Joseph S. Butts

Glycogen Formation After Alanine Administration in Adrenalectomized Animals.

According to Evans, 1 the metabolism of endogenous protein is interfered with in the adrenalectomized, rat. In our present studies the metabolism of an orally administered amino acid, alanine, has been investigated in animals with the adrenal gland removed. Butts 2 has shown that this amino acid is converted to glycogen in the organism, the maximum storage being 8 hours after the oral administration of the acid. To 7 normal male and a like number of normal female rats 1.6 mg. alanine per square centimeter of body surface, was administered by stomach tube. A like amount was given to the paired brothers and sisters of the above animals which had been bilaterally adrenalectomized 6 days previously and had received salt solution since. All animals were fasted 24 hours before the alanine was fed. Three animals of each sex and of each group were given an amount of water by stomach tube equivalent in volume to the alanine solution fed in the other group. In the groups of normal and adrenalectomized males, blood sugars were determined on blood obtained from the tails just before alanine administration and at the periods after administration shown in the table. At the end of 8 hours the livers of all animals were removed under amytal anesthesia and glycogen determined in the organ. A summary of the results is given in Table I. Apparently adrenalectomy does interfere with the conversion of alanine to glycogen. The blood sugar changes are also less marked. Not only this but all of the adrenalectomized animals showed a drop in the sugar level after the peak which was reached one hour after alanine administration, while the normal rat showed a high level throughout the 4-hour period.

Effect of Diet on Ketonuria in the Rat

It was noted 1 that the fasting ketonuria in the rat rarely exceedel 2 mg. per day over periods of several days. More recently, instances of higher values in the excretion of acetone bodies in fasting rats fed sodium chloride solution have become increasingly frequent, so that at least 80% give higher results. Thus, the average values of acetone bodies for fasting male rats were 7.7 mg. (0.0–45.3) for 34 rats on the first fast day and 12.3 mg. (0.6–27.2) for 11 animals on the second one. The corresponding averages on 19 female animals fasted for 3 consecutive days were respectively 2.8 mg. (0.0–18.8), 10.4 mg. (0.0–33.0), and 9.6 mg. (0.2–35.9). Moreover, it was found that although the same sex difference in the excretion of ketone bodies followed the administration of diacetic acid as was previously observed, the levels in our recent experiments during 5 days of fasting were consistently higher than those previously obtained. The only variation in experimental regime from that earlier employed to which these alterations could be ascribed, was a change in the stock diet. Because of the outbreak of pellagra in our colony, our previous high carbohydrate diet (II) had been fortified in Vitamin G several months earlier by the inclusion of 5% of desiccated liver therein (Diet III). It was found that the fasting ketonuria observed over periods of several days in rats previously on Diet III was gradually reduced to a low level when the animals were returned to Diet II (liver-free) for 60 days. The original level of fasting ketonuria of 4.9 (0.0–17.7) for 9 rats on the first fast day, 17.6 mg. (2.7–29.2) for 8 animals on the second day, and 15.1 mg. (3.5–30.3) for 5 rats on the third one was reduced after 60 days on Diet II to a mean of 0.9 mg. (0.6–1.2) for 9 rats on the first and 1.7 mg. (0.5–3.1) for the same animals on the second fast day.

Sexual variation in carbohydrate metabolism. 7. Effect of alkalosis on fasting ketonuria in the rat.

Booher and Killian 1 first reported that abnormally large amounts of acetone bodies were associated in human subjects with conditions of uncompensated alkalosis which arose either from an excessive alkali administration or from the loss of HCl by excessive vomiting. Butts and Deuel 2 previously showed that no sex differences occurred in the slight ketonuria which occurs in fasting rats previously fed on a high carbohydrate liver-free diet although a definite sex variability was demonstrated in rats to which diacetic acid was administered. In the present tests sodium bicarbonate was fed to fasting rats in a dose of 2.17 mg. per sq. cm. of body surface by stomach tube in 3 divided doses daily. This corresponds with the alkali intake in tests in which sodium acetoacetate was administered in an amount of 1.5 mg. per sq. cm. per day (calculated as acetone). The acetone body excretion with the male rats over a 4-day experimental period averaged 5.3, 8.6, 5.8, and 6.1 mg. per day for 14 rats on the first day and 11 animals on the other days which corresponds with an excretion of 0.14, 0.24, 0.16, and 0.17 gm. per sq. meter of body surface respectively. The females developed an appreciable ketonuria in distinction to the almost blank values on the males. Thus, the acetone body excretion in the urine gave a mean of 21.3, 39.6, 39.2, and 37.4 mg. of acetone of 14 rats on the first day and 11 on each of 3 following days. The values calculated on the basis of grams per square meter of body surface per day were 0.77, 1.39, 1.39, and 1.32 gm. respectively. A normal response to diacetic acid administration was demonstrated on a control day. The experiments show the greater susceptibility of the female rat to alkalosis than the male.

