Jap..7. Pharrnacol. 1, 9 (1951)

EFFECT OF THE SALTS OF MESO- ON ALLOXAN MELLITUS YOSHITO KOBAYASHI, SHIGERU OHASHI AND SETSUYA TAKEUCHI

Department of Pharmacology, Faculty of Medicine, University of Tokyo Received for publication April 10, 1951 Since the discovery of , various new compounds having hypoglycemic action other than the pancreatic hormone have been reported. In 1922 Collip (1) reported Glucokinin and in 1927 Allen (2) Myrtillin, both of which are extracted from plants or yeast, and have hypoglycemic action. Best and Scott (1923) (3) stated that there are substances in the thyroid gland, and muscles that act in much the same way as insulin. Synthalin, a derivative of guanidin is effective but not in practical use, because of its marked side-action. That a kind of fatty acid containing seventeen C-atoms is effective against diabetes mellitus was reported by Kahn (1923) (4), but there was little evidence to support it. In 1945 Nath (6) reported he had succeeded in decreasing the acetone bodies in the blood of diabetics by using amellin. In Japan, proof has been found that there are substances which have hypogly cemic action in seaweed, yeast and certain wild plants, but their chemical com position is totally unknown. In 1948 Kumagaya (5) reported he had discovered an acidic substance extracted from the Lathyrus Palustris L. var. Macranthus (white) Fernald and effective against diabetes mellitus. These findings so far reported lec, us to explore some unknown acidic com pounds probably contained in plants, which might be efficacious in producing a marked decrease in the blood sugar level. This report deals with the hypoglycemic effect produced by the salts of meso-oxalic acid on the rabbit and dog having alloxap diabetes mellitus (10-18).

METHODS Rabbit : White rabbits (2.5-3.0 kilograms body weight) were housed in separate cages and were maintained on a constant diet consisting of 300g of bean curd residue and 50-100g of carrots daily. After few weeks observations of general health and blood sugar determination were made on each animal , they were given intravenous injections of alloxan in doses of 100 mg per kilogram in 5% solution with distilled water. For 20-30 weeks following the injection of alloxan, the dia betic state of each rabbit-Was studied by determining the sugar and acetone bodies content in the blood before daily feeding , as well as the twenty-four hour volume and sugar and noil-protein nitrogen in it . Frequently diabetes induced by alloxan is completely cured spontaneously within few weeks , but this is very rare if the rabbit continues to show glycosuria for more than 4 weeks . 10 Y. KOBAYASHI, S. OHASHI & S. TAKEUCHI When it was found the rabbit was ready to be tested, calcium salt or sodium salt of meso-oxalic acid was administered by mouth, and the aforementioned examination made. Acetone-bodies in the blood were determined according to Ravin (7) (1936), blood-sugar according to Hagedorn-Jensen (8) (1923), non-proteinnitrogen in urine by Kjeldahl's method, sugar in urine by Benedict-Fujii's method (9) (1937). Dog: Generally young dogs (1-1.5 years) become afflicted with moderate dia betes mellitus when injected with 60-65 mg per kilogram of alloxan . The tendency for diabetes is more severe in the case of aged dogs. In view or the nature of the experiments, extreme caution was taken in giving the dogs moderate diabetes. Unlike the rabbit, the diabetes mellitus induced by alloxan in dogs is apt to deteriorate and become critical . It is very difficult to maintain any dog showing about 10 grams of daily urinary sugar for an extended period. Usually the symptoms grew worse after approximately 3 weeks, culminating in severe diabetes mellitus. In addition to the experiments with moderate and severe alloxan diabetic dogs, tests using depancreatized dogs were made. The dogs were kept and fed in cages specially constructed to collect all the urine and they were fed once a day on a diet consisting of rice (100-150 grams), fish (50-100 grams) and n:iso (soy bean paste) (10 grams) boiled in 300-500 grams of water with 2-3 grams of salt added. The urine and blood tests were made along the lines of the method outlined in the case of the rabbit. The drug was administered by mouth for several days running, with the interval varying between drug and feeding times. RESULTS Rabbit: To facilitate ascertaining the effect of the drugs, tests were conducted on two groups of rabbits the first with severe diabetes mellitus (total daily amount of urinary sugar over 5 grams, fasting blood sugar over 300mg,/100 cc), and the other with moderate diabetes mellitus (total daily amount of urinary sugar 2-5 grams, fasting blood sugar 100-200mg/100 cc). The following results were obtained : 1) Effect on moderate alloxan diabete?, mellitus in rabbits: When given 100 mg per kilogram of either sodium salt or calcium salt of meso oxalic acid twice a day, the state of the rabbits were apparently improved: the amount of sugar in the urine decreased or entirely disappeared from the very day the drug was given, the body weight increased, and the blood sugar content diminished in some cases. However, during this perid glycosuria occasionally appeared but only in a small amount, which can be stopped altogether if care is taken to give the drug just at the right moment after feeding. That is to say, the drug is first given 3 MESO-OXALIC ACID SALTS ON ALLOXAN DIABETES i1 hours before feeding and then 7-8 hours afterwards. When the drug was stopped, glycosuria reappeared from the self-some day and the body weight and blood sugar content returned to their respective levels before the drug was given (see Figs. I and 2, Tables I and II).

