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Effects of insulin on thiamine phosphorylation and dephosphorylation in liver homogenates of normal, thiamine-deficient, and alloxan-diabetic rats
Effects of Insulin on Thiamine Phosphorylation and Dephosphorylation in Liver Homogenates of Normal, Thiamine-Deficient, and Alloxan-Diabetic Rats* Pier0 P. Foa, Harriet R. Weinstein, Jay A. Smith and Milton Greenberg From the Department
of Physiology
and Pharmacology, The Chicago Medical
School,
Chicago 1.2, Illinois
Received May 13, 1952 INTRODUCTION
Diabetes mellitus is frequently associated with signs suggesting improper utilization of available thiamine. For example, the severity of diabetic neuritis, which is very similar to neuritis of thiamine de& ciency, seems to be proportional to the level of fasting blood sugar (1) and does not respond to thiamine therapy unless the patient is well controlled with diet and insulin (2). Diabetic retinopathy has been linked to a defect in pyruvate metabolism (3), and a high concentration of blood pyruvate, which is characteristic of thiamine deficiency, haa been found in diabetics (4). In diabetics, however, even large intravenous doses of thiamine do not bring the blood pyruvate down to normal, although this can be accomplished by the administration of thiamine phosphate [cocarboxylase (5)]. This seems to indicate that the diabetic cannot convert thiamine into its active phosphorylated form, a conclusion suggested also by the observation that the respiratory quotient of alloxan-diabetic rats rises following the administration of glucose and cocarboxylase, but not if glucose is given with thiamine hydrochloride (6). On the other hand, according to other investigators, pancreatic diabetes does not accelerate the development of thiamine deficiency (7), nor does it seem to alter pyruvate metabolism (8, 9).. The possibility of an altered thiamine metabolism in diabetes was investigated in our I Assisted by grants-in-aid from the Upjohn Company of Kalamazoo, Michigan, and the U. S. Public Health Service.
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FOA, WEINSTEIN,
SMITH AND GREENBERG
laboratory (10). Our experiments showed that the intravenous injection of thiamine into a normal dog is followed by its rapid phosphorylation, as indicated by a rise in the concentration of diphosphothiamine in the blood, and that the ability to phosphorylate thiamine is enhanced by the simultaneous administration of insulin, lost after removal of the pancreas, and restored once more by insulin. These results were confirmed recently by other investigators using rat livers (ll), and are consistent with the hypothesis that the phosphorylation of thiamine in the blood is depressed in diabetes and promoted by insulin. As insulin increases the synthesis of adenosine triphosphate (ATP) and of creatine phosphate in muscle (12, 13) and liver (14), and thus, directly or indirectly, influences phosphorylation reactions (9, 15, IS), we wondered if the observed effects of insulin on the phosphorylation of thiamine in viva were by-products of this turnover of high-energy phosphate or if they were the result of a direct action of insulin on the thiamine-phosphorylating enzymes. The in vitro experiments described in this paper represent a first step in the attempt to answer this question. Preliminary reports have appeared elsewhere (17, 18). METHODS
A. Alloxan Diabetes Adult albino rats, kept on diets of Purina Dog Chow and fasted 48 hr. (19), received an intraperitoneal injection of alloxan monohydrate (Eastman; 200 mg./kg.). After the injection, the animals were given free access to food and water, and 1 hr. later received an intraperitoneal injection of 5 ml. of a 200/osolution of glucose. About 30% of the animals thus treated survived, with no apparent illness . other than constant and severe glycosuria and polyuria. After 1 week or more, they were used for the experiments. Some of the remaining animals died within 2 or 3 days with oliguria and hematuria, and the rest developed only mild or no diabetes, and were discarded.
B. Thiamine Dejiciency This was produced by feeding an 18% casein diet (20), except that instead of yeast the following supplements were added to 100 g. of diet: riboflavin 3 mg., niacin 5 mg., pyridoxine hydrochloride 2 mg., calcium pantothenate 10 mg., paminobenzoic acid 19 mg., ascorbic acid 30 mg., choline hydrochloride 20 mg., and inositol 3 mg. The rats, housed in individual cages, were fed this diet for no less than 3 weeks, at which time the appearance of the animal and the loss of weieht indicated a severe thiamine deficiency.
INSULIN
AND
THIAMINE
325
C. Experiments All animals were fasted for 24 hr. preceding the experiment, and stunned. The livers were removed immediately, chilled in ice-cold, isotonic potassium chloride-potassium phosphate buffer at pH 7.5, minced with scissors, and washed with several portions of ice-cold buffer to remove the blood. The liver was then weighed and homogenized in a Waring blendor with four parts, by volume, of the same buffer to give a 20% homogenate, the suspension was filtered through several layers of gauze and dialyzed in a Visking bag stirred in a beaker containing 1 1. of the same buffer. After 1 hr., the buffer was changed and the dialysis continued for another hour. One milliliter of homogenate was placed in the main compartment of a Warburg flask. In some flasks 50 pg. thiamine hydrochloride and/or 0.3 unit of amorphous insulin powder (Lilly) dissolved in buffer was added. The side arm of the flask contained magnesium 2 X 10-a M, creatine (Eastman) 5 X 10-‘&f, glutamic acid 0.2 n&f, potassium citrate 0.015 M, cytochrome c (Sigma) 2 X 1Cr4 M, and adenosine triphosphate (Sigma) 5 X l(r4 M. In addition, the central well contained 0.2 ml. of 30% potassium hydroxide. The air in the flasks was replaced with oxygen. In some exploratory experiments 0.2 ml. of 0.3 M potassium fluoride was added. The results were not modified significantly by this addition, and since fluoride may actual.ly inhibit the phosphorylation of thiamine (21, 22), it was omitted from subsequent experiments. The incubation mixture was thus essentially the same as the one used by other authors in the study of creatine phosphorylation coupled with the oxidation of various substrates, including glutamate (23, 24, 25). Potassium salts were used exclusively since potassium has been shown to have an accelerating effect and sodium an inhibiting effect on several phosphorylation reactions, including the phosphorylation of the adenylic acid system (26, 27, 28), which is the main phosphorylating agent of thiamine (29). All the preceding operations were carried out in a cold room at O-3’%. Manometers and flasks were then transferred to a constant temperature bath at 37.5”C., equilibrated for 10 min., the content of the side arm was then tipped into the main compartment of the Warburg flask, and manometric readings were made every 20 min. for 1 hr. The interval also served as incubation period. Before and at the end of incubation, the pH was measured, 0.5-ml. aliquots of the mixture were delivered with shaking into a 15-ml. centrifuge tube containing 2 ml. of 1% acetic acid, and the tube was immersed in boiling water for 10 min. Thiamine and thiamine phosphates were determined in duplicate using the thiochrome method as described elsewhere (lO).t Apparent recoveries of thiamine added to control samples of homogenate varied between 90 and 105%, and were similar to those recently obtained by others using manometric methods (30). The statistical significance of the results was calculated with the method of Fisher (31) and expressed as “P,” the probability of the results being due to chance. 2 We have used the term “thiamine phosphates” rather than “cocarboxylase” because the materials were not analyzed enzymatically, but by means of the thiochrome method which determines both thiamine mono- and pyrophosphate.
