THE EFFECT OF INSULIN UPON THE QUANTITATIVE RELA-

TIONSHIP BETWEEN &HYDROXYBUTYRIC ACID

AND ACETOACETIC ACID IN BLOOD AND URINE*
BY IRENE E. STARK AND MICHAEL SOMOGYI
(From the Laboratory of the Jewish Hospital of St. Louis, St. Louis)

(Received for publication, January 5, 1943)

In a preceding report (1) we described cases of diabetic ketosis in which

glucose feeding fails to produce appreciable changes either in the distribu-

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tion of ketone bodies between blood corpuscles and plasma or in the fl ratio
(100 X ,&hydroxybutyric acid)/(total ketone bodies) of blood and urine.
These subjects, in contrast to those with non-diabetic ketosis, tend to
respond to glucose feeding with an increase of the ketonemic level and of
ketonuria and require massive doses of insulin for the suppression and aboli-
tion of ketosis. Their condition is attributed to the impaired ability of the
liver to store glycogen and to burn carbohydrate, a deficiency that can be
corrected only by the administration of adequate amounts of insulin which
acts by inhibiting hepatic glycogenolysis (2). The purpose of our experi-
ments was to compare the effects of insulin (combined with carbohydrate
feeding) upon the distribution and /3 ratios in this type of diabetic ketosis
with the effects of glucose feeding in fasting ketosis.
The insulin requirement in severe diabetic ketosis is always large; in-
dividual variations, however, are considerable and unpredictable, so that
overinsulinization may entail hypoglycemic episodes. Since hypoglycemia
causes an increase in ketosis, and thus reverses the antiketogenic effect of
insulin (3), we were careful to forestall hypoglycemia by always keeping
the blood sugar well above normal postabsorptive levels by timely admin-
istration of glucose. At the same time care was taken to maintain a posi-
tive balance between carbohydrate administered and glucose lost in the
urine in order to ascertain that substantial amounts of carbohydrate were
utilized by the organism.
Under these conditions the distribution of ketone bodies between corpus-
cles and plasma as well as the 0 ratio changed in diabetic patients in the
same general direction as in non-diabetic subjects after glucose feeding
(without insulin injections). Evidence to illustrate this fact is presented
in Table I. As regards the distribution of ketone bodies between corpuscles
and plasma in the postabsorptive state, it may be seen that, as in healthy
individuals, the concentration was markedly higher in the plasma than in
the corpuscles. But after glucose utilization had started, within some
* This work was aided by the Helen Yonkers Research Fund.
731
732 DISTRIBUTION OF KETONE BODIES

hours after the injection of insulin, the distribution ratio shifted in favor
of the cells; i.e., the decline was faster in the plasma than in the cells, just
as in non-diabetic ketosis. This response was considerably slowed down
in the instance of patient L. L., in deep coma, who was much more resistant
to insulin action than were the other two patients.
The p ratio, as may be noted, decreased substantially after insulin action
had taken effect; in other words, the concentration of &hydroxybutyric
acid decreased faster than the *concentration of acetoacetic acid. The
shift in the relationship between the two acids was more prominent in the

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TABLE I
E$ect of Insulin upon Distribution of Acetoacetic Acid and P-Hydroxybutyric Acid
in Blood of Diabetic Subjects

Corpuscles Plasma

Time
Patient and remarks after Aceto-
1 nsulin acetic 3 ratio
acid i
__-.
hrs. w. w. ?w. WR. mg. mR.
xr cm 1Per cent per cent per cent per cent $%r cev

L. L. Admitted in 0 63.4 82.9 142.3 57 92.8 84.3 177.0 48

coma; received 200 6 43.1 58.4 101.5 58 115.0 93.9 209.0 45
units insulin during 13 32.6 3.21 35.8 9 21.2 3.22 24.4 13
first 4 hrs. after ad- 19 14.7 3.00 17.7 17 9.28 2.98 12.3 24
mission
M. R. Severe ketosis; 0 40.0 48.3 88.2 55 44.4 98.6 143.0 69
received 45 units in- 2 52.6 28.2 80.8 35 40.5 41.2 81.7 51
sulin on admission 6 30.0* 4.70* 34.7 13*
S. B.
received
Severe ketosis;
55 units in-
0
6.5 60.0 14.0 74.0 19
57.6*93.7*
40.8 22.6
104.0
63.4
*I 66*
36
sulin during 4 hrs.
after admission

* These data represent the analysis of whole blood; the samples were not sufficient
for the analysis of the plasma.

