lucki Kyoritsu Hospital, I waki Many attempts have be - Medicina ...

Tohoku J. exp. Med .., 1968, 96, 127-141
Studies on Anti-diabetic Effect of Sodium Oxaleacetate
Kiyohiko Yoshikawa
Department of Internal Medicine (Chief: K. Yosh-ikawa),
lucki Kyoritsu Hospital, I waki
Many attempts have been made to treat diabetes mellitus with oral medic
ation, but none of them seems to have been successful. It has been known that a
decoction of Eunymus alata sieb growing in Japan has a blood sugar lowering ac
tion. The effective component was isolated, crystallized, and identified as
oxaloacetic acid (OAA) by infrared spectral analysiṣ Stable salt of O AA (OAA
sodium) was prepared. The administration of OAA-sodium brought about lowering
of the blood sugar level in normal and alloxan diabetic animals. The same drug
was given to diabetic patients, and it was found effective in all of 10 of Type I and
in 6 of 11 of Type II diabetics according to Yamakawand Kurokawa's classifica
tion
Ȧ long-term administration of OAA-sodium induced hyperplasiand prolifera
tion of the islet cells of the pancreas in rats, but no mentionable changes were
found in the other organs.
It is strongly suspected that OAA-sodium stimulates insulin secretion through
some unknown mechanism.
The standard treatment for diabetic patients has been well established, and
especially both the importance of insulin and diet control are generally accepted.
However, the necessity of oral medication is strongly felt on acount of the hazards
of insulin injection and the obnoxiousness of diet control. Collip1 extracted
glucokinin from yeast and other plants, and Allen2 extracted myritillin from blue
berries, and John3 reported that jukalic leaves contained a certain effective sub
stance. All of these extracts are said to have an effect to lower blood sugar concen
tration. Kumagai et al.`s,' also investigated the blood sugar lowering effect of a
variety of Lathyrus paalustris, a native plant in Sakhalin island.
In my previous paper,' we reported the same effect of the decoction of
Eunymus alata sieb which is an ordinary shrub growing in the mountainous
districts of Japan. Further investigation has revealed that the effective component
in the decoction is oxaloacetic acid. The present report describes the experimental
and clinical studies of the effect of oxaloacetic acid on the diabetic condition.
MATERIALS AND METHODS
Preparation
A decoction was prepared by adding 200 ml of 2N HCl to 100 g of dried powder of
Eunymus alata sieb. Then the extract was evaporated in vacuo and further extraction was
Received for publication, May 28, 1968.
127

128 K. Yoshikawa
made with a Soxhlet's extractor using alcohol. The final extract thus obtained was tested
and proved to be effective in lowering the blood sugar in rabbits. After this procedure,
further extraction was made with ether and finally a crystalline substance was obtained.
Infrared spectral analysis demonstrated that the crystal was oxaloacetic acid. Since
oxaloacetic acid is unstable, its sodium salt was used in the animal experiment as well as in
the clinical investigation.
Measurement of O AA concentration
The principle of measurement of OAA concentration is based on the fact that OAA is an
acid and dissociates when anilin or Co2+ is added, while ketoglutaric acid, pyruvic acid
and others have no such reaction.
Method of analysis
1) Five ml of 10% aqueous solution of trichloroacetic acid was added to 2-3g of the
sample to remove proteinous components. After centrifugation the supernatant was
filtered. After washing with 5 ml of acid solution, the precipitate was filtered again.
Furthermore, the precipitate on filter paper was rinsed with a small quantity of water.
2) The filtrate and the washings were mixed together and the mixture was divided into
halves, each of which was poured onto a 50 ml funnel-shaped separator.
3) A 15% aqueous solution of CoC12.6H2O was added to each of the solutions at a
ratio of 1:5, and the mixture was left to stand for 30 minutes.
4) One ml of 2-4-dinitrophenyl hydrazine solution (0.1 g/2N HCl 100ml) was added
to the solution and the mixture was left to stand for 30 minutes.
5) Then both the solutions were treated according to the following procedures; 10 ml
of ethyl acetate was added to the solution and the mixture was shaken enough to make keto
acid, from which hydrazone and excess hydrazine were extracted. The water layer was
discarded and the ester layer was washed 2-3 times with water. Ten ml of Na2C03-NaHCO3
solution (50 g Na2CO3 anhydride and 5g NaHCO3 per liter) was added and the mixture was
shaken vigorously. Hydrazone alone diffused into the water phase. After the phase separa
tion, most of the ester layer was sucked away with a pipet. The water phase was
washed 3-4 times with 10 ml of carbon tetrachloride until the carbon tetrachloride phase
became colorless. Then the water phase was transferred into a 25 ml measuring flask,
and Na2CO3-NaHCO3 solution was added to make it accurately 25 ml. The mixture was
poured into a cell 1 cm in depth, and the optical density at 384 m,u was measured.
6) The difference in the reading between both mixtures was proportionate to the
quantity of oxaloacetic acid. (The mixture including Co2+ contained more oxaloacetic acid
than the other, and the optical density lowered, when no difference in the reading was noted
in the absence of oxaloacetic acid). Oxaloaeetic acid was determined on the basis of the
standard curve of oxaloacetic acid-hydrazine derivative.
Two hundred mg of sodium oxaloacetate, measured by the above procedures, were
given orally to two diabetic patients and a healthy subject. The values before administra
tion, at 30 minutes and at one hour after administration were obtained as follows:
Subject Before At 30 min At I hour
K.S. (diabetic) 0 0 3.1ƒÊg/100 ml
Y.O. (normal) 0 0 2.2ƒÈg/l00ml
M.Y. (diabetic) 0 0 1.5ƒÊg/100 ml
RESULTS
1) Insulin-like activity (ILA) of oxaloacetic acid
Cross-bred male Wistar rats 110-120 g in weight were fed. on high-carbo
hydrate diet consisting mainly of wheat for at least one week under the same condi
tion. After fasting for 24 hours, 3 rats were given a blow against the head, and