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FOA,
WEINSTEIN,
SMITH
AND
GREENBERG
RESULTS
The amounts of total, free, and esterified thiamine found in the liver of normal, thiamine-deficient, and alloxan-diabetic rats are presented in Table I. The liver of normal rats contains an average of 10.2 pg. total thiamine/g. fresh tissue. Of this, about 82% is in the form of thiamine phosphates and 18% in the form of free thiamine. Thiamine deficiency redu.ces the total thiamine content of the liver to an average of 2.4 pg./g. TABLE I Free, Phosphorylated and Total Thiamine Content of Rat Liver Homogenates Migrograms per gram of fresh tissue Nomml (11 rats) AVerage -~-~-~
Free thiamine Thiamine
(FT) _.__...,,..
phosphates (TP) . .._
Total thiamine
(TT) .,.._._.,_
Thiamine-deficient (9 rats)
Range
AVerage
Range
Allo;,y;f&&eti AVerage
Range
1.9
l.l-
2.4
0.4
O.l- 1.0
7.0
5.4- 9.6
8.3
6.5-12.8
2.0
0.8- 4.2
3.7
2.1- 5.6
10.2
7.7-15.0
2.4
0.9- 5.2 10.8
7.5-15.2
Percent phosphorylation (z
X 100> . . . . . . . . . . . . . . . . 81.9 ,75.0-85.9 85.7 i76.9-90.9 ! Pa
>0.05
34.3 24.141.9
o P indicates the probability of the difference in per cent phosphorylation from normal being due to chance. If P ? 0.01 the difference is statistically “highly significant”; if P? 0.05 the difference is “significant”; if P > 0.05 the difference is “not significant.”
fresh tissue, but it does not change the relative’ amounts of the free (14%) and phosphorylated (86%) fractions. Liver tissues from well-fed alloxan-diabetic rats contain a normal amount of total thiamine (average 10.8 H./g. fresh tissue), but only 36% of it is phosphorylated. Table II shows that incubation does not affect the total thiamine content of liver homogenates from normal, thiamine-deficient, or alloxan-diabetic rats. The phosphorylated fraction, however, decreases from 82 to 45y0 in the normal homogenate and from 86 to 43% in the thiamine-deficient one. The addition of insulin to the incubation mixture prevents the decrease in thiamine phosphates in both the normal
INSULIN
AND
TABLE Effect
of Incubation
327
THIAMINE
II
and of Insulin
on the Free, Phosphorylated Content of Rat Liver Homogenates
Micrograms
per 3 ml. of incubation Before incubation Average
and Total Thiamine
mixture
After incubation Y Average
Range
Range
~ a~%ig;i*
1
Average
I
Range
Normal (11 rats) T
Free thiamine .............. Thiamine phosphate ........ Total thiamine ............. Thiamine phosphate x loo Total thiamine
1.1 5.0 6.1
0.7- 1.7 3.9- 7.7
81.9
-
T
I
P
..
i 1.4- 7.2 1.9 5.8 4.2-10.8
1.2 4.7 5.9
0.6- 2.0 3.5 8.6 4.1-10.1
24.6-72.0
79.6
71.4-98.0
/ 0.4- 2.1 0.3- 0.9 0.7- 2.8
0.4 1.0 1.4
O.l- 1.5 0.6 1.7 0.7- 3.2
1.6- 4.6 2.0- 3.8 4.0- 8.4~
r0.08 +-----A
Thiamine-deficient Free thiamine.. Thiamine phosphate. Total thiamine. Thiamine phosphate Total thiamine
3.6 3.0
0.3 1.2 1.5
(9 rats)
O.l- 0.6 0.6- 2.5 0.7- 3.1
0.8 0.6 1.4
P
Alloxan-diabetic Free thiamine., 4.2 Thiamine phosphate. 2.2 Total thiamine.. . 6.4 Thiamine phosphate Total thiamine x 100 34.3 T-
(10 rats)
3.2- 5.8 1.S 3.4 4.4 9.2
4.0 1.9 5.9
1 2.P 5.6 ; 0.5- 3.2 4.6 8.8
3.1 3.0 6.1
24.1-41.9
32.2
8.4-55.5
49.1
>0.6
t
P
i
31.2-60.0
328
FOA,
WEINSTEIN,
SMITH
AND
TABLE E$ect of Thiamine
GREENBERG
III
and Insulin
on the Free, Phosphorylated Content of Rat Liver Homogenates
and Total
(Fifty micrograms thiamine hydrochloride added to the incubation Micrograms ner 3 ml. of incubation mixture
~ Before incubation Average 1
Range
1
Range
mixture)
After incubation with 0.3 unit amorphous insulin
After incubation lx
Thiamine
Average
(
Range
Normal (11 rats) Free thiamine Thiamine phosphate.. . Total thiamine .._..... Thiamine phosphate x loo p Total thiamine
48.0 5.8 53.2
42.3-52.1 47.6 4.0- 7.4 5.1 49.6-56.2 52.7
g1 7
7H48
I
Thiamine-deficient Free thiamine 49.8 Thiamine phosphate.. 1.2 Total thiamine .._ 50.8 Thiamine phosphate x 1OO 2 3 Total thiamine
6;;1.9
,9;
42.7-51.0 4.0- 6.4 48.3-56.9 ,74-125 . .