corpuscles than in the plasma, especially in the more advanced stages of

the process. These changes are of the same character as those observed
in healthy subjects, with the sole difference that they proceed at a lower
rate in diabetic ketosis, and in particular in the state of coma. It is due
to this diminished rate that in no case of diabetic ketosis has the eomplete
disappearance of /3-hydroxybutyric acid been observed, whereas in non-
diabetic ketosis this occurs within 3 to 4 hours after glucose feeding.
These changes in diabetic and non-diabetic ketosis then are due to the
same cause; namely, to the fat- and protein-sparing effect of carbohydrate
 I. E. STARK AND M. SOMOGYI 733

in the metabolism of the liver. Ketosis in the diabetic organism is due to

an unbridled hepatic glycogenolysis. Hand in hand with the inability of
the liver to store glycogen goes its inability to use carbohydrate to cover its
own fuel requirement. As a result, fats and proteins are used and abnormal
amounts of ketone bodies are transmitted to the blood. Then, when
insulin begins to inhibit hepatic glycogenolysis (besides enhancing the
passage of carbohydrate into muscle cells), the liver is again in a position to
burn its preferential fuel, carbohydrate, and the oxidation of excessive
amounts of fats and proteins, the source of ketone bodies, is suppressed.
This is the same process that occurs in fasting ketosis as the result of carbo-

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hydrate feeding alone.
Changes in the urines of diabetic patients, when ketonemia decreases
under the action of insulin, are illustrated in Table II which contains the
results of observations on two patients whose blood also had been studied.
Urine samples were collected at brief intervals of time, without the loss of
any fraction that was excreted over the entire period of the observations.
It may be noted in both cases that after insulin action had become effective
(as evidenced by utilization of carbohydrate) ketonuria began to diminish
rapidly. Simultaneously the p ratio showed a moderate initial decrease.
But whereas in fasting ketosis the /? ratio was reduced to zero within 2
hours after glucose feeding (Le., @-hydroxybutyric acid completely disap-
peared from the urine), in the two diabetic patients it has not dropped
below 55 and 60, respectively. After this initial decline the p ratio rose
again and reached the preinsulin level, despite the fact that at the same
time ketonuria continued to decrease substantially. The p ratio, then,
changed here in the same manner as in fasting ketosis, with only a quanti-
tative difference, inasmuch as the changes were less extensive and pro-
ceeded at a diminished rate.
In urine samples that were collected after the period in which the @ratio
had returned to the high basic level, a second drop occurred, which was
considerably greater than the first. In both patients M. R. and S. B. the
second drop has taken place during the 7th hour after the first insulin
injection, the fi ratio having decreased in the two cases ‘to as low as 23 and
33, respectively. This drop was again followed by a rise, notwithstanding
the fact that the rate of excretion of total ketone bodies has not increased.
We consistently obtained similar results in studies of several other diabetic
patients.
These findings are at variance with the view (4) that the p ratio in the
urine decreases whenever the rate of excretion of ketone bodies decreases.
The fact seems to be that an initial decrease of the p ratio is followed by an
increase and subsequently by another decrease. This is equally true for
fasting ketosis under the influence of glucose feeding and for diabetic
734 DISTRIBUTION OF KETONE BODIES

ketosis under the influence of insulin action. In our study of fasting

ketosis, however, we could record only one drop and the subsequent increase
of the /3 ratio, because the experiments were confined to 4 hour periods.

TABLE II
E$ect of Insulin upon Relationship between Acetoacetic Acid and P-Hydroxybutyric
Acid in Urine of Diabetic Subjects

VOL Total B
Path t Time Remarks ume retone
bodies rati0
- __-

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tc. @. w. %. fw.
per hr. per hr. per hr. fier hr.

M. R 4 .OO- 5.30 Vomited 4-5 times be- 570 13.3 510.0 1824.0 2:234.0 77
fore insulin was given
5.30- 7 .OO 25 units insulin at 5.30 330 6.6 438.0 851.0 289.0 66
7.00- 7.45 20 “ “ “ 7.30 100 5.3 275.0 333.0 606.0 55
7.45- 8.30 15 gm. glucose intra- 65 1.7 130.0 449.0 579.0 68
venously at 7.45
8.30-10 .OO 15 units insulin at 9.05, 115 0.5 72.7 160.0 233.0 69
24 gm. CHO at 9.30
10.00-11 .OO 5 units insulin and 28.5 45 0 9.60 33.0 42.6 77
gm. CHO at 10.15
11 .OO-12.00 57 gm. CHO at 11.00 75 0 a.40 2.1( 10.4 23
12.00- 2.00 66 “ “ “ 1.00 110 0 11.5 5.1( 16.6 31
2.00- 3.00 200 0 34.1 5.5( 39.6 14
3.00- 5.00 33 “ “ ‘( 3.05 775 0 47.4 6.2( 53.7 21
5.00- 7.00 135 0 4.50 6.5( 11.0 59
S. B. 10.30-12.30 40unitsinsulinat11.30 450 11.3 236.5 951.0 187.0 80
12.30- 1.30 24 gm. CHO at 1.30 450 13.5 795.0 1292.0 087.0 62
1.30- 2.30 18 I‘ “ ‘( 2.00 450 5.8 663.0 995.0 658.0 60
2.30- 3.30 15 units insulin at 3.00 320 11.2 426.0 703.0 129.0 62
3.30- 4.30 140 5.6 108.0 203.0 311.0 65
4.30- 5.30 28.5 gm. CHO at 5.00 85 0.9 25.6 69.3 94.9 73
5.30- 6.30 28.5 “ “ “ 6.05 190 0 43.7 21.9 65.6 33
6.30- 8.00 48 gm. CHO at 6.45 165 0.6 25.6 59.0 85.6 69
8.00- 9.00, 74 “ “ “ 9.00 280 2.8 17.6 73.9 91.5 81
9.00-10.00’ 25 “ I‘ (‘ 9.10 275 11.0 31.1 32.2 63.3 51
10.00-11.00 300 15.0 70.5 49.2 119.7 41
11.00-12.00, 24 “ ‘I “ 11.40 175 8.8 39.2 20.7 59.9 35

