We reported recently (1) the formation of creatine from glycocyamine by rat liver slices; and that 40 to 50 per cent more creatine was formed when methionine was added with the glycocyamine to the Ringer’s solution in which the slices were immersed. Among some thirty odd amino acids, methylated amines, a methylated purine, and betaine only methionine gave this increased rate of methylation. The rate of creatine formation under these conditions is sufficient, if it is of the same order of magnitude in vivo, to make good the entire loss as urinary creatinine

Borsook, Henry

Dubnoff, Jacob W.

English

Caltech Authors - Main

CREATINE FORMATION IN LIVER AND IN KIDNEY* 
BY HENRY BORSOOK AND JACOB W. DUBNOFF 
(Prom the William G. Kerckhoff Laboratories of the Biological Sciences, 
California Institute of Technology, Pasadena) 
(Received for publication, April 12, 1940) 
We reported recently (1) the formation of creatine from glyco- 
cyamine by rat liver slices; and that 40 to 50 per cent more crea- 
tine was formed when methionine was added with the glycocya- 
mine to the Ringer’s solution in which the slices were immersed. 
Among some thirty odd amino acids, methylated amines, a 
methylated purine, and betaine only methionine gave this in- 
creased rate of methylation. The rate of creatine formation 
under these conditions is sufficient, if it is of the same order of 
magnitude in vivo, to make good the entire loss as urinary creati- 
nine. 
If the methylation of glycocyamine in the liver is important 
both as a mechanism and as a site of creatine formation in animals, 
one may expect to find it in the livers of most other animals. 
We accordingly examined the livers of a number of animals for 
their ability to carry out this reaction. At the same time the 
possibility of its occurring in their kidneys was investigated also. 
The findings in this survey, summarized in Table I, were that 
glycocyamine can be methylated by the liver of every animal 
examined; the stimulating effect of methionine observed with rat 
liver was found with some but not all the other animals; the kid- 
ney appears to be unimportant (except in the pigeon) for the 
methylation of glycocyamine without or with methionine. 
The experimental and analytical procedure was the same as was 
described in our previous communication, except that the experi- 
ments with frog liver and kidney were carried out at 25’ instead 
* Presented before the meeting of the American Society of Biological 
Chemists at New Orleans, March 13-16, 1940 (Proc. Am. Sot. Biol. Chenk., 
J. Biol. Chem., 133, p. xv (1940)). 
635 
636 Creatine Formation 
of 38”. All the creatine figures given represent differences in 
chromogenic material in the Jaffe reagent before and after digestion 
with the NC bacteria of Miller and Dubos (4). 
Baker and Miller (3) have reported that creatine is formed 
from unidentified precursors in rat liver and kidney (slices and 
mash). Our findings on this point are that small amounts of 
creatine (beyond the experimental error) were formed from pre- 
TABLE I 
Pormation of Creatine in Liver and Kidney Slices 
Temperature 38”; glucose-Ringer’s solution. The creatine is given as 
mg. per 100 gm. of wet weight of tissue. 
Animal 
cat. ............ 
Dog ............ 
Guinea pig .... 
Frog (25”). ..... 
Pigeon. ......... 
Rabbit. ......... 
Rat. ............ 
i 
Creatine in liver 
After 4 hrs. incubation 
Creatine in kidney 
- 
After 4 hrs. incubation 
cyan- dl-methi- cyam- dl-methi- 
ine mine he mine 
-__ __-__ 
12.0 12.2 21.1 23.2 1.9 2.5 2.4 2.3 
2.9 2.9 3.5 10.3 17 2B 26 26 
1.7 3.4 12.4 12.1 3.0 4.5 8.5 6.6 
2.2 2.2 4.8 8.4 36 44 47 45 
1.0 2.0 11.8 16.0 10.8 10.7 17.9 22.3 
5.8 5.7 . 26.4 12.0 9.4 15.2 15.0 15.4 
1.4 2.8 20.6 28.4 10.8 18.6 16.3 18.2 
_____ 
The initial values are lower than those given by Baker and Miller (2, 3) 
for rat liver and kidney. In our procedure the slices are washed in a large 
volume of Ringer’s solution for at least 3 minutes before they are trans- 
ferred to the reaction vessels or submitted to analysis. Preformed creatine 
undoubtedly diffuses out of the slices during this washing. 
existing precursors in the liver slices of the guinea pig, pigeon, and 
rat. Kidney slices gave significant increases in every case except 
in the pigeon. 
After the cell structure of rat liver and kidney was destroyed, no 
creatine was formed from preexisting precursors nor from glyco- 
cyamine without or with methionine. The homogenizing proce- 
dure of Potter and Elvehjem (5) was used to disrupt the cells. 
It would seem that the creatine formed by chopped rat liver and 
H. Borsook and J. TV. Dubnoff 637 
kidney from preexisting precursors (Baker and Miller (3)) must 
be ascribed to the intact cells in the mash rather than to a liberated 
enzyme. 
It is difficult to account for the large (absolute) amounts of 
creatine formed from preexisting precursors in the kidney slices 
in every case except in the pigeon, in view of the inability of cat, 
dog, rabbit, and rat kidney slices to methylate added glyco- 
cyamine, even in the presence of added methionine. The one 
animal which was negative in this respect-the pigeon-was the 
only animal whose kidney gave a positive effect with glycocya- 
mine and a further increase with methionine. 
When the Ringer’s solution contained glycocyamine, there were 
large increases in creatine with the liver slices of every animal 
except the dog. In the interpretation of this negative effect in 
the case of the dog the positive effect observed when methionine 
was added with the glycocyamine must be taken into account. 
One possible interpretation is that the preexisting concentration 
of the glycocyamine-methylating agent is very low in dog liver- 