(9 rats)
45.3-52.5 47.6 0.5- 2.0 4.4 46.4-53.9 52.0 l.O- 4.0
44.S51.3 47.8 3.6- 7.2 5.2 49.5-58.1 53.2
8.4
r
1’
45.5-51.3 47.1 3.8- 5.7 4.3 49.8-55.1 51.4 6.8-10.7
>o.g
8.3
45.0-48.8 3.8- 5.2 47.0-53.0 7.6-10.3
11
P
I
Alloxan-diabetic Free thiamine .._..__..._._ 54.2 .Thiamine phosphate. 2.2 Total thiamine . . . . . . . . . 56.4 Th;o;&F?ye
x 100
(10 rats)
53.2-55.8 45.0 1.2- 3.4 4.4 54.4-59.2 49.6
3.9
2.2 T
I
5.7
8.8
40.349.6 44.3 2.5- 5.3 4.8 45.1-54.9 49.1 5.2-10.5
9.7
T
1
I
P
I
40.3-48.7 3.8- 5.5 45.8-54.1 7.7-12.0
INSULIN
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329
and the thiamine-deficient homogenates. The thiamine phosphates content of the alloxan-diabetic homogenates, which is already low before incubation, does not decrease following incubation without insulin, but increases significantly when insulin is added to the incubation mixture. Table III shows that when thiamine is added to normal rat liver homogenates, the amount of thiamine phosphates found at the end of the incubation period is the same as that present before incubation. The further addition of insulin has no significant effect. When thiamine is added to thiamine-deficient liver homogenates, the amount of thiamine phosphates not only does not decrease, but it actually increases, sometimes reaching but never surpassing the values found in normal homogenates. The further addition of insulin has no effect. When thiamine is added to alloxan-diabetic liver homogenates there is again a significant increase in the amounts of thiamine phosphates which now approach normal. Insulin produces a further small, but significant increase. There were no significant differences in the oxygen consumption of the normal, thiamine-deficient, or alloxan-diabetic rat liver homogenates, nor was respiration modified by the addition of thiamine and/or insuiin. DISCUSSION
The normal values reported here agree with those reported by others using the thiochrome (32) or the manometric method (11, 33) and confirm the fact that most of the thiamine present in liver tissue is phosphorylated (29). The low thiamine content of the liver in the rat fed a thiamine-deficient diet proves that a severe deficiency had actually been obtained. In these animals all fractions were about equally reduced so that, as in the normal animals, most of the liver thiamine was phosphorylated. Alloxan diabetes did not influence the total thiamine content of the liver, but lowered the percentage of thiamine phosphates. This low percentage of phosphorylation confirms the results obtained in viva (10, 11) and supports the hypothesis that the diabetic animal is either unable to phosphorylate thiamine at a normal rate, or that, in diabetes, there is an increase in the rate of thiamine dephosphorylation. A prevalence of glucose dephosphorylation over glucose phosphorylation in alloxan diabetes has been reported (34, 35). Under our experimental conditions, incubation of normal, thiaminedeficient, and alloxan-diabetic rat liver homogenates did not result in
330
FOA,
WEINSTEIN,
SMITH
AND
GREENBERG
a destruction of thiamine, as recently reported (36). Incubation did, however, decrease the phosphorylated fraction in the normal and thiamine-deficient homogenates. This phenomenon may be a direct effect of specific “vitamin B1 pyrophosphatases” (37, 38), or it may be an indirect consequence of a reduction in available ATP brought about by liver ATPases. The adenosinetriphosphatase (ATPase) activity of the homogenates used in these experiments could have been stimulated by the presence of magnesium ions and by the absence of inhibiting fluorides (3942). The amount of thiamine phosphates present in liver homogenates from alloxan-diabetic rats did not decrease after incubation, perhaps because it was already as low before incubation as it was in normal and thiamine-deficient homogenates after incubation, and the rate of dephosphorylation had decreased sharply. The addition of 50 pg. thiamine to normal rat liver homogenates caused a shift in the equilibrium between phosphorylation and dephosphorylation, either as a result of mass action or perhaps as a result of the inhibition of specific phosphatases (37,38). The addition of thiamine, however, did not result in the formation of more thiamine phosphates than were present before incubation, in agreement with the finding that “the amount of cocarboxylase synthesized by the liver rarely surpasses that normally present in the tissue; and, consequently, little synthesis can be observed with tissues of normal animals” (29). On the other hand, normal livers appear to synthesize more than a normal amount of cocarboxylase from thiamine administered in tivo (43). The addition of 50 pg. thiamine to thiamine-deficient or to alloxandiabetic rat liver homogenates promoted the phosphorylation of thiamine to values approaching normal. It appears that both phosphorylation and dephosphorylation of thiamine may occur simultaneously in liver homogenates. Under the conditions of our experiments dephosphorylation prevails. The addition of thiamine reverses the equilibrium, and the amounts of thiamine phosphates return toward those normal values which seem to represent a ceiling above which no further phosphorylation occurs no matter how much thiamine is available. Insulin also reverses the process of dephosphorylation of preformed thiamine phosphates in normal and thiamine-deficient homogenates, and brings back toward normal the thiamine phosphate content of alloxan-diabetic homogenates. In other words, the addition of insulin, like the addition of thiamine, probably tends to inhibit thiamine dephosphorylation and/or favor its phosphorylation. The addition of insulin and thiamine together results in greater phosphorylation than the addi-
INSULIN
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331
tion of either alone only in the homogenates from alloxan-diabetic rats. Again, the “normal ceiling” cannot be surpassed.The decreasedthiamine phosphorylation in alloxan diabetes and its stimulation by insulin can be explained by the fact that insulin increasesthe rate of ATP formation (44, 45) and ATP is a phosphorylating agent for thiamine (29). Other reactions requiring ATP, such as the acetylation of p-aminobenzoic acid (46) and the phosphorylation of creatine (24) are decreased in diabetes and restored to normal by insulin. It is interesting to note that the acetylation of p-aminobenzoic acid is also decreased in thiamine deficiency (46). The fact that an increase in thiamine phosphorylation is not accompanied by an increase in oxygen consumption parallels the observations that adenosine can be phosphorylated by ATP in anaerobic as well as aerobic conditions (47, 48) as long as sufficient ATP is available. Other reactions, such as the synthesis of glycogen (49,50) and the phosphorylation of glucose in vitro (51), can be stimulated by insulin without an over-all increase in oxygen consumption. Perhaps insulin influences the equilibrium between phosphorylation and dephosphorylation of thiamine by stimulating the anaerobic oxidations through which high-energy bonds are formed (48) or by promoting a more efficient coupling between the processes of phosphorylation and oxidation (24, 51, 52). In this and in our preceding papers the effect of insulin on the phosphorylation of thiamine has been observed in the intact animal and in the presence of intact liver cells. The possibility that insulin may catalyze directly the enzyme systems responsible for the phosphorylation of thiamine has, therefore, not been ruled out and is being investigated wit,h the use of cell-free liver extracts. ACKNOWLEDGMENTS
Amorphous insulin powder was a gift of Eli Lilly and Company. The guidance of Dr. Andrew H. Ryan in the calculation and interpretation the statistical analyses is gratefully acknowledged.