FIG. 1. Effect of meso-oxalic acid on moderate alloxan diabetes mellitus in rabbits

Fig. 2. Effect of meso-oxalic acid on moderate alloxan diabetes mellitus in rabbits 1.2 Y. KOBAYASHI, S. OHASHI & S. TAKEUC;HI

TABLE I. Blood sugar value and daily amount of sugar in urine of moderate alloxan diabetic rabbit receiving calcium salt of meso-oxalic acid

TABLE II. Blood-sugar value and daily amount of sugar in urine of moderate alloxan diabetic rabbit receiving calcium salt of meso-oxalic acid

2) Effect on severe alloxan diabetes mellitus in rabbits : After giving the same dose of either sodium or calcium salt of rneso-oxalic acid as in the former cases, the daily amount of sugar in urine decreased to one MESO-OXALIC ACID SALTS ON ALLOXAN DIABETES Y3 half or at times to as little as one third. Also there was a decline in the amount of non-protein nitrogen in the urine as well as an increase in body waight. Occa sionally the sugar level in the blood fell, the acetone bDdies decreased or vanished completely (see Figg. 3 and 4, Tables III and IV).

FIG. 3. Effect of meso-oxalic acid on severe alloxan diabetes mellitus in rabbits

FIG. 4. Effect of meso-oxalic acid on severe alloxan diabetes mellitus in rabbits 14 Y. KOBAYASHI, S. OHASHI & S. TAKEUCHI

TABLE III. Blood-sugar value and daily amount of sugar in urine of severe alloxan diabetic rabbit receiving calcium salt of meso-oxalic acid

TABLE IV. Experimental result of blood-sugar value, acetone-bodies in blood, daily amount of urine, amount of sugar and N P N in urine of severe alloxan diabetic rabbit given calcium salt of meso-oxalic acid MESO-OXALIC ACID SALTS ON ALLOXAN DIABETES 15

In these tests we made, approximately half of the severe diabetic experimental rabbits showed absolutely no reaction whatsoever to the drug. The results of the tests are shown in Table V :

