THE TlUNSFORMATION OF CREATINE INTO CREATi

ININE BY THE MALE AND FEMALE HUMAN

ORGANISM.

BY WILLIAM C. ROSE, RUTH H. ELLIS, AND OSCAR C. HELMING.

(From the Laboratory of Physiological Chemistry, University of Illinois,
Urbana.)

(Received for public&ion, February 16, 1928.)

Since the publication of the classical paper of Folin (1906), in

which he concluded that creature and creatinine are independent
of each other in metabolism, a number of articles have appeared
indicating that, contrary to Folin’s views, a limited amount of
creatine may be transformed into its anhydride in the animal or-
ganism. (For a review of the literature see Hunter, 1922.) The
most noteworthy contribution dealing with this problem is the
recent paper of Benedict and Osterberg (1923). These authors
have sho.wn that in dogs the long continued oral administration of
creatine leads to a very gradual increase in creatinine excretion
which, after several weeks, may exceed the normal output by 22 to
33 per cent. This observation permits of no reasonable doubt
that dogs can dehydrate creatine; but the remarkable feature is
the fact that days or even weeks are required for the completion
of the transformation.
In man no investigations of a similar sort have been reported.
In experiments conducted several years ago, and involving the
administration of large doses of creatine over brief periods of
time, Rose and Dimmitt (1916) observed unmistakable increases
in creatinine elimination. The paper of Benedict and Osterberg
suggested to us the desirability of determining whether the reac-
tion is as slow and difl?icult for the human organism to accomplish
as it is for the dog. But we were especially interested in comparing
the behavior of the male and female human subjects. Such a
comparison should be of interest in connection with the well known
fact that women are more subject to creatinuria than are men.
171

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172 Creatine Metabolism

If the creatinuria of the female is due to a failure in creatine de-

hydration, this fact should be manifested following the prolonged
administration of creature. On the other hand, if the difference
between the sexes is due to unlike powers of retention, this also
should be capable of demonstration. With these points in mind,
two prolonged experiments were carried out, one upon a male and
the other upon a female. The results, which are outlined below,
are so clear cut and agree so closely with the data secured by Bene-

TABLE I.
Composition of Diets.

Male subject. Female subject.

om. am.
Bread (whole wheat). . . . . . . . . . . . . . . . . . . . . . . . . . 200 200
Butter .................................... . ... 75 75
Eggs (raw weight). ........................ . ... 100 100
Milk. ..................................... .. .. 1200 1200
Potato (boiled). ........................... . 200 150
Beans (canned Lima, cooked). ............. . 125 125
Lettuce ................................... . .. . 100 100
Mayonnaise. ........................... . .. . . . . 20 20
Bran ...................................... .. 25 25
Sugar ..................................... .. .. 15 15
Jelly. ..................................... . .. 40 40
Orange .................................... .. . 300 300

Nitrogen, content,* gm .......................... 14.75 14.55

Energy value,* calories ......................... 2970 2920
I I
* Most of the nitrogen and energy data are taken from Atwater, W. O.,
and Bryant, A. P., U.S. Dept. Agric., Office Exp. Stations, Bull. 18 (Revised),
1906; but the milk, mayonnaise, jelly, beans, and bran were analyxedfor
total nitrogen.

diet and Osterberg upon dogs, that it seemedto us unnecessary to

make additional experiments of this kind.
The subjects were normal graduate students in this laboratory,
and were selected for the purpose in question becauseof their simi-
larity in body weight. Both were subjected to exactly the same
treatment as regards dosage and length of time of the creatine
administration. The diets employed are shown in Table I. As
will be observed, they are identical with the exception that the
 Rose, Ellis, and Helming 173