The waves in.which preponderance of /3-hydroxybutyric acid alternates

with the preponderance of acetoacetic acid can be observed only when urine
samplesare collected at brief (1 to 2 hour) intervals of time. Collection of
samples during longer periods necessarily blurs the picture. With the
example presented in Table III we wish to demonstrate how misleading
the analytical data of urines can be if t,his fact is ignored. In the instance
 I. E. STARK AND M. SOMOGYI 735

of patient L. G. blood samples, which were taken at 0, 6, and 10.5 hours

after the first injection of insulin, showed the regularly recurrent picture;
namely, a consistent decrease of the fi ratio when the ketonemic level
declined. Urine samples which were excreted during 1 hour periods co-
incident with the sampling of blood would indicate that the changes in the
urine ran parallel with the changes in the blood. However, the analysis
of intermediate hourly urine samples, presented in parentheses in Table
III, clearly shows the wave-like fluctuations of the ,B ratio in the urine.

TABLE III
Effect of Insulin on Ketone Bodies in Whole Blood and Urine of Diabetic Subject

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L. G., patient admitted in coma; received 285 units of insulin during the first 5
hours following admission. The parentheses designate intermediate hourly urine
samples.

Whole blood Urine

I
Blood sugar
Aceto-
acetic acid

hrs. nzg. ger cent mg. per cent mg. per cent mg. fier cent mg. per hr.
822 99.0 198.0 297.0 64 791t
(i.5) (1860)
(3.5) (2474) (56)
6 520 98.0 68.0 166.0 41 131 74
7.5 390 80.0 31.0 111.0 28 62.9 54
10.5 244 44.2 2.80 47.0 6 17.7 40
-
* These p ratios were determined on specimens excreted during 1 hour periods.
t This is a postabsorptive sample collected at the time of admission; since the
length of time during which it was excreted was unknown, the value 791 represents
mg. per cent and not mg. per hour.

SUMMARY

In severe ketosis of diabetic patients glucose feeding has no effect upon

the quantitative relationship of ketone bodies in blood and urine. When
such patients are enabled to utilize carbohydrate (circulating endogenous
glucose or glucose administered either parenterally or orally) by the injec-
tion of adequate amounts of insulin, the ketone bodies show changes of the
same general nature as those in healthy subjects after glucose feeding. As
the ketonemic level decreases, the distribution between corpuscles and
plasma shifts in favor of the corpuscles, and the /3 ratio decreases gradually
and consistently. The decrease is faster in the plasma than in the cor-
puscles.
In the urine the /3 ratio first decreases, then increases, as it does in non-
diabetic persons. Extension of the observations over sufficiently long
736 DISTRIBUTION OF KETONE BODIES

periods of time (8 to 12 hours), however, reveals the fact that after an

initial decrease the /? ratio again rises and then drops for a second time, and
this wave-like change repeats itself irrespective of the fact that the excre-
tion of total ketone bodies diminishes. This finding is at variance with the
view of previous workers according to which the ,8 ratio always decreases
when ketonuria diminishes.

BIBLIOGRAPHY

1. Stark, I. E., and Somogyi, M., J. Biol. Chem., 147, 721 (1943).
2. Somogyi, M., and Weichselbaum, T. E., J. Biol. Chem., 146, 567 (1942).
3. Somogyi, M., J. Biol. Chem., 141, 219 (1941).

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4. Friedemann, T. E., J. Biol. Chem., 142, 635 (1942).
 THE EFFECT OF INSULIN UPON THE
QUANTITATIVE RELATIONSHIP
BETWEEN β-HYDROXYBUTYRIC ACID
AND ACETOACETIC ACID IN BLOOD
AND URINE
Irene E. Stark and Michael Somogyi
J. Biol. Chem. 1943, 147:731-736.

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