of
CONCLUSIONS
1. Normal rat liver contains about 10 pg. thiamine/g. fresh tissue, more than 80% in the form of thiamine phosphates. 2. Thiamine-deficient rat liver contains an average of 2.4 pg. thiamine/g. fresh tissue, more than 8501,phosphorylated. 3. Livers from well-fed, alloxan-diabetic rats contain a normal amount of thiamine, of which only 36% is phorphorylated. 4. Normal and t’hiamine-deficient rat liver homogenates are capable
332
FOA, WEINSTEIN,
SMITH
AND GREENBERG
of splitting thiamine phosphates on incubation. This process of dephosphorylation is reversed by thiamine. 5. Incubation does not decrease the thiamine phosphates content of alloxan-diabetic homogenates which is already low. The addit,ion of thiamine causes an increase in thiamine phosphates. 6. Insulin, like thiamine, reverses the process of thiamine dephosphorylation in normal and thiamine-deficient homogenates and causes thiamine phosphates to return toward, but not above, the normal preincubation values. In these two types of homogenates, the effects of insulin and thiamine are not additive. 7. The addition of insulin to alloxan-diabetic rat liver homogenates causes an increase in the thiamine phosphates fraction to values greater than those found before incubation. In this type of homogenate the increase is more pronounced if thiamine is also added. 8. Under no circumstances have more thiamine phosphates been obtained than are found in normal, nonincubated liver homogenates. 9. Changes in the rate of thiamine phosphorylation are not accompanied by significant changes in oxygen consumption. 10. The results are consistent with the hypothesis that the phosphorylation and, therefore, utilization of thiamine are below normal in diabetes and are increased by insulin. REFERENCES 1. BONKALO, A., Arch. Internal.
Med. 86, 944 (1950). 24, 111 (1945). NELSON, R. A., Am. J. Digestive Diseases 14, 352 (1947). HORWITT, M. K., AND KREISLER, O., J. Nutrition 37, 411 (1949). MARKEES, S., Schweiz. med. Wochschr. 81, 1145 (1951). SILIPRANDI, N., Boll. sot. ital. biol. sper. 26, 1508 (1950). STYRON, C. W., TUFKER, H. ST. G., JR., RHODES, A. F., SMITH, T. C., AND MARBLE, A., Proc. Sot. &ptZ. BioZ. Med. 50,242 (1942). BUEDINO, E., FAZEKAS, J. F., HERRLICH, H., AND HIMWICH, H. E., J. BioZ. Chem. 148, 97 (1943). MILLER, M., DRUCKER, W. R., OWENS, J. E., CRAIQ, J. W., AND WOODWARD, H., JR., J. CZin. Invest. 31, 115 (1952). FOA, P. P., SMITH, J. A., AND WEINSTEIN, H. R., Arch. Biochem. 13,449 (1947). SILIPRANDI, D., AND SILIPRANDI, N., Boll. sot. ital. biol. sper. 26, 1510 (1950). SACKS, J., Am. J. Physiol. 143, 157 (1945). GORANSON, E. S., HAMILTON, J. E., AND HAIST, R. E., J. BioZ. Chem. 174, 1
2. RUNDLES, R. W., Medicine 3. 4.
5. 6.
7. 8.
9. 10. 11. 12. 13.
(1948). 14. KAPLAN, N. O., AND GREENBERG, D. M., J. BioZ. Chem. 166,525 (1944). 15. FOA, P. P., Chicago Med. School @art. 7,s (1946). 16. FOA, P. P., Chicago Med. School Quart. 13, 1 (1951).
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17. FOA, P. P., WEINSTEIN, H. R., SMITH, J. A., AND GREENBERG, M., Am. J. Physio2. 167, 784 (1951). 18. WEINSTEIN, H. R., FOA, P. P., SMITH, J. A., AND GREENBERG, M., Am. J. Physiol. 167, 835 (1951). 19. KASS, E. H., AND WAISBREN, B. A., PTOC.Sot. Exptl. Biol. Med. 60,303 (1945). 20. FOA, P. P., Arch. Biochem. 6,215 (1945). 21. LIPSCHITZ, M. A., POTTER, V. R., AND ELVEHJEM, C. A., J. BioZ. Chem. 134, 147 (1938). 22. OCHOA, S., Biochem. J. 33, 1262 (1939). 23. PARDEE, A. B., AND POTTER, V. R., J. BioZ. Chem. 181,739 (1949). 24. GORANSON, E. S., AND ERIJLKAR, S. D., Arch. Biochem. 24, 40 (1949). 25. MCILWAIN, H., BUCHEL, L., AND CHESHIRE, J. D., Biochem. J. 43, 12 (1951). . 26. OHLMEYER, P., AND OCHOA, S., Biochem. 2. 293,338 (1937). 27. BOYER, P. D., LARDY, H. A., AND PHILLIPS, P. H., J. BioZ. Chem. 146, 673 (1942); ibid. 149, 529 (1943). 28. WIEBELHAUS, V. D., AND LARDY, H. A., AT&. Biochem. 21, 321 (1949). 29. OCHOA, S., in EVANS, E. A., JR., The Biological Action of the Vitamins. University of Chicago Press, 1942. 30. BLANCHAER, M. C., AND COHEN, L. H., Can. J. Research, Ea7, 275 (1949). 31. FISHER, R. A., Statistical Methods for Research Workers. Oliver and Boyd, Edinburgh, 1938. 32. WESTENBRINK, H. G. K., AND GOUDSMIT, J., Enzymologia 6,307 (1938). 33. OCHOA, S., AND PETERS, R. A., Biochem. J. 32,1501 (1938). 34. DRABKIN, D. L., Proc. Am. Diabetes Assoc. 8,171 (1948). 35. BROH-KAHN, R. H., AND MIRSKY, J. A., Arch. Biochem. 16,87 (1948). 36. SOMOGYI, J. C., Helv. Physiol. et PharmacoZ. Acta 8, 75 (1950). 37. MELNICK, D., AND FIELD, H., JR., Proc. Sot. Exptl. Biol. Med. 39, 317 (1938). 38. WESTENBRINK, H. G. K., VAN DORP, D. A., GRUBER, M., AND VELDMAN, H., Enzymologia 9, 73 (1941). 39. SWANSON, M. A., J. Biol. Chem. 191,577 (1951). 40. NOVIKOFF, A. B., HECHT, L., AND PODBER, E., Federation PTOC.10,230 (1951). 41. KAI~CKAR, H., Enzymologia 2, 47 (1937). 42. GORE, M. B. R., Biochem. J. 60, 18 (1951). 43. SILIPRANDI, D., AND SILIPRANDI, N., Acta Vitaminologica 6, 1 (1951). 44. KAPUN, N. O., AND GREENBERG, D. M., J. BioZ. Chem. 166, 525, 553 (1944); Science 102, 447 (1945). 45. GORANSON, E. S., HAMILTON, J. E., AND HAIST, R. E., J. BioZ. Chem. 174, 1 (1948). 46. CHARALAMPOUS, F. C., AND HEGSTED, D. M., J. BioZ. Chem. 180, 623 (1949). 47. CANZANELLI, A., GUILD, R., AND RAPPORT, D., Am. J. Physiol. 162,168 (1950). 48. HUNTER, F. E., JR., J. BioZ. Chem. 177,361 (1949). 49. GEMMILL, C. L., AND HAMMAN, L., JR., Bull. Johns Hopkins Hosp. 68, 50 (1941) 50. STADIE, W. C., AND ZAPP, J. A., JR., J. BioZ. Chem. 170.55 (1947). 51. POLIS, B. D., POLIS, E., KERRIGAN, M., AND JEDEIKIN, L., Arch. Biochem. 23, 505 (1949). 52. STADIE, W. C., YaZe J. BioZ. Med. 16, 539 (1944).