male subject ingested a somewhat larger quantity of potatoes than

did the female. The creatine was a commercial product which
had been purified by repeated recrystallizations until entirely free
of creatinine, and then dried in vocuo. Three such preparations
were employed. Analyses showed the presence of traces of water
of crystallization in each, for which allowance was made in calculat-
ing the creatine intake. As indicated in Tables II and III, the
daily dosage of anhydrous creatine (present in 1 gm. quantities of
the three preparations) amounted to 0.97,0.95, and 0.96 gm. The
creatine was taken in two equal amounts mixed with the food at
the morning and noon meals. Great care was exercised in the
urine analyses to insure the maintenance of identical conditions
throughout. But for a few unavoidable exceptions the volumes
were always made up to the same quantity. Total nitrogen was
determined by the Kjeldahl-Gunning method, while total and
preformed creatinine were estimated by the Folin (1914) proce-
dures. In the latter, creatinine of known purity was employed
as the standard. The subjects ingested the constant diets for
several days preceding the collection of the first urines.
The results of the two experiments are detailed in Tables II and
III. In each experiment the creatine administration was begun
after two preliminary periods of 7 days each and was continued
for seven periods of 7 days each. Five after periods followed
during which no creatine was given. In the male subject (Table
II), the remarkable fact is brought out that no unchanged crest-
tine was excreted during the entire experiment. Perhaps this
would not occur invariably in males, inasmuch as the power of
retention might depend somewhat upon the degree of saturation
of the tissues when the creatine administration was inaugurated.
The creatinine output showed no appreciable increase until the
third period of creatine feeding, and from that time slowly but
steadily increased until the end of the first after period. Indeed,
the average daily output was slightly larger for the first after period
than for the preceding weeks during which creatine was adminis-
tered. The maximum for a single day was on tbe last day of the
first after period when the output amounted to 2.22 gm., or an
increase over the average for the preliminary periods of 0.44 gm.
(24.7 per cent).
During the second after period the creatinine began to decline
 TABLE II.
Fate of Ingested Creatine.
Subject 1, Male.
This subject excreted no creatine as creatinine throughout the experi-
ment.
- - -
urine. C ‘r&E
1Body nine
D&R‘. v,reight mef- Ramarb.
3&i-
e nine. B cient
-- _ -- --
kg. gm. gm.
Feb. 28 72.7 1.82 First preliminary period.
Mar. 1 1.84
“ 2 1.80
“ 3 1.80
“ 4 1.76
“ 5 1.70
“ 6 1.75
-- -- -- - _ -
C1 1.78 14.5 Average of 7 days.
-- -- . - _-
Mar. 7 72.3 1.74 Second preliminary period.
“ 8 1.73
“ 9 1.77
“ 10 1.78
“ 11 1.80
“ 12 1.82
“ 13 1.73
-- -- - _ --
(I 1.77 24.5 , Average of 7 days.
-_ -- -- - --
Mar. 14 72.3 1.72 First period of creatine
“ 15 1.74 administration.
“ 16 1.75
“ 17 1.68
“ 18 1.77
“ 19 1.72
“ 20 1.81
- -- - _- -.
o.97f 1.74 1 24.1 1 Average of 7 days.
-. -- -- - -.
Mar. 21 72.1 1 1.81 Second period of creatine
“ 22 1.7( i administration.
LL 23 Sample 10st.
” 24 1.71i
“ 25 1.811
“ 26 1.8( )
4c 27 1.8~I
- -. -- -
0.97 11.81 1.8 1 25.0 Average of 6 days.
- - -
* Same amount for each day of period.
174
 Rose, Ellis, and Helming 175
TABLE II~ontinued.

i hati-
Body nine Remarke.
Data.
P might hati- coef-
nine. icient.
--

kg. gm. gm. om.

Mar. 28 72.7 1.84 Third period of creatine
“ 29 1.87 administration.
“ 30 1.89
“ 31 1.89
Apr. 1 1.87
“ 2 1.81
“ 3 1.34
-_ -
0.97’ 11.2 1.86 25.7 Average of 7 days.
-_ -
Apr. 4 72.2 0.97 1.85 Fourth period of areatine
“ 5 0.97 1.90 administration.
“
6 0.95 1.77
“
7 0.95 2.05
“
8 0.95 1.99
‘6
9 0.95 2.00
“ 10 0.95 1.92
-_

0.96 11.9 1.93 26.7 Average of 7 days.

-. - A
Apr. 11 72.8 1.88 Fifth period of creatine
“ 12 2.02 administration.
“ 13 1.98
“ 14 1.96
“ 15 1.98
“ 16 2.09
“ 17 2.11
-_

0.95 12.2 2.00 27.5 Average of 7 days.

-. -
Apr. 18 72.6 2.14 Sixth period of creatine
“ 19 1.95 administration.
“ 20 1.9s
“ 21 2.01
(( 22 1.95
” 23 2.01
“ 24 2.11
-.

0.95 11.i 2.a 27.9 Average of 7 days.

-

* Same amount for each day of period.

176 Creat,ine Metabolism
TABLE II-continued.

I
-

Urine.
Zreati-
Body nine Remarks.
Date. weight CWJ&
Creati- Bcient.
‘i? nine.
.-

kg. !Jm. gm. gm.