lower than in the livers of any of the other animals investigated. 
With kidney slices the added glycocyamine was not methylated 
or was methylated only to an insignificant extent in every case 
except in the guinea pig and pigeon. 
Bodansky (6) found an increase in the creatine content of rat 
kidney after glycocyamine was fed. Our interpretation of this 
increase was that it arose from temporary storage in the kidney 
of creatine synthesized in the liver from the glycocyamine fed 
(1). This interpretation is based on the finding that rat kidney 
slices do not methylate glycocyamine under conditions in which 
methylation readily occurs in liver. Bodansky concurs in this 
interpretation (personal communication). 
Methionine accelerated the methylation of glycocyamine by 
liver slices of the dog, frog, pigeon, and rat. This stimulation was 
not observed with the cat, guinea pig, and rabbit. Two possible 
reasons for this failure are that the livers of these animals con- 
tained a plethora of the methylating agent, or that the relatively 
high concentration of the unnatural isomer in the dl-methionine 
used was inhibitory (as in the rabbit liver slices). 
The pigeon was the only animal whose kidney slices were 
stimulated by methionine in their methylation of glycocyamine. 
Creatine Formation 
The pigeon is exceptional as far as creatine formation is concerned 
in that its kidney appears to share this function with its liver and 
resembles in this respect the livers rather than the kidneys of the 
other animals. 
The negative effect of methionine wit,h the kidney slices of all 
the animals tried except the pigeon is a little unexpected. Oxida- 
tive deaminat’ion is more active in kidney than in liver slices (7); 
and after oxidativc deamination the lability of the S-methyl 
group is increased (8). It might have been expected therefore 
that the methylation of glycocyamine would be more active in 
kidney than in liver. This is not the case. 
Our finding in this survey, that methylation of glycocyamine 
is an active function of the livers of all the animals examined, 
strengthens the conclusion that creatine is formed normally in the 
liver by the methylation of glycocyamine. It would be surprising 
if this is a fortuitous coincidence of little or no physiological 
significance. Other mechanisms and other sites of creatine form- 
ation are? of course, not excluded by these findings. 
The significance of methionine here is less clear. Our findings 
suggest that it is the methylating agent of glycocyamine in liver 
or its precursor. We are hesitant at present to accept methionine 
itself as the methylating agent, because its effect is small consider- 
ing the concentration used. It may be that the actual methylating 
agent is the product of the oxidative deamination of methionine. 
This and other possibilities are now under investigation. 
Du Vigneaud and coworkers (9) have shown that the presence 
of choline in the diet enables the animal to utilize homocystine OI 
homocysteine in place of methionine. They have suggested that 
choline enables the body to methylate the sulfhydryl group of 
homocysteine by furnishing the required methyl group, and 
further that methionine may furnish the methyl groups of choline. 
The reality of the latter relationship is now established by direct 
proof recently presented by du Vigneaud.’ 
Our observations suggest that the S-methyl group of methionine 
may participate also in the formation of the N-methyl group of 
creatine. In this connection and in view of our findings with dog 
liver slices it is interesting that Stekol and Schmidt (10) found 
1 Verbal communication at the meeting of the American Society of 
Biological Chemists at New Orleans, March 13-16, 1940. 
H. Borsook and J. W. Dubnoff 639 
in the dog an increase in urinary creatinine after dl-methionine was 
fed, and none after glycine, glutamic acid, or Z-pyrrolidonecar- 
boxylic acid. 
SUMMARY 
1. A survey was made of the possibility of creatine formation 
in vitro by slices of the livers and kidneys of the cat, dog, guinea 
pig, frog, pigeon, rabbit, and rat. 
2. Evidence of the existence of small amounts of creatine pre- 
cursors in the livers of the guinea pig, pigeon, and rat, and in the 
kidneys of all of t.he above animals except the pigeon was found. 
3. This finding appears to be much less significant quantita- 
tively than the much more active glycocyamine-methylating 
function of the liver slices of all animals investigated. Only 
guinea pig and pigeon kidney slices possessed this ability. 
4. The above findings are interpreted as indicating that creatine 
normally is formed by the methylation of glycocyamine in the 
liver. 
BIBLIOGRAPHY 
1. Borsook, H., and Dubnoff’, J. W., J. Biol. Chem., 132, 559 (1940). 
2. Baker, Z., and Miller, B. F., J. Biol. Chem., 130, 393 (1939). 
3. Baker, Z., and Miller, B. F., J. Biol. Chem., 132, 233 (1940). 
4. Miller, B. F., and Dubos, R., PTOC. Sot. Exp. Biol. and Med., 36, 335 
(1936). Dubos, R., and Miller, B. F., J. Biol. Chem., 121,429 (1937). 
5. Potter, V. R., and Elvehjem, C. A., J. Biol. Chem., 114, 495 (1936). 
6. Bodansky, M., J. Biol. Chew, 116, 641 (1936). 
7. Krebs, H. A., Z. physiol. Chem., 217, 191 (1933). 
8. Waelsch, H., and Borek, E., J. Am. Chem. Sot., 61, 2252 (1939). 
9. du Vigneaud, V., Chandler, J. P., Moyer, A. W., and Kcppcl, 1). M., J. 
Biol. Chem., 131, 57 (1939). 
10. Stekol, J. A., and Schmidt, C. I,. A., Univ. California. Pub. I’hysiol., 8, 
31 (1933). 


Creatine formation in liver and in kidney

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Creatine formation in liver and in kidney

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