Anti-diabetic Effect of Sodium Oxaloacetate 120
killed by cutting both the carotid arteries. The adipose tissue of the epididymis
was quickly resected with minimal damage to the tissue. Here, care must be taken
to leave the thick root adjacent to the epididymis and the large blood vessels on the
side of the epididymis. The resected tissue was soaked in Krebs-Ringer bicarbonate
buffer (pH 7.4, 21°C), and after 15-20 minutes the tissue on the side of the peripheral
lobulation was divided into eighteen pieces with even thickness. The water on the
surface of these pieces was sucked up with filter paper and the wet tissue was weigh
ed on a torsion balance. Each piece was 80 to 90 mg in weight.
Two kinds of medium, KRG and KRBG, were prepared. The former con
sisted of Krebs-Ringer bicarbonate buffer (pH 7.4) and glucose at a concentration
of 200 mg/100 ml, and the latter was ten-fold dilution of serum in KRG solution.
Two ml of KRG solution was poured into flasks each and the same amount of KRBG
solution into other 3 flasks. Six pieces of the tissue were put into those 6 flasks.
In order to provide aerobic condition a gas mixture of 95% 02 and 5% CO2 was
filled in the flasks which were connected with the Warburg apparatus. Then,
the contents in. the flasks were incubated for 120 minutes at 37.5°C at a shak
ing frequency of 110/min. Glucose was measured before and after incubation
by Fujita-Iwatake's method. On the assumption that glucose mg/g wet tissue
weight is the amount of glucose taken up in the tissue, the difference in the
glucose uptake between KRG and KRBG was considered to be due to insulin-like
activity of the sample serum.
To examine the effect of oxaloacetic acid on d-glucose uptake, 0.2 nil of OAA
diluted with KRB buffer at a concentration of 1 mg/100 ml was added to 2 ml of
KRBG solution. After incubation, the glucose uptake was calculated for the
determination of OAA effect.
So far as 10 diabetic cases were concerned, the d-glucose uptake ranged gener
ally from 0.62 to 5.43 mg/100 ml/g in fasting stage and from 3.72 to 9.29 when OAA
was added to the serum (Table 1).
On the other hand, 15 normal subjects showed an increase in the d-glucose
uptake ranging from 0.27 to 12.36 mg/100 ml/g in fasting stage and from 0.67 to
11.57 mg/100 ml/g when OAA was added to the serum, but so far as ILA was con
cerned, 11 among 15 cases showed an increase, while the other 4 cases showed no
change (Table 2).
TABLE 1. Effect of OAA upon glucose uptake in diabetics (mg/100 ml/s)