Apr. 25 72.4 0.95 2.10 0
,
Seventh period of creatine
“ 26 0.95 2.17 administration.
” 27 0.95 2.05
” 28 0.95 2.16
“ 29 0.96 1.98
“ 30 0.96 2.17
May 1 0.96 2.03
- _-

0.95 11.1’ 2.09 28.! 9 Average of 7 days.

-
May 2 72.9 2.06 First after period.
“ 3 2.10
“ 4 2.01
I‘ 5 2.17
“ 6 2.21
“ 7 2.21
“ 8 2.22
_ --
0 11.7 2.14 29.4 Average of 7 days.
- -- -
May 9 72.4 2.17 Second after period.
“ 10 2.13
“ 11 1.93
“ 12 1.94
“ 13 1.85
“ 14 2.02
“ 15 2.04
_ --
0 12.2 2.01 27. 8 Average of 7 days.
- - -,
May 16 72.2 1.95 Third after period.
‘I 17 2.01
“ 18 1.99
“ 19 1.88
“ 20 1.83
“ 21 1.97
u 22 1.89
-- --
0 11.7 1.93 26.7 Average of 7 days.
-
 Rose, Ellis, and Helming 177
TABLE II--conchded.

keati-
Body nine
might coef-
&nt.

hl. 8”.
May 23 72.4 1.96 Fourth after period.
“ 24 1.94
“ 25 1.88
“ 26 1.90
‘( 27 1.75
“ 28 1.84
“ 29 1.75

1.86 25.7 Average of 7 days.

May 30 72.2 1.88 Fifth after period.

“ 31 1.53
June 1 1.91
“ 2 1.80
“ 3 1.75
“ 4 Sam] Ple lost ;.
“ 5 1.79
~- _-
11.04 1.83 25.3 Average of 6 days.
-

and continued to diminish slowly and somewhat irregularly to

the end of the experiment. On the last day of the fifth after period
the output was practically identical with the average daily excre-
tion of the fore periods.
The results of the experiment upon the female (Table III)
manifest some interesting variations from the data secured with
the male subject. During the preliminary periods, the female
excreted small and variable amounts of creatine. With the be-
ginning of the creatine feeding, no alteration in the creatine out-
put was observed until the third period. It then began to increase
quite rapidly and reached a maximum in the fourth period, when
approximately one-third of that administered reappeared un-
changed in the urine. During the last three periods of creatine
feeding the average output remained fairly constant although the
figures from day to day show rather wide fluctuations. On the
2nd day of the first after period the creatine excretion dropped to
178 Creatine Metabolism
TABLE III.
Fate of Ingested Creatine.
Subject 2, Female.

Body
I
%z- -
T
- ( h3ti
nine
Date. I veighl ‘30%
in-
:ested.
(hesti tine &I3 ’me5-
oient.
‘P nine. cresti,
nine.
_ _ --
k0. 0m. m. m. 07%
Feb. 28 72.9 1.25 0.09 First preliminary
Mar. 1 1.27 0.12 period.
“ 2 1.27 0.08
“ 3 1.25 0.08
“ 4 1.25 0.07
“ 5 Lt.26 0.10
“ 6 1.25 0.08
- _- .- _-
0 11.97 1.26 0.09 17.3 Average of 7 days.
- _ _-
Mar. 7 72.1 1.26 0.05 Second preliminary
“ 8 1.26 0.06 period.
“ 9 1.26 0.05
“ 10 1.28 0.07
“ 11 1.30 0.03
“ 12 1.31 0
“ 13 1.26 0.07
- .- _-
1.52 I 1.28 0.05 17.8 Average of 7 days.
.- - _-
Mar. 14 72.7 1.25 0.06 First period of crea-
“ 15 1.26 0.07 tine administration.
“ 16 1.36 0.05
‘( 17 1.31 0.05
“ 18 1.37 0.03
“ 19 1.28 0.05
“ 20 1.33 0.08
- - _-
0.97' k 10.89 I 1.31 0.06 18.0 Average of 7 days.
-- _- _-
Mar. 21 73.3 1.33 0.05 Second period of crea-
“ 22 1.36 0 tine administration.
“ 23 1.37 0
“ 24 1.38 0.10
“ 25 1.37 0.08
“ 26 1.42 0.10
“ 271 1
- - _-
0.97' r 11.47 1.37 0.06 18.7 Average of 6 days.
- - - -
* Same amount for each day of period.
t Mens’truation; urine not collected.
 Rose, Ellis, and Helming 179
TABLE I~-~On~~?W3d.
-

Creati.
Date. Ebd nine Fbmarks.
vWI 2 t coeffi-
cient.

--
kg. gm. m. gm. m.
