Lois F. Hallman

A comparison of the ketolytic effect of succinic acid with glucose.

Korányi and Szent-Györgyi 1 have reported recently that succinic acid given as the calcium salt in daily amounts of 5 to 10 gm. or less is able to decrease the ketosis in diabetics. In the present investigations we have compared the ketolytic action of succinic acid with that of glucose when administered twice daily to fasting male rats weighing approximately 200 gm., in which the endogenous ketonuria was induced by the previous administration of a high butter-fat diet low in protein, as reported earlier. 2 The studies on ketonuria were made on the second to fifth fast days inclusive during which sodium chloride solution (fasting controls), sodium succinate in doses from 9.95 to 149.2 mg. per 100 gm. rat daily, or glucose in equivalent amounts (7.55 to 113.2 mg. per 100 gm. rat) was fed. Urine collections were made daily. The ketonuria of the fasting controls usually rises to a maximum on the third fast day, after which it diminishes rapidly. In Table I a summary of the data is given. No ketolytic effect obtained when as much as 149.2 mg. of succinic acid per 100 gm. rat was given although glucose at a comparable level gave a marked decrease in the acetonuria. This amount of succinic acid on a surface area basis is 50% greater than the largest dose employed by Korányi and Szent-Györgyi. As slight decrease in ketonuria appears when only 7.55 mg. of glucose is given while a dose of 37.8 mg. caused an average decrease of acetone-bodies in the urine from 33.7 mg. per 100 gm. rat in the control tests to 19.4 mg. There was no evidence of diarrhea in any experiments nor of other toxic effects ascribable to the succinic acid.

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.

Deviation from the "Beer Law" in the Kuttner-Cohen Method for Determination of Phosphorus.

Recently, when determinations of inorganic phosphate in mixtures containing sodium glycerophosphate (used as a substrate in plasma phosphatase determinations) were required, the use of heat involved in the Benedict-Theis method rendered it obviously unsuitable. The many advantages of the Kuttner-Cohen method, 1 with the modifications later suggested by Kuttner and Lichtenstein 2 and Raymond and Levene, 3 recommended themselves to us. We found this method capable of yielding very accurate results (within ±2% of the known quantities), after correction for the considerable deviations from the “Beer law”. These deviations were determined by a large number of analyses, charts were constructed and tables compiled for more convenient reading. We use 1 cc. of plasma when it is expected to contain 5 mg. of phosphorus per 100 cc. or more, and 2 cc. of plasma when it is expected to contain less. Ten cc. of water are added and 5 cc. of 10% trichloracetic acid. About 14 cc. of filtrate are available. Four cc. of the acid-molybdate mixture is added to an aliquot diluted to 5 cc, the tube is mixed by tapping, and 1 cc. of stannous chloride solution∗ is added. Several standards of different concentrations may be employed in a series of tests. We found it convenient to employ a single standard containing 0.02 mg. per 5 cc. We usually include in one series about 18 tests, triplicate 0.02 mg. standards, and, as a check upon all foreseen and unforeseen sources of error, several solutions of known concentration covering approximately the same range as the unknowns. After calculation of the results the correction is applied.

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.