of Physiology
and Pharmacology, The Chicago Medical
School,
Chicago 1.2, Illinois
Received May 13, 1952 INTRODUCTION
Diabetes mellitus is frequently associated with signs suggesting improper utilization of available thiamine. For example, the severity of diabetic neuritis, which is very similar to neuritis of thiamine de& ciency, seems to be proportional to the level of fasting blood sugar (1) and does not respond to thiamine therapy unless the patient is well controlled with diet and insulin (2). Diabetic retinopathy has been linked to a defect in pyruvate metabolism (3), and a high concentration of blood pyruvate, which is characteristic of thiamine deficiency, haa been found in diabetics (4). In diabetics, however, even large intravenous doses of thiamine do not bring the blood pyruvate down to normal, although this can be accomplished by the administration of thiamine phosphate [cocarboxylase (5)]. This seems to indicate that the diabetic cannot convert thiamine into its active phosphorylated form, a conclusion suggested also by the observation that the respiratory quotient of alloxan-diabetic rats rises following the administration of glucose and cocarboxylase, but not if glucose is given with thiamine hydrochloride (6). On the other hand, according to other investigators, pancreatic diabetes does not accelerate the development of thiamine deficiency (7), nor does it seem to alter pyruvate metabolism (8, 9).. The possibility of an altered thiamine metabolism in diabetes was investigated in our I Assisted by grants-in-aid from the Upjohn Company of Kalamazoo, Michigan, and the U. S. Public Health Service.
323
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SMITH AND GREENBERG
laboratory (10). Our experiments showed that the intravenous injection of thiamine into a normal dog is followed by its rapid phosphorylation, as indicated by a rise in the concentration of diphosphothiamine in the blood, and that the ability to phosphorylate thiamine is enhanced by the simultaneous administration of insulin, lost after removal of the pancreas, and restored once more by insulin. These results were confirmed recently by other investigators using rat livers (ll), and are consistent with the hypothesis that the phosphorylation of thiamine in the blood is depressed in diabetes and promoted by insulin. As insulin increases the synthesis of adenosine triphosphate (ATP) and of creatine phosphate in muscle (12, 13) and liver (14), and thus, directly or indirectly, influences phosphorylation reactions (9, 15, IS), we wondered if the observed effects of insulin on the phosphorylation of thiamine in viva were by-products of this turnover of high-energy phosphate or if they were the result of a direct action of insulin on the thiamine-phosphorylating enzymes. The in vitro experiments described in this paper represent a first step in the attempt to answer this question. Preliminary reports have appeared elsewhere (17, 18). METHODS
A. Alloxan Diabetes Adult albino rats, kept on diets of Purina Dog Chow and fasted 48 hr. (19), received an intraperitoneal injection of alloxan monohydrate (Eastman; 200 mg./kg.). After the injection, the animals were given free access to food and water, and 1 hr. later received an intraperitoneal injection of 5 ml. of a 200/osolution of glucose. About 30% of the animals thus treated survived, with no apparent illness . other than constant and severe glycosuria and polyuria. After 1 week or more, they were used for the experiments. Some of the remaining animals died within 2 or 3 days with oliguria and hematuria, and the rest developed only mild or no diabetes, and were discarded.
B. Thiamine Dejiciency This was produced by feeding an 18% casein diet (20), except that instead of yeast the following supplements were added to 100 g. of diet: riboflavin 3 mg., niacin 5 mg., pyridoxine hydrochloride 2 mg., calcium pantothenate 10 mg., paminobenzoic acid 19 mg., ascorbic acid 30 mg., choline hydrochloride 20 mg., and inositol 3 mg. The rats, housed in individual cages, were fed this diet for no less than 3 weeks, at which time the appearance of the animal and the loss of weieht indicated a severe thiamine deficiency.
INSULIN
AND
THIAMINE
325
C. Experiments All animals were fasted for 24 hr. preceding the experiment, and stunned. The livers were removed immediately, chilled in ice-cold, isotonic potassium chloride-potassium phosphate buffer at pH 7.5, minced with scissors, and washed with several portions of ice-cold buffer to remove the blood. The liver was then weighed and homogenized in a Waring blendor with four parts, by volume, of the same buffer to give a 20% homogenate, the suspension was filtered through several layers of gauze and dialyzed in a Visking bag stirred in a beaker containing 1 1. of the same buffer. After 1 hr., the buffer was changed and the dialysis continued for another hour. One milliliter of homogenate was placed in the main compartment of a Warburg flask. In some flasks 50 pg. thiamine hydrochloride and/or 0.3 unit of amorphous insulin powder (Lilly) dissolved in buffer was added. The side arm of the flask contained magnesium 2 X 10-a M, creatine (Eastman) 5 X 10-‘&f, glutamic acid 0.2 n&f, potassium citrate 0.015 M, cytochrome c (Sigma) 2 X 1Cr4 M, and adenosine triphosphate (Sigma) 5 X l(r4 M. In addition, the central well contained 0.2 ml. of 30% potassium hydroxide. The air in the flasks was replaced with oxygen. In some exploratory experiments 0.2 ml. of 0.3 M potassium fluoride was added. The results were not modified significantly by this addition, and since fluoride may actual.ly inhibit the phosphorylation of thiamine (21, 22), it was omitted from subsequent experiments. The incubation mixture was thus essentially the same as the one used by other authors in the study of creatine phosphorylation coupled with the oxidation of various substrates, including glutamate (23, 24, 25). Potassium salts were used exclusively since potassium has been shown to have an accelerating effect and sodium an inhibiting effect on several phosphorylation reactions, including the phosphorylation of the adenylic acid system (26, 27, 28), which is the main phosphorylating agent of thiamine (29). All the preceding operations were carried out in a cold room at O-3’%. Manometers and flasks were then transferred to a constant temperature bath at 37.5”C., equilibrated for 10 min., the content of the side arm was then tipped into the main compartment of the Warburg flask, and manometric readings were made every 20 min. for 1 hr. The interval also served as incubation period. Before and at the end of incubation, the pH was measured, 0.5-ml. aliquots of the mixture were delivered with shaking into a 15-ml. centrifuge tube containing 2 ml. of 1% acetic acid, and the tube was immersed in boiling water for 10 min. Thiamine and thiamine phosphates were determined in duplicate using the thiochrome method as described elsewhere (lO).t Apparent recoveries of thiamine added to control samples of homogenate varied between 90 and 105%, and were similar to those recently obtained by others using manometric methods (30). The statistical significance of the results was calculated with the method of Fisher (31) and expressed as “P,” the probability of the results being due to chance. 2 We have used the term “thiamine phosphates” rather than “cocarboxylase” because the materials were not analyzed enzymatically, but by means of the thiochrome method which determines both thiamine mono- and pyrophosphate.