Metabolism of Ethyl Esters of Fatty Acids

It has been demonstrated 1 that the conversion of caproic, butyric, and beta-hydroxy butyric acids to acetone bodies in fasting rats by beta oxidation is quantitative, whereas greater amounts of ketone bodies originate after sodium caprylate than after isomolecular quantities of sodium acetoacetate are fed. The latter phenomenon suggests that delta oxidation occurs in the latter case. No ketone bodies are formed when the sodium salts of the fatty acids with an odd carbon chain, as propionic, valeric, heptoic or nonylic acids were fed. It was later demonstrated 2 that the conversion of the odd chain fatty acids into glycogen must represent an approximately quantitative transformation by beta oxidation into propionic acid. The fatty acids with an even number of carbon atoms were entirely ineffective as glycogen formers. However, it was impossible to feed the soaps of the fatty acids having a greater number of carbon atoms than 9 in an equimolecular dose to that found effective with the shorter chain fatty acids because of the relatively large quantities of solution which must be administered. In the present experiments, by administering the fatty acids as their ethyl esters it has been possible to feed isomolecular quantities of all of the fatty acids up to stearic acid in similar doses to those employed in the earlier work—namely, 15 gm. per square meter of body surface per day, calculated as acetone. The average excretion of acetone bodies in fasting male rats, second to fourth days, calculated as acetone in grams per square meter of body surface per day after feeding the ethyl esters was as follows: acetoacetate, 2.01 (57)∗; butyrate, 1.37 (27); caproate, 1.95 (31); caprylate, 7.29 (12); caprate, 6.74 (16); laurate, 6.75 (17); myristate, 5.21 (9); palmitate, 2.94 (10); stearate, 2.40 (11); oleate, 5.07 (11). This indicates that the excretion of acetone bodies after the caproate and butyrate is an approximately quantitative one, whereas the acetonuria after the administration of the ethyl esters of the fatty acids with 8 or more carbon atoms is greater than that of the acetoacetate controls. This would indicate that more than one acetoacetate residue originates from the oxidation of one molecule of fatty acid having 8 or more carbon atoms.

Glycogen formation after various fatty acids.

The transformation of the even-chained fatty acids into the acetone bodies has been demonstrated to be a quantitative one. 1 It was shown that such odd-chained fatty acids as propionic, valeric and heptoic did not give rise to appreciable amounts of the acetone bodies. With the exception of the experiments of Ringer 2 on phlorhizinized dogs and the negative results of Eckstein 3 on glycogen formation in the white rat, no experimental evidence is on record regarding the glycogenic ability of the various fatty acids. In the present tests the sodium salts of propionic, diacetic, butyric, valeric, caproic, heptoic, caprylic and nonylic acids were fed, by stomach tube, to rats previously fasted for 48 hours in doses equivalent to 1 mg. (calculated as acetone) per sq. cm. of body surface. In series 1 the rats were killed 6 hours after the administration of fatty acids: in series 2, seven hours after such administration. The average results on liver glycogen in series 1 are as follows: Control, (10 animals) 0.27% (Range 0.11–0.71); Propionic (9) 1.36% (0.21–1.86); Valeric (10) 0.69% (0.51–1.06); Diacetic (10) 0.18% (0.12–0.22); Butyric (9) 0.30% (0.20–0.46); and Caproic (10) 0.16% (0.09–0.22). In series 2 the results were as follows: Control (9) 0.18% (0.07–0.35); Valeric (10) 0.64% (0.27–1.16); Heptoic (10) 1.02% (0.63–1.54); Nonylic (10) 0.83% (0.43–1.50); Caprylic (10) 0.25% (0.13–0.45). It is apparent that the odd-chained fatty acids, valeric, heptoic and nonylic, give rise to approximately the same amount of glycogen in the liver as propionic acid. This indicates that the process of beta-oxidation of the odd-chained fatty acids is fairly quantitative. On the other hand, the even-chained fatty acids such as diacetic, butyric, caproic and caprylic are unable to form appreciable amounts of glycogen.

Relation of Anterior Pituitary to Sexual Differences in Ketosis in the Rat

Burn and Ling 1 have reported that the administration of an alkaline extract of the anterior pituitary gland to female rats fed on a butter fat diet brought about a marked rise in the level of ketonuria. The experiments on males were uniformly negative. In order to determine whether the sexual differences in ketonuria reported in the previous paper 2 might be traced directly or indirectly to this keto-genetic hormone, further experiments under varying conditions seemed advisable. The experimental procedure was similar to that used in the previous work. 2 The extracts of anterior pituitary were made from fresh glands of steers according to the procedure of Burn and Ling. 1 The administration of this extract to normal or castrated male or female rats which were receiving diacetic acid resulted in a marked rise in the level of acetone bodies in the urine over that of the controls. The response was as prompt and as great in male as in female rats and in the castrated as the normal animals. No glycosuria was observed. The following is the number of experiments which were made on the anterior pituitary extract and all were strongly positive; normal males, 9; castrate males, 14; normal females, 11; oophorectomized females, 14. When the anterior pituitary extract was injected in normal male and female rats which were fasted but to which 10% NaCl was administered instead of diacetic acid, the excretion of acetone bodies in the urine was promptly increased from an average of about 1 mg. per day to 30-65 mg. No sexual difference in response was noted. Boiled extract was inactive as was the filtrate after an alcoholic precipitation of the active extract. Negative results were likewise obtained when an active extiract was fed by stomach tube instead of being injected subcutaneously. It seems that the greater susceptibility of the female to ketosis may be associated with a larger production of the ketogenetic substance in the anterior lobe as the result of the stimulation of some substance produced in the ovary other than theelin.