From Table V, in the case of severe diabetic rabbits, the salts of m3so-oxalic acid caused the sugar content of the urine to decrease in 40% of the total number of rabbits subjected to the test, whereas they had no effect at all on the remaining 60%. In the case of moderate diabetic rabbits, there was reaction in all 20 cases tested. In 8 cases the urinary sugar vanished entirely, in 4 cases the amount decreased by 75%, and in the remaining 8 cases the rate of decrease was 50°0. Dog : 1) Effect of calcium salt of m3so-oxalic acid on moderate alloxan diabetes mellitus in dogs: Dogs showing 5 10 grams of sugar on a daily diet of 100 150 grams of rice were included in this category (moderate). Table VI. Fig. 5 illustrates a case of a dog showing about 5 grams of sugar in the urine on a daily diet of 150 grams of rice. In this case, the animal was given 50 mg per kilogram of calcium salt of m so-oxalic acid mixed with a small quantity of fish one hour before feeding time, whereupon the sugar amount in the urine decreased by half and no trace of sugar could be found on the following day. For two whole days after the drug was stopped, there was no increase in the amount of urine and no presence of sugar was found. However, from the third day on, the amount of urine began to increase once more with sugar gradually reappearing. As the blood sugar remained at the normal level, no change was noticed. The dog in Tab VII was given 50 mg per kilogram of the drug four hours 16 Y. KOBAYASHI, S. OHASHI & S. TAKEIJCH1 before feeding time. In this case, the sugar content of the urine decreased but did not disappear altogether. Usually, with mDderate diabetic dogs the sugar begins to reappear in the urine when the drug is stopped, but thereafter the sugar amount varies for a while.

FIG. 5. Effect of meso-oxalic acid on moderate alloxan diabetes mellitus in dogs

TABLE VI. Blood sugar value and daily amount of sugar in urine of moderate alloxan diabetic dog receiving calcium salt of meso-oxalic acid MESO-OXALIC ACID SALTS ON ALLOXAN DIABETES 17,

TABLE VII. Blood sugar value and daily amount of sugar and N P N in urine of moderate alloxan diabetic dog receiving calcium salt of meso-oxalic acid

2) Effect of calcium salt of meso-oxalic acid on severe alloxan diabetes mellitus in dogs: a) Despite administering of 50 mg per kilogram of calcium salt of meso-oxalic acid three times a day to dogs showing 30-50 grams of sugar on a daily diet of L00-150 grams of rice, there was no decrease in the sugar in the urine (see Table VIII). b) If the drug is given while the amount of urinary sugar is 'maintained at an approximately same level as that of moderate alloxan diabetic dogs by injecting insulin in proper doses daily after each feeding time, the sugar content of the urine decreases or totally disappears, and in some cases the non-protein nitrogen in the urine diminishes (see Tables IX and X). It must be noted, however, the effect of the drug on severe alloxan, diabetic dogs as in (b) is less than that on moderate diabetic dogs. 18 Y. KOBAYASHI, S. OHASHI & S. TAKEUCHI

TABLE VIII. Blood sugar value and daily amount of sugar and NPN in urine of severe alloxan diabetic dog receiving calcium salt of meso-oxalic acid

TABLE IX. Blood sugar value and daily amount of sugar in urine in severe alloxan diabetic dog given calcium salt of meso-oxalic acid in conjunction with doses of insulin MESO-OXALIC ACID SALTS ON AT,LOXAN DIABETES 19

TABLE X. Blood sugar value and daily amount of N P N in urine in severe alloxan diabetic dog given calcium salt of meso-oxalic acid in conjunction with doses of insulin

3) Effect of calcium salt of meso-oxalic acid on the depancreatized dog: As shown in Table XI, doses of 100-200 mg per kilogram of calcium salt of meso-oxalic acid given to depancreatized dogs injected with protamin-zinc insulin daily before feeding time to regulate the daily amount of urinary sugar at a constant level did not produce any drop in the sugar content of the urine.

TABLE XI. Blood sugar value and daily amount of sugar and N P N in urine of depancreatized dog receiving calcium salt of meso-oxalic acid 20 Y. KOBAYASHI, S. OHASHI & S. TAKEUCHI