326
FOA,
WEINSTEIN,
SMITH
AND
GREENBERG
RESULTS
The amounts of total, free, and esterified thiamine found in the liver of normal, thiamine-deficient, and alloxan-diabetic rats are presented in Table I. The liver of normal rats contains an average of 10.2 pg. total thiamine/g. fresh tissue. Of this, about 82% is in the form of thiamine phosphates and 18% in the form of free thiamine. Thiamine deficiency redu.ces the total thiamine content of the liver to an average of 2.4 pg./g. TABLE I Free, Phosphorylated and Total Thiamine Content of Rat Liver Homogenates Migrograms per gram of fresh tissue Nomml (11 rats) AVerage -~-~-~
Free thiamine Thiamine
(FT) _.__...,,..
phosphates (TP) . .._
Total thiamine
(TT) .,.._._.,_
Thiamine-deficient (9 rats)
Range
AVerage
Range
Allo;,y;f&&eti AVerage
Range
1.9
l.l-
2.4
0.4
O.l- 1.0
7.0
5.4- 9.6
8.3
6.5-12.8
2.0
0.8- 4.2
3.7
2.1- 5.6
10.2
7.7-15.0
2.4
0.9- 5.2 10.8
7.5-15.2
Percent phosphorylation (z
X 100> . . . . . . . . . . . . . . . . 81.9 ,75.0-85.9 85.7 i76.9-90.9 ! Pa
>0.05
34.3 24.141.9
o P indicates the probability of the difference in per cent phosphorylation from normal being due to chance. If P ? 0.01 the difference is statistically “highly significant”; if P? 0.05 the difference is “significant”; if P > 0.05 the difference is “not significant.”
fresh tissue, but it does not change the relative’ amounts of the free (14%) and phosphorylated (86%) fractions. Liver tissues from well-fed alloxan-diabetic rats contain a normal amount of total thiamine (average 10.8 H./g. fresh tissue), but only 36% of it is phosphorylated. Table II shows that incubation does not affect the total thiamine content of liver homogenates from normal, thiamine-deficient, or alloxan-diabetic rats. The phosphorylated fraction, however, decreases from 82 to 45y0 in the normal homogenate and from 86 to 43% in the thiamine-deficient one. The addition of insulin to the incubation mixture prevents the decrease in thiamine phosphates in both the normal
INSULIN
AND
TABLE Effect
of Incubation
327
THIAMINE
II
and of Insulin
on the Free, Phosphorylated Content of Rat Liver Homogenates
Micrograms
per 3 ml. of incubation Before incubation Average
and Total Thiamine
mixture
After incubation Y Average
Range
Range
~ a~%ig;i*
1
Average
I
Range
Normal (11 rats) T
Free thiamine .............. Thiamine phosphate ........ Total thiamine ............. Thiamine phosphate x loo Total thiamine
1.1 5.0 6.1
0.7- 1.7 3.9- 7.7
81.9
-
T
I
P
..
i 1.4- 7.2 1.9 5.8 4.2-10.8
1.2 4.7 5.9
0.6- 2.0 3.5 8.6 4.1-10.1
24.6-72.0
79.6
71.4-98.0
/ 0.4- 2.1 0.3- 0.9 0.7- 2.8
0.4 1.0 1.4
O.l- 1.5 0.6 1.7 0.7- 3.2
1.6- 4.6 2.0- 3.8 4.0- 8.4~
r
Thiamine-deficient Free thiamine.. Thiamine phosphate. Total thiamine. Thiamine phosphate Total thiamine
3.6 3.0
0.3 1.2 1.5
(9 rats)
O.l- 0.6 0.6- 2.5 0.7- 3.1
0.8 0.6 1.4
P
Alloxan-diabetic Free thiamine., 4.2 Thiamine phosphate. 2.2 Total thiamine.. . 6.4 Thiamine phosphate Total thiamine x 100 34.3 T-
(10 rats)
3.2- 5.8 1.S 3.4 4.4 9.2
4.0 1.9 5.9
1 2.P 5.6 ; 0.5- 3.2 4.6 8.8
3.1 3.0 6.1
24.1-41.9
32.2
8.4-55.5
49.1
>0.6
t
P
i
31.2-60.0
328
FOA,
WEINSTEIN,
SMITH
AND
TABLE E$ect of Thiamine
GREENBERG
III
and Insulin
on the Free, Phosphorylated Content of Rat Liver Homogenates
and Total
(Fifty micrograms thiamine hydrochloride added to the incubation Micrograms ner 3 ml. of incubation mixture
~ Before incubation Average 1
Range
1
Range
mixture)
After incubation with 0.3 unit amorphous insulin
After incubation lx
Thiamine
Average
(
Range
Normal (11 rats) Free thiamine Thiamine phosphate.. . Total thiamine .._..... Thiamine phosphate x loo p Total thiamine
48.0 5.8 53.2
42.3-52.1 47.6 4.0- 7.4 5.1 49.6-56.2 52.7
g1 7
7H48
I
Thiamine-deficient Free thiamine 49.8 Thiamine phosphate.. 1.2 Total thiamine .._ 50.8 Thiamine phosphate x 1OO 2 3 Total thiamine
6;;1.9
,9;
42.7-51.0 4.0- 6.4 48.3-56.9 ,74-125 . .
