains four per cent glycine which is not supplied by the food. On the other hand, if glycine synthesis plays an all-important rôle in hippuric acid formation when benzoic acid is administered, then we should not find any difference in the hippuric acid elimination when the protein supplied contains preformed glycine. The toxic effect noted after large quantities of benzoic acid are fed should occur always when a certain dosage is administered, that is, after the rate of glycine synthesis has reached its maximum and the amount produced is insufficient to counteract the toxic effect of benzoic acid. Our results do not confirm these theoretical arguments. The hippuric acid elimination when casein, together with large quantities of benzoic acid, is given to a pig is practically identical with that when benzoate is given during starvation. Under like conditions the administration of meat protein results in a small increase in hippuric acid formation, while administration of gelatin, with its content of 25 per cent of glycine, causes a marked increase in the output of the substance.

Proteins of the cotton seed.

By extracting finely ground cottonseed kernels (hull-free) with benzene (C6H6) nearly all of the fatty and resinous substances and much of the coloring material is eliminated. For this purpose benzene is far superior to ether. Such a thorough removal of the above substances from the flour greatly facilitates a satisfactory subsequent extraction of the proteins by different solvents. The high percentage of nitrogen extracted by sodium chloride solution (Table I) is doubtless due to the method of preparation of the flour. We were able to separate from the salt extract two globulins. Of these one can be precipitated directly at 0.4 to 0.5 of saturation with ammonium sulfate. The other flocculates at a saturation of 0.7 to 0.8, but only after it is diluted with water so that the ratio of the final volume to the weight of the original cottonseed flour extracted is as 50:1. A fraction having a relatively low nitrogen content, but containing a constant and very high ash percentage was obtained from the salt extract by coagulation; the composition of this preparation points to a nucleic acid. The examination of this product is under way and the results will be published later. The globulins were prepared by reprecipitation with ammonium sulfate followed by dialysis. The identification of glutelin and a nucleo protein present in the meal, and also the chemical analyses and determination of certain physical constants of the different protein fractions are under way.