Ames, Stanley R.

Elvehjem, C. A.

Sarma, P. S.

English

Publications of the IAS Fellows

TRANSAMINASE AND PYRIDOXINE DEFICIENCY* 
BY STANLEY R. AMES,t P. S. SARMA, AND C. A. ELVEHJEM 
(From the Department of Biochemistry, College of Agriculture, University of 
Wisconsin, Madison) 
(Received for publication, October 14, 1946) 
The r&e of vitamins in enzymatic systems has been examined fre- 
quently by the determination of enzymatic activities in animals maintained 
on a vitamin-deficient ration. The indication that pyridoxal phosphate 
functions as the prosthetic group of transaminase offered an excellent 
opportunity to test this relationship in the case of another vitamin and to 
devise more stringent criteria for the deficiency method of relating vita- 
mins and enzymes. This study of the effect of a pyridoxine deficiency on 
transaminase was facilitated by the use of an improved vitamin Bs-de- 
ficient ration developed in this laboratory. 
While studying the reversible in vitro interconversion of pyridoxal and 
pyridoxamine, Snell (1) first suggested that vitamin Ba was concerned in 
biological transamination. Schlenk and Snell (2) suggested that vitamin 
Bg may function as cotransaminase, owing to the fact that the tissues of 
vitamin Bs-deficient rats were low in transaminase and also that pyridoxal 
and pyridoxamine plus adenosine triphosphate (ATP) had the ability to 
reactivate partially the deficient system in six out of nine trials. These 
conclusions and the experimental method employed have been strongly 
criticized (3), but the experimental basis for this criticism was later shown 
to be in error (4). Schlenk and Fisher (5), on the basis of isolation ex- 
periments, indicated that vitamin B, was involved in glutamic-aspartic 
transaminase. Leloir and Green (6) isolated two transaminase systems 
and reported that there was no vitamin Bg present, but later Green et al. 
(7) presented evidence which points to pyridoxal phosphate as cotrans- 
aminase .
This investigation confirms and extends the findings of Schlenk and Snell 
(2). Observations are made concerning transaminase and succinic oxidase 
in normal and vitamin BG-deficient rats, both with and without the addi- 
tion of various members of the vitamin Bs complex and their phosphates. 
* Published with the approval of the Director of the Wisconsin Agricultural Ex- 
periment Station. These studies were aided by a grant from the Rockefeller Founda- 
tion. Presented before the Division of Biological Chemistry of the American Chemi- 
cal Society at Chicago, September, 1946. 
t Present address, Research Laboratories, Distillation Products, Inc., Rochester, 
New York. 
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136 TRANSAMINASE AND VITAMIN Be 
Methods 
Albino rats, obtained from Sprague-Dawley, were placed on experi- 
ment at weaning age. One group of rats was fed the vitamin Be-deficient 
ration developed by Sarma, Snell, and Elvehjem (8), and a group of con- 
trol animals was fed the same diet plus 150 y of pyridoxine hydrochloride 
per 100 gm. of ration. The dry weights of samples were determined by evap- 
orating to constant weight in open crucibles in an electric oven at 110”. 
Transaminase values were determined by the method of Ames and Elve- 
hjem (9), which measures the oxalacetic acid formed in a reaction mixture 
containing I-aspartic acid, cu-ketoglutaric acid, and tissue homogenate. 
The components of the fIna1 reaction mixture are 0.50 ml. of 0.25 M sodium 
potassium phosphate buffer (pH 7.3), 0.50 ml. of 0.20 M sodium aspartate, 
0.50 ml. of 0.10 M sodium cu-ketoglutaratel (one side arm), the desired 
amount of tissue homogenate in 0.10 M sodium phosphate buffer (pH 7.3), 
and a sufficient quantity of this same 0.10 M buffer to yield a total reaction 
volume of 2.50 ml. The transaminating reaction was allowed to proceed 
for exactly 10 minutes and was stopped by the addition of 0.50 ml. of 
aniline citrate reagent from a second side arm. The carbon dioxide evolved 
is a measure of the oxalacetic acid formed in this reaction. Corrections 
were applied for both the carbon dioxide arising because of the addition 
of tissue homogenate and the carbon dioxide evolution at zero tissue con- 
centration obtained by extrapolation. In experiments in which the ex- 