(9 rats)
45.3-52.5 47.6 0.5- 2.0 4.4 46.4-53.9 52.0 l.O- 4.0
44.S51.3 47.8 3.6- 7.2 5.2 49.5-58.1 53.2
8.4
r
1’
45.5-51.3 47.1 3.8- 5.7 4.3 49.8-55.1 51.4 6.8-10.7
>o.g
8.3
45.0-48.8 3.8- 5.2 47.0-53.0 7.6-10.3
11
P
I
Alloxan-diabetic Free thiamine .._..__..._._ 54.2 .Thiamine phosphate. 2.2 Total thiamine . . . . . . . . . 56.4 Th;o;&F?ye
x 100
(10 rats)
53.2-55.8 45.0 1.2- 3.4 4.4 54.4-59.2 49.6
3.9
2.2 T
I
5.7
8.8
40.349.6 44.3 2.5- 5.3 4.8 45.1-54.9 49.1 5.2-10.5
9.7
T
1
I
P
I
40.3-48.7 3.8- 5.5 45.8-54.1 7.7-12.0
INSULIN
AND
THIAMINE
329
and the thiamine-deficient homogenates. The thiamine phosphates content of the alloxan-diabetic homogenates, which is already low before incubation, does not decrease following incubation without insulin, but increases significantly when insulin is added to the incubation mixture. Table III shows that when thiamine is added to normal rat liver homogenates, the amount of thiamine phosphates found at the end of the incubation period is the same as that present before incubation. The further addition of insulin has no significant effect. When thiamine is added to thiamine-deficient liver homogenates, the amount of thiamine phosphates not only does not decrease, but it actually increases, sometimes reaching but never surpassing the values found in normal homogenates. The further addition of insulin has no effect. When thiamine is added to alloxan-diabetic liver homogenates there is again a significant increase in the amounts of thiamine phosphates which now approach normal. Insulin produces a further small, but significant increase. There were no significant differences in the oxygen consumption of the normal, thiamine-deficient, or alloxan-diabetic rat liver homogenates, nor was respiration modified by the addition of thiamine and/or insuiin. DISCUSSION
The normal values reported here agree with those reported by others using the thiochrome (32) or the manometric method (11, 33) and confirm the fact that most of the thiamine present in liver tissue is phosphorylated (29). The low thiamine content of the liver in the rat fed a thiamine-deficient diet proves that a severe deficiency had actually been obtained. In these animals all fractions were about equally reduced so that, as in the normal animals, most of the liver thiamine was phosphorylated. Alloxan diabetes did not influence the total thiamine content of the liver, but lowered the percentage of thiamine phosphates. This low percentage of phosphorylation confirms the results obtained in viva (10, 11) and supports the hypothesis that the diabetic animal is either unable to phosphorylate thiamine at a normal rate, or that, in diabetes, there is an increase in the rate of thiamine dephosphorylation. A prevalence of glucose dephosphorylation over glucose phosphorylation in alloxan diabetes has been reported (34, 35). Under our experimental conditions, incubation of normal, thiaminedeficient, and alloxan-diabetic rat liver homogenates did not result in
330
FOA,
WEINSTEIN,
SMITH
AND
GREENBERG
a destruction of thiamine, as recently reported (36). Incubation did, however, decrease the phosphorylated fraction in the normal and thiamine-deficient homogenates. This phenomenon may be a direct effect of specific “vitamin B1 pyrophosphatases” (37, 38), or it may be an indirect consequence of a reduction in available ATP brought about by liver ATPases. The adenosinetriphosphatase (ATPase) activity of the homogenates used in these experiments could have been stimulated by the presence of magnesium ions and by the absence of inhibiting fluorides (3942). The amount of thiamine phosphates present in liver homogenates from alloxan-diabetic rats did not decrease after incubation, perhaps because it was already as low before incubation as it was in normal and thiamine-deficient homogenates after incubation, and the rate of dephosphorylation had decreased sharply. The addition of 50 pg. thiamine to normal rat liver homogenates caused a shift in the equilibrium between phosphorylation and dephosphorylation, either as a result of mass action or perhaps as a result of the inhibition of specific phosphatases (37,38). The addition of thiamine, however, did not result in the formation of more thiamine phosphates than were present before incubation, in agreement with the finding that “the amount of cocarboxylase synthesized by the liver rarely surpasses that normally present in the tissue; and, consequently, little synthesis can be observed with tissues of normal animals” (29). On the other hand, normal livers appear to synthesize more than a normal amount of cocarboxylase from thiamine administered in tivo (43). The addition of 50 pg. thiamine to thiamine-deficient or to alloxandiabetic rat liver homogenates promoted the phosphorylation of thiamine to values approaching normal. It appears that both phosphorylation and dephosphorylation of thiamine may occur simultaneously in liver homogenates. Under the conditions of our experiments dephosphorylation prevails. The addition of thiamine reverses the equilibrium, and the amounts of thiamine phosphates return toward those normal values which seem to represent a ceiling above which no further phosphorylation occurs no matter how much thiamine is available. Insulin also reverses the process of dephosphorylation of preformed thiamine phosphates in normal and thiamine-deficient homogenates, and brings back toward normal the thiamine phosphate content of alloxan-diabetic homogenates. In other words, the addition of insulin, like the addition of thiamine, probably tends to inhibit thiamine dephosphorylation and/or favor its phosphorylation. The addition of insulin and thiamine together results in greater phosphorylation than the addi-
INSULIN
AND
THIAMINE
331
tion of either alone only in the homogenates from alloxan-diabetic rats. Again, the “normal ceiling” cannot be surpassed.The decreasedthiamine phosphorylation in alloxan diabetes and its stimulation by insulin can be explained by the fact that insulin increasesthe rate of ATP formation (44, 45) and ATP is a phosphorylating agent for thiamine (29). Other reactions requiring ATP, such as the acetylation of p-aminobenzoic acid (46) and the phosphorylation of creatine (24) are decreased in diabetes and restored to normal by insulin. It is interesting to note that the acetylation of p-aminobenzoic acid is also decreased in thiamine deficiency (46). The fact that an increase in thiamine phosphorylation is not accompanied by an increase in oxygen consumption parallels the observations that adenosine can be phosphorylated by ATP in anaerobic as well as aerobic conditions (47, 48) as long as sufficient ATP is available. Other reactions, such as the synthesis of glycogen (49,50) and the phosphorylation of glucose in vitro (51), can be stimulated by insulin without an over-all increase in oxygen consumption. Perhaps insulin influences the equilibrium between phosphorylation and dephosphorylation of thiamine by stimulating the anaerobic oxidations through which high-energy bonds are formed (48) or by promoting a more efficient coupling between the processes of phosphorylation and oxidation (24, 51, 52). In this and in our preceding papers the effect of insulin on the phosphorylation of thiamine has been observed in the intact animal and in the presence of intact liver cells. The possibility that insulin may catalyze directly the enzyme systems responsible for the phosphorylation of thiamine has, therefore, not been ruled out and is being investigated wit,h the use of cell-free liver extracts. ACKNOWLEDGMENTS
Amorphous insulin powder was a gift of Eli Lilly and Company. The guidance of Dr. Andrew H. Ryan in the calculation and interpretation the statistical analyses is gratefully acknowledged.
of
CONCLUSIONS
1. Normal rat liver contains about 10 pg. thiamine/g. fresh tissue, more than 80% in the form of thiamine phosphates. 2. Thiamine-deficient rat liver contains an average of 2.4 pg. thiamine/g. fresh tissue, more than 8501,phosphorylated. 3. Livers from well-fed, alloxan-diabetic rats contain a normal amount of thiamine, of which only 36% is phorphorylated. 4. Normal and t’hiamine-deficient rat liver homogenates are capable
332
FOA, WEINSTEIN,
SMITH
AND GREENBERG
of splitting thiamine phosphates on incubation. This process of dephosphorylation is reversed by thiamine. 5. Incubation does not decrease the thiamine phosphates content of alloxan-diabetic homogenates which is already low. The addit,ion of thiamine causes an increase in thiamine phosphates. 6. Insulin, like thiamine, reverses the process of thiamine dephosphorylation in normal and thiamine-deficient homogenates and causes thiamine phosphates to return toward, but not above, the normal preincubation values. In these two types of homogenates, the effects of insulin and thiamine are not additive. 7. The addition of insulin to alloxan-diabetic rat liver homogenates causes an increase in the thiamine phosphates fraction to values greater than those found before incubation. In this type of homogenate the increase is more pronounced if thiamine is also added. 8. Under no circumstances have more thiamine phosphates been obtained than are found in normal, nonincubated liver homogenates. 9. Changes in the rate of thiamine phosphorylation are not accompanied by significant changes in oxygen consumption. 10. The results are consistent with the hypothesis that the phosphorylation and, therefore, utilization of thiamine are below normal in diabetes and are increased by insulin. REFERENCES 1. BONKALO, A., Arch. Internal.
Med. 86, 944 (1950). 24, 111 (1945). NELSON, R. A., Am. J. Digestive Diseases 14, 352 (1947). HORWITT, M. K., AND KREISLER, O., J. Nutrition 37, 411 (1949). MARKEES, S., Schweiz. med. Wochschr. 81, 1145 (1951). SILIPRANDI, N., Boll. sot. ital. biol. sper. 26, 1508 (1950). STYRON, C. W., TUFKER, H. ST. G., JR., RHODES, A. F., SMITH, T. C., AND MARBLE, A., Proc. Sot. &ptZ. BioZ. Med. 50,242 (1942). BUEDINO, E., FAZEKAS, J. F., HERRLICH, H., AND HIMWICH, H. E., J. BioZ. Chem. 148, 97 (1943). MILLER, M., DRUCKER, W. R., OWENS, J. E., CRAIQ, J. W., AND WOODWARD, H., JR., J. CZin. Invest. 31, 115 (1952). FOA, P. P., SMITH, J. A., AND WEINSTEIN, H. R., Arch. Biochem. 13,449 (1947). SILIPRANDI, D., AND SILIPRANDI, N., Boll. sot. ital. biol. sper. 26, 1510 (1950). SACKS, J., Am. J. Physiol. 143, 157 (1945). GORANSON, E. S., HAMILTON, J. E., AND HAIST, R. E., J. BioZ. Chem. 174, 1
2. RUNDLES, R. W., Medicine 3. 4.
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THIAMINE
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17. FOA, P. P., WEINSTEIN, H. R., SMITH, J. A., AND GREENBERG, M., Am. J. Physio2. 167, 784 (1951). 18. WEINSTEIN, H. R., FOA, P. P., SMITH, J. A., AND GREENBERG, M., Am. J. Physiol. 167, 835 (1951). 19. KASS, E. H., AND WAISBREN, B. A., PTOC.Sot. Exptl. Biol. Med. 60,303 (1945). 20. FOA, P. P., Arch. Biochem. 6,215 (1945). 21. LIPSCHITZ, M. A., POTTER, V. R., AND ELVEHJEM, C. A., J. BioZ. Chem. 134, 147 (1938). 22. OCHOA, S., Biochem. J. 33, 1262 (1939). 23. PARDEE, A. B., AND POTTER, V. R., J. BioZ. Chem. 181,739 (1949). 24. GORANSON, E. S., AND ERIJLKAR, S. D., Arch. Biochem. 24, 40 (1949). 25. MCILWAIN, H., BUCHEL, L., AND CHESHIRE, J. D., Biochem. J. 43, 12 (1951). . 26. OHLMEYER, P., AND OCHOA, S., Biochem. 2. 293,338 (1937). 27. BOYER, P. D., LARDY, H. A., AND PHILLIPS, P. H., J. BioZ. Chem. 146, 673 (1942); ibid. 149, 529 (1943). 28. WIEBELHAUS, V. D., AND LARDY, H. A., AT&. Biochem. 21, 321 (1949). 29. OCHOA, S., in EVANS, E. A., JR., The Biological Action of the Vitamins. University of Chicago Press, 1942. 30. BLANCHAER, M. C., AND COHEN, L. H., Can. J. Research, Ea7, 275 (1949). 31. FISHER, R. A., Statistical Methods for Research Workers. Oliver and Boyd, Edinburgh, 1938. 32. WESTENBRINK, H. G. K., AND GOUDSMIT, J., Enzymologia 6,307 (1938). 33. OCHOA, S., AND PETERS, R. A., Biochem. J. 32,1501 (1938). 34. DRABKIN, D. L., Proc. Am. Diabetes Assoc. 8,171 (1948). 35. BROH-KAHN, R. H., AND MIRSKY, J. A., Arch. Biochem. 16,87 (1948). 36. SOMOGYI, J. C., Helv. Physiol. et PharmacoZ. Acta 8, 75 (1950). 37. MELNICK, D., AND FIELD, H., JR., Proc. Sot. Exptl. Biol. Med. 39, 317 (1938). 38. WESTENBRINK, H. G. K., VAN DORP, D. A., GRUBER, M., AND VELDMAN, H., Enzymologia 9, 73 (1941). 39. SWANSON, M. A., J. Biol. Chem. 191,577 (1951). 40. NOVIKOFF, A. B., HECHT, L., AND PODBER, E., Federation PTOC.10,230 (1951). 41. KAI~CKAR, H., Enzymologia 2, 47 (1937). 42. GORE, M. B. R., Biochem. J. 60, 18 (1951). 43. SILIPRANDI, D., AND SILIPRANDI, N., Acta Vitaminologica 6, 1 (1951). 44. KAPUN, N. O., AND GREENBERG, D. M., J. BioZ. Chem. 166, 525, 553 (1944); Science 102, 447 (1945). 45. GORANSON, E. S., HAMILTON, J. E., AND HAIST, R. E., J. BioZ. Chem. 174, 1 (1948). 46. CHARALAMPOUS, F. C., AND HEGSTED, D. M., J. BioZ. Chem. 180, 623 (1949). 47. CANZANELLI, A., GUILD, R., AND RAPPORT, D., Am. J. Physiol. 162,168 (1950). 48. HUNTER, F. E., JR., J. BioZ. Chem. 177,361 (1949). 49. GEMMILL, C. L., AND HAMMAN, L., JR., Bull. Johns Hopkins Hosp. 68, 50 (1941) 50. STADIE, W. C., AND ZAPP, J. A., JR., J. BioZ. Chem. 170.55 (1947). 51. POLIS, B. D., POLIS, E., KERRIGAN, M., AND JEDEIKIN, L., Arch. Biochem. 23, 505 (1949). 52. STADIE, W. C., YaZe J. BioZ. Med. 16, 539 (1944).