130 . Yoshikawa K
TABLE 2. d-Glucose uptake in normal subjects (mg/100 ml/g)
Fig. 1. Effect of OAA upon glucose uptake (mg/100 ml/g).
Significant differences were observed between the two cases, i.e., when OAA
alone was added to the buffer, and when serum and OAA were added. In the
former case, there was no change in ILA. This suggests that OAA has no ILA
increasing effect by itself, but has an effect on the insulin-like activity in the
serum (Fig. 1).
2) Hypoglycemic effect in normal dogs
According to the method reported by Kobayashi,7 healthy adult dogs were
anesthetized by nembutal administration, and 480 ml of 2% OAA-Na solution
was infused into the pancreaticoduodenal artery with constant infusion technic at
a rate of 1 ml per minute. Repeated sampling of the blood was made and blood
sugar was measured. The blood sugar level lowered precipitously from the pre
infusion level of 92 mg/100 ml to 46 mg/100 ml in 6 hours and to 30 mg/100 ml in
8 hours. Control animals showed a pre-infusion value of 98 mg/100 ml on the
average, and 80 and 76 mg/100 ml respectively 6 and 8 hours after infusion of
saline only (Fig. 2).

Anti-diabetic Effect of Sodium Oxaloacetate 131
Fig. 2. Hypoglycemic effect of continued infusion of OAA-Na into the pancreatico
duodenalis in normal dogs.
3) Effect of intravenous administration of OAA-Na to rabbits
In 6 fasting healthy rabbits, OAA-Na was injected in a dose of 20 mg into the
auricular vein, and serial determinations of blood glucose were done
As shown in Table 1, normal rabbits fasting for 24 hours showed a decrease of
blood glucose at 8 hours of fasting ranging from 8 to 21% and averaging 12% of
the control level. In the rabbits treated with OAA, the rate of decrease was 24%
to 45%, averaging 33%. The difference between these two groups is statistically
significant. It is also noted that there were two types of the blood glucose curve,
one showing initial temporal increase followed by a rapid decrease and the other
showing a gradual decrease (Table 3).
TABLE 3. Hypoglycemic effect of intravenous administration of OAA-Na
4) Effect of OAA-Na administration in alloxan diabetic rabbits
Six rabbits treated with alloxan were fed on standard diet and urinary sugar
excretion was measured. When the daily urinary output of sugar reached a
constant level, OAA-Na was given orally in a dose of 200 mg per kg of body
weight. As shown in Table 2, significant decrease of blood sugar and urine sugar

132 K. Yoshikawa
TABLE 4. Effect of OAA-Na administration in alloxan diabetic rabbits
output as well as the increase of body weight was observed in all but one rabbit
(Table
4)_
5) Clinical trial of OAA-Na (Table 5)
As various factors are known to influence the status of diabetic patients , the
evaluation of any treatment will be difficult if these factors are unstable. There
fore, clinical trial of OAA-Na was made in patients who had been treated by diet
and insulin administration and their symptoms were stabilized. A dose from 200
mg to 1,000 mg per day in three divided doses was orally given after meal.
The criteria for the evaluation were as follows:
a) For Type I diabetics according to Yamakawa and Kurokawa's classifica
tion,' fasting blood sugar less than 120 nig/100 ml and 24-hour urinary output of
TABLE 5.
Clinical effect of OAA-Na

Anti-diabetic Effect of Sodium Oxaloacetate 133
sugar less than 10 g were considered to show an excellent effect of OAA-Na. For
Type II diabetics, fasting blood sugar less than 150 mg/ 100 nil and urinary output
of sugar less than 10 g/day were evaluated. Blood sugar was measured by Fujita
Iwatake's method.9
b) A considerable improvement below the criteria stated above was regarded
as being effective.
The result of the clinical trials is summarized in Table 5. Namely, 15 male
and 6 female diabetic patients, from 15 to 70 years of age, were given OAA-Na
orally, and the administration was remarkably effective in II patients, effective in
5 patients and ineffective in 5 patients, As shown in Table 6, all 10 patients of
Type I diabetes responded favorably to OAA-Na administration. On the other
hand, in cases of Type II it was remarkably effective in 2, effective in 5 and
ineffective in 5 cases. As shown in Table 7, it is noteworthy that the patients who
showed a remarkable effect were all over 40 years of age.
When the constitution of patients was divided into slim, normal and obese,
the administration of OAA-Na was most effective in the obese type as shown in
Table 8. The relationship between the effectiveness of OAA-Na administration and
the fasting blood sugar level before the administration is summarized in Table 9.
As naturally expected, the more severe the diabetic condition, the less effective the
administration of OAA-Na.
The patients were divided into three groups depending upon the duration of
the clinical course of the disease. As shown in Table 10, the administration of
on diabetics /oral administration)