tent of reactivation effected by various members of the vitamin Bs com- 
plex was determined, these solutions were added at the expense of the 
0.10 M phosphate buffer. 
Succinic oxidase was determined by the method of Schneider and Potter 
(10) and solutions to be tested for possible reactivation were added at the 
expense of the water. 
Solutions were made up in glass-redistilled water as follows: pyridoxine 
monohydrochloride, Merck, 100 y per ml.; pyridoxal monohydrochloride, 
Merck, 100 y per ml.; pyridoxamine dihydrochloride, Merck, 100 y per 
ml. ; pyridoxal phosphate (as dibarium salt),2 100 y per ml. ; pyridoxamine 
phosphate,3 100 y per ml.; ATP, 0.75 M (weighed as dibarium salt but with 
barium removed by precipitation as sulfate). 
1 Appreciation is expressed to Professor R. H. Burris for a generous sample of OI- 
ketoglutaric acid. 
* Obtained from Professor I. C. Gunsalus through the courtesy of Professor E. E. 
Snell. The dibarium salt was weighed out and the barium removed by precipitation 
with sodium sulfate. After acid hydrolysis, the solution contained 34 y  per ml. 
of pyridoxal, as determined microbiologically by Jesse C. Rabinowitz. 
3 Prepared from the above solution of pyridoxal phosphate by autoclaving with 
glutamic acid, as proposed by Snell in a private communication, on the basis of the 
analogous reaction which occurs with pyridoxal (1). 
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0 I I I r 
0 2 4 6 
WEEKS ON RATION 
FIG. 1. Effect of vitamin Be-deficiency on transaminase activities and dry weight 
determinations of heart muscle from albino rats. Curves I and IV, 0, normal; 
transaminase activities and dry weights respectively of tissues from rats receiving 
adequate vitamin Be. Curves II and V, 0, deficient; transaminase activities and 
dry weights respectively of tissues from rats placed at weaning age on a diet deficient 
in vitamin Be. Curves III and VI, A, converted; transaminase activities and dry 
weights respectively of tissues from rats receiving adequate vitamin Be from 4 to 
6 weeks after being depleted for a period of 4 weeks. Average standard deviations 
of the experimental determinations are Curve I, Q = 16.9; Curve II, c = 11.9; Curve 
III, g = 5.3; and Curves IV, V, and VI, c = 0.42. 
0 2 4 6 
WEEKS ON RATION 
FIG. 2. Effect of vitamin Be deficiency on transaminase activities and dry weight 
determinations of kidney cortex from albino rats. Curves I and IV, 0, normal; 
transaminase activities and dry weights respectively of tissues from rats receiving 
adequate vitamin Be. Curves II and V, q , deficient; transaminase activities and 
dry weights respectively of tissues from rats placed at weaning age on a diet deficient 
in vitamin Ba; Curves III and VI, A, converted; transaminase activities and dry 
weights respectively of tissues from rats receiving adequate vitamin Be from 4 to 6 
weeks after being depleted for a period of 4 weeks. Average standard deviations 
of the experimental determinations are as follows: Curve I, (r = 4.2; Curve II, ,T = 
2.5; Curve III, o = 4.3; and Curves IV, V, and VI, c = 0.57. 
137 
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138 TRdNSAMINASE AND VlTAMIN Bg 
Results 
Preliminary experiments indicated that rats maintained 6 weeks on a 
vitamin Be-deficient ration had low levels of transaminase in all tissues 
examined; i.e., heart, kidney, liver, brain, and skeletal muscle. Heart 
muscle and kidney cortex were arbitrarily selected as the tissues to be 
utilized in a careful study of the effect of a vitamin Bc-deficient diet on 
transaminase activity. 
The results obtained by determining the transaminase activity in each of 
the two experimental groups at 2 week intervals are indicated in Figs. 1 
and 2 for heart and kidney tissue respectively. Over t.he 6 weeks experi- 
mental period, the animals receiving a vitamin Bg supplement maintained 
their initial transaminase activity, whereas the animals on the vitamin Be- 
deficient ration had transaminase activities of about 40 per cent of the con- 
TABLE I 
Succinic Oxidase and Pyridoxine Dejkiency 
Description of animals* 
IL--- 
Adequate pyridoxine ........ 6 
Convertedt ................. 6 
Pyridoxine-deficient ......... 6 
“ ......... 4-5 
“ ......... 10-12 
Kidney cortex Hear1 muscle 
Qoz No;nJ,mi- 
198 4 
143 4 
183 2 
002 No.;o,flni- 
148 3 
143 3 
128 4 
13s 4 
127 4 
* Albino rats were placed on indicated diets at weaning age. 
t Albino rats receiving adequate pyridoxine from 4 to 6 weeks, after being pre- 
viously depleted on a deficient ration for a period of 4 weeks. 
trol values. The decrease in enzymatic activity is most rapid during the 
initial 2 week period. 
One group of animals was transferred at the end of 4 weeks from the vita- 
min Be-deficient ration to the ration supplemented wiith pyridoxine. A 
marked increase in transaminase activity was noted but the level of activ- 
ity observed in these rats did not reach the level for those rat.s supplemented 
with pyridoxine from the beginning. These observations hold for both 
heart and kidney tissue. 
In order to determine whether transaminase was being affected spe- 
cifically by a vitamin Bg deficiency or whether other enzymatic activities 
were being depressed as well, the activity of succinic oxidase was deter- 
mined on both vitamin Bc-deficient rats and those receiving a pyridoxine 
supplement. Table I shows that the succinic oxidase activity was lower 
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AMES, SARMA, 24ND ELVEHJEM 139 
than that in the normal animals but was not depressed nearly as much as 
in the case of transaminase. 
The ability of various members of the vitamin Be complex to reactivate 
the depressed enzymatic activities was determined. Pyridoxal and pyri- 
doxamine have been previously reported to be inactive, but the addition 
of ATP to either of the above compounds is reported partially to reactivate 
the system (2). The data included in Table II essentially confirm the 
TABLE II 
Effect of Additional Components on Transaminase and Succinic Oxidase Activities of 
Heart Muscle from Pyridoxine-Deficient Albino Rats 
Additional component* 
Pyridoxal. 
“ + ATP.. . 
“ phosphate. 
Pyridoxamine. 
“ + ATP..... 
“ phosphate:. 
I- 
@$ transaminase, 
normal = 2.50 
Control Plus component 
146 146 
164 164 
152 165 
165 160 
146 187 
164 191 
92 112 
147 147 
152 130 
165 175 
147 181 
89 135 
92 145 
@oo, succinic oxidase, 
nomlal = 148 
Control sYei-- 
age 
147 
150 
151 
124 
Plus 
component 
average 
I- 
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57t 
3136 
* Additional components were added at the following levels: pyridoxal and pyri- 
doxamine, 0.30 ml. of 100 y per ml.; ATP, 0.20 ml. of 0.75 M; and pyridoxal phosphate 
and pyridoxamine phosphate, 0.50 ml. of 100 y per ml. 
t This depression of the succinic oxidase system was not observed when ATP 
alone was added to the reaction vessel. 
$ See footnote 3 in the text. 
5 The preparation of pyridoxamine phosphate contained 10 mg. of glutamic acid 
per ml., which itself causes a stimulation of the succinic oxidase systems. 
previous findings concerning pyridoxal and pyridoxamine, but, at the low 
levels added, no significant increase could be shown on the further addi- 
tion of ATP. However, the addition of pyridoxal phosphate and pyridox- 
amine phosphate resulted in substantial increases in the transaminase 
activity. No stimulation was observed when tissues from control animals 
were used and none of the compounds tested were effective in reactivating 
the succinic oxidase system from either deficient or control animals. 
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140 TRANSAMINa4SE AND VITAMIN 136 
DISCUSSION 
The study of transaminase activity as a function of the age of the animal 
showed that in the normal animals no significant change was apparent in 
either heart or kidney within the 6 weeks experimental period. Trans- 
aminase in the vitamin Bs-deficient animals exhibited an initial rapid de- 
crease followed by a plateau in the activity curve. On adding vitamin B6 
to the ration the animals showed an immediate rapid gain in transaminase 
activity. The albino rat apparently has only a small reserve of vitamin 
Bc complex available for conversion into cotransaminase, since the enzy- 
matic activity parallels the growth in showing rapid fluctuations when vita- 
min Bg is removed or returned to the diet. 
Some workers have questioned the results obtained from vitamin-de- 
ficient animals on the basis of inanition and presumed variation in water, 
fat, carbohydrate, and protein content of the deficient tissues, While 