DISCUSSION

Our experiments corroborated that when sodium and calcium salts of meso oxalic acid are given orally to alloxan diabetic rabbits and dogs, the urine volume falls, the urinary sugar either decreases or disappears entirely, the amount of NPN in the urine also diminishes, the blood sugar level falls, and generally the animals gain weight. And it was found that the potency of the effects is closely correlated with the interval between the administration of drug and feeding. In order to produce moderate diabetes mellitus, 100 mg per kilogram of alloxan were injected intravenously into grown-up rabbits and 125 mg per kilogram into young rabbits respectively, while in the case of dogs intravenous doses of 60-65 mg per kilogram were given. Whereas with rabbits, the sugar content of the urine is apt to remain con stant for quite a while and their condition does not grow worse, with dogs their diabetic condition gets worse in most cases, in the urine increases, the blood sugar level rises, and they finally succumb. It should be noted here that, taking the carbohydrate content of the food into consideration, rabbits eliminating 2-5 grams of sugar in the urine daily and dogs whose sugar content of the urine was 5 -10 grams were classified as mode rate diabetes mellitus, while animals showing more than the above amount were classified as severe diabetes mellitus. Comparing the effect of meso-oxalic acid on the dog and the rabbit as outlined above, a lesser quantity is needed to take effect for the dog than for the rabbit, and moreover the effect lasts longer. From the fact that the use of insulin in combination with meso-oxalic acid produced more marked effect, it is presumed this drug enhances the activity of insulin. However, the fact this drug used together with insulin in the tests with the depancreatized dog produced little effect leads us to believe this drug does not aid the activity of insulin itself. Furthermore, it is assumed the presence of is essential.

SUMMARY 1. When 100 mg per kilogram of sodium or calcium salt of meso-oxalic acid is given by mouth twice a day to the alloxan diabetic rabbit (3 hours before and 7 hours after feeding time) for several days running, the sugar in the urine either falls to less than one third or completely disappears. Body-weight, on the other hand, increases, MESO-OXALIC ACID SALTS ON ALLOXAN DIABETES 21

Administering the drug to the moderate diabetic dog in the amount of 50 mg per kilogram once a day (one hour prior to being fed) results in total disyppear ance of sugar in the urine. If the dru.J is stopped being given, in the case of the rabbit, sugar reappears from the esjf-same day, whereas in the case of the dog, it does not unti! a couple of days later. 2. The more serious the diab~Aes in both the rabbit and dog, the less the effect of the drug. However, in some cases of the severe alloxan diabetic rabbit there is a de crease or total disappearance of acetone bodies in the blood, a decrease in the sugar and NPN content of the urine, accompanied by a marked dryp in the blood sugar lvel and by gain in weight. 3. When the drug is given to severe alloxan diabetic dogs wnose daily sugar content of the urine is maintained at about 5 grams by injecting the proper dose of insulin, its effect comes into evidence and the sugar in the urine decreases or disappears entirely. 4. The salt of meso-oxalic acid have no effect whatsoevor on the depancreatized dog.

REFERENCES

1) COLLIP : Tr. Roy. Soc. Canada 17, 39 (1922) : J. Biol. Chem. 56, 513 (1-923); 57, 65 (1923). 2) ALLEN: J. A. M. A. 89, 1577 (1927). 3) BEST, SCOTT: Ibid. 81, 382 (1.23). 4) KAHN : Am. J. M. Sc. 166, 823 (1923). 5) KUMAGAYA: Sogo-igaku 3, 12 (1936). 6) NATH : Ann. Biochem. & F_xper. Med. 5,101 (1945). 7) RAVIN : J. Biol. Chem. 115, 511 (1936). 8) HAGEDORN.JENSENEN : Biochem. Ztschs. 135, 57 (1923). 9) BEDICT-FUJII: J. Biochem. 25, 237 (1937). 10) GOI DNER AND GOMORI : Endocrinology 33 ,297 (1943; 35, 241 (1944). 11) THOROGOOD AND Z IMMERMANN : Ibid. 57,191 (1935). 12) BAILEY, C. C. AND BAILEY, O. T.: J. A. M A. 122, 1165 (194'3). 13) KENDELL, MEYER, LEWIS AND VICTOR : Proc. Exper. Biol. & Med. 60, 190 (1945). 14) JACOBS : Ibid. 37, 407 (1937). 15) DUNN, SHEEHAN AND MCLETCHIE : Lancet 1, 484 (1943). 16) DUNN AND Me LETCHIE : Ibid. 2, 384 (1943). 17) WAISBREN : Proc. Soc. Exper. Biol. & Med. 67, 154 (1938). 18) ARCHIBALD : I. Biol. Chem. 158, 387 )1945).