134 K. Yoshikawa
TABLE 6. Clinical effect and diabetic types
TABLE 7. Clinical effect and age
TABLE 8.
Clinical effect and the fasting blood sugar
FBS: fasting blood sugar
TABLE 9. Body types and clinical effect

Anti-diabetic Effect of Sodium Oxaloaeetate 135
TABLE 10. Period of the disease and clinical effect
OAA-Na was remarkably effective in 8 of 9 cases under one year, 3 of 9 cases of 1 to
3 years, and none of 3 cases over 3 years. The administration of OAA-Na had no
influence on the liver function tests and the blood acetone or cholesterol level.
6) Results of administration of OAA-Nce in rats
To 40 rats of Wistar strain OAA-Na was given in an oral dose of 5 mg per day
by mouth for 40 days and the effect was examined. The pancreas of these rats
demonstrated proliferation and hyperplasia of the islet cells (Figs. 3 and 4). In
another 40 rats of the same strain given OAA-Na in an oral dose of 10 mg per day
for four months, the same results were obtained (Fig. 5). On the other hand, the
pancreatic islets showed various changes. Some islets were decreased in size and
hyperemic, alpha cells being atrophic, while beta cells were hypertrophic and
stained densely (Figs. 6 and 7). In the animal group, to which OAA-Na had been
given subcutaneously in a dose of 2 mg for a week, alpha cells were atrophic, but
beta cells showed no change (Fig. 8). In 40 Wistar strain rats, to which OAA-Na
was given for 24 hours, alpha cells showed atrophy and vacuoles and beta cells
hypertrophy (Fig. 9). The liver, hypophysis, adrenals and gonadal glands showed
no particular changes.
DISCUSSION
A number of investigations have been reported of blood sugar lowering
substances which are contained in plants.
In my previous communications we have reported the blood sugar lowering
effect of the decoction of Euaymus alata sieb. A further analysis of the decoction
has led to the conclusion that OAA contained in the decoction was the effective
substance. OAA was stabilized when its sodium salt was made.
The critical evaluation of effectiveness of a new anti-diabetic agent is in general
based on a decrease in blood sugar, urinary sugar output and insulin requirement.
OAA-Na was found to be effective in treating diabetics, especially those belong
ing to the so-called. Type I. In our clinical investigation, OAA-Na was given only
to those patients with a long-standing diabetic condition, and therefore a higher

136 K. Yoshikawa
rate of effectiveness will be expected when milder cases are subjected to the treat
ment.
The mechanism of the effectiveness of OAA-Na is still obscure. Rice and
Evance10 reported that the addition of oxaloacetic acid to the homogenized major
pectoral muscle of pigeon enhanced the metabolism of pyruvic acid.
Acetyl-CoA produced from pyruvic acid condenses with oxaloacetate to form
citrate, which is then oxidized in the TCA cycle to water and C02. It is also well
established that in diabetic patients the intermediates of the TCA cycle, especially
oxaloacetate, are decreased. Frohman et al.11 reported that intermediates of the TCA
cycle are decreased in the muscle of alloxan diabetic rats. In our experiment, the
administration of OAA-Na to alloxan diabetic rabbits caused a decrease of blood
sugar and urine glucose and an increase of body weight. Acetone body disappeared
in these diabetic animals when administered with OAA-Na. In normal dogs, the
administration of OAA-Na induced significant hypoglycemia. Long-term admin
istration of OAA-Na in rats caused hypoglycemia and proliferation of the islet cells
of the pancreas. These findings suggest that OAA-Na stimulates the islet cells,
regulates the abnormal metabolic process, and enhances the secretion of insulin,
and thereby controls the diabetic condition. Further investigation is needed
in determining whether OAA-Na per se stimulates the beta cells or other inter
mediates of the TCA cycle are responsible.
According to Maske,12 insulin possesses a specific and strong ability to form
chelate complexes with zinc and with some other metals, and insulin is stored by the
interaction of zinc within the granules. Various metabolites, such as citrate,
oxaloacetate, glutathione, cysteine, histidine and organic phosphorus compounds,
form a stronger complex with zinc than they do with insulin. This reaction was
considered, according to Maske, to consist in bringing insoluble insulin into soluble
one by releasing the hormone from insoluble zinc-insulin complexes. The anti
diabetic effect of the decoction of Eunymus alata sieb and of OAA-Na isolated there
from can be understood from this point of view, although the exact mechanism
needs further investigation.
Acknowledgment
My gratitudes are due to Emeritus Prof. Toshio Kurokawa, Tohoku University,
Director of the Cancer Institute Hospital, Tokyo for his direction throughout this study.
References
1) Collip, J.B. Glucokinin. A new hormone present in plant tissue. J. biol. Chem.,
1923, 56, 513-543.
2) Allen, F.M. Blueberry leaf extract. Physiologic and clinical properties in relation to
carbohydrate metabolism. J. Amer. Med. Ass., 1927, 89, 1577-1581.
3) John, H.J. A trial of eucalyptus infusion in diabetes. J. Metab. Res., 1922, 1, 489-495.
4) Kumagai, T., Fujise, S., Yagi, S., Moriya, M. & Endo, H. On the effect of Lathyrus
in the treatment of diabetes mellitus and on the effective fraction of its extract. Sogo
Igaku (Jap.), 1946, 3, 385-389.
5) Kumagai, T., Kumagai, K., Oikawa, Y. & Endo, H. On the blood sugar lowering

Anti-diabetic Effect of Sodium Oxaloacetate 137
effect of oxalic acid. Sogo Igaku (Jap.), 1949, 6, 768-770.
6) Yoshikawa, K. Studies on the treatment of diabetes mellitus by the decoction of
eunymus alata sieb. Jap. J. Gastroent. (Jap.), 1958, 55. 23.
7) Kobayashi, Y. Chemical treatment of diabetes mellitus. Collection of submitted
reports of government sponsored scientific research by Japanese Ministry of Education.
Supplement (Jap.), 1953.
8) Kurokawa, T. On the diabetes mellitus. Folia endocr. jap. (Jap.), 1951, 27, 141-166.
9) Fujita, S. & Iwatake, D. Quantitative determination of true blood sugar by enzymatic
method. Biochem.. Z., 1931, 242, 43-60.
10) Rice, L. & Evans, E.A.Jr. The in vitro effect of insulin in pigeon breast muscle.
Science, 1943, 97, 470-471.
11) Frohman, C.E., Orten, J. M. & Smith, A.H. Levels of acids of the citric acid cycle
in tissues of normal and diabetic rats. J. biol. Chena., 1951, 193, 803-807.
12) Maske, H. Role of zink in the insulin secretion. Diabetes, edited by R.H. Williams,
Paul B. Hoeber, New York, 1962, pp. 46-63.

138 K. Yoshikawa
Fig. 3. The pancreatic islet of a normal rat shows significant hypertrophy after a long term
administration of OAA-Na. Gomori stain, 10•~60.
Fig. 4. The pancreatic islets of normal rats after long term administration of OAA-Na.
The islet's cells increased in number and size. Gomori stain, 10•~10.
Fig. 5. The pancreatic islets of normal rats after long term administration of OAA-Na
are shown. Gomori, 10•~10. Two islets were conjugated to form a larger islet.

Anti-diabetic Effect of Sodium Oxaloacetate 130

140 K. Yoshikawa
Fig. 6. The pancreatic islets of normal rats after oral administration of OAA-Na, with
daily doses of 2 mg for 4 months. # cells are dense and hypertrophied.
Fig. 7. The pancreatic islet of normal rats after oral administration of OAA-Na with
daily doses of 10 mg for 4 months. The islet are small. ƒÀ cells are hypertrophied and
j granules are stained densely. a cells are decreased in number and atrophic.
Fig. 8. The pancreatic islet of normal rats after subcutaneous administration of OAA-Na
for 4 months. a, and fi cells are atiopbic and scme nuelei are destroyed.
Fig. 9. The pancreatic islet of normal rats after subcutaneous administration of OAA-Na
for 4 months. The islet is hyperemic. ƒÀ cells decrease in size and encircle the islet.
ƒÀ cells are densely stained.

Anti-diabetic Effect of Sodium Oxaloacetate 141