the many secondary effects possible in a study of this type are not over- 
looked, it is believed that this variation has been overemphasized. In 
this investigation, the dry weights were carefully determined for each 
group and, while the dry weight varied a little from the normal in the case 
of kidney cortex, no variation was observed in the case of heart muscle. It 
is important to note that the small variations observed can by no means 
account for the large changes noted in the enzymatic activity. Snell, 
Guirard, and Williams (11) have investigated the concentrations of biotin 
and pantothenic acid in normal and vitamin BG-deficient animals and ob- 
served that there were no changes in the concentration of the first two 
vitamins, while the concentration of vitamin Be was greatly depressed. 
It is evident from these observations that, while the tissues from vitamin- 
deficient animals may exhibit slight alterations in properties not directly 
related to the deficiency syndrome, these are of secondary importance in 
studying the primary effect of the vitamin deficiency. A priori criticisms 
of the vitamin-deficient animal technique for the study of enzymatic 
structure which are based on these secondary changes are not entirely 
justified. 
While an enzyme depending on a particular vitamin for its activity is 
markedly inactivated in the vitamin-deficient animal, it has been shown 
that other enzymes may exhibit some depression of activity. In the at- 
tempt to circumvent these secondary effects, the vitamin or one of its deriv- 
atives could be added to the reaction mixture to determine whether any 
reactivation takes place. A successful experiment constitutes evidence 
that a relationship between the vitamin and the enzyme is probable. When 
the vitamin-deficient animal is used to study enzymatic structure, it is 
suggested that wherever possible two criteria be imposed before a relation- 
ship is postulated; first, the activity of the enzyme must show a marked 
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AMES, SARMA, AND ELVEHJEM 141 
depression in the deficient animal and, on feeding the deficient-animal the 
particular factor, the enzymatic activity must be rapidly regained; and, 
secondly, the addition in vitro of the vitamin or a derivative thereof must 
result in a specific reactivation. 
SUMMARY 
1. The heart and kidney tissues of albino rats maintained on a vitamin 
B6-deficient ration exhibited about 40 per cent of the transaminase activ- 
ity of similar tissues from animals receiving adequate vitamin Bs. 
2. Succinic oxidase was somewhat depressed in the deficient animals, 
but only to 80 to 90 per cent of normal. 
3. The addition of pyridoxal and pyridoxamine with or without added 
adenosine triphosphate was without effect at the low levels tested, but the 
addition of pyridoxal phosphate or pyridoxamine phosphate at approxi- 
mately the same low level resulted in a substantial reactivation of the trans- 
aminase activity. None of the compounds tried had any effect in reac- 
tivating the succinic oxidase system. 
4. The changes in transaminase activity parallel the changes in growth 
when vitamin Bg is withheld or returned to the diet. 
It is a pleasure to thank Professor E. E. Snell for his helpful advice and 
criticism. 
BIBLIOGRAPHY 
1. Snell, E. E., J. Am. Chem. Sot., 6’7, 194 (1945). 
2. Schlenk, F., and Snell, E. E., J. Biol. Chem., 167,425 (1945). 
3. Cohen, P. P., and Lichstein, H. C., J. Biol. Chem., 169, 367 (1945). 
4. Lichstein, H. C., Gun&us, I. C., and Umbreit, W. W., J. Biol. Chem., 161, 311 
(1945). 
5. Schlenk, F., and Fisher, A., Arch. Biochem., 8, 337 (1945). 
6. Leloir, L. F., and Green, D. E., Federation Proc., 4, 96 (1945). 
7. Green, D. E., Leloir, L. F., and Nocito, V., J. Biol. Chem., 161, 559 (1945). 
8. Sarma, P. S., Snell, E. E., and Elvehjem, C. A., J. BioZ. Chem., 166, 55 (1946). 
9. Ames, S. It., and Elvehjem, C. A., J. Biol. Chem., 166, 81 (1946). 
10. Schneider, W. C., and Potter, V. R., J. BioZ. Chem., 149, 217 (1943). 
11. Snell, E. E., Guirard, B. M., and Williams, R. J., J. BioZ. Chem., 143, 519 (1942). 
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Transaminase and pyridoxine deficiency

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Transaminase and pyridoxine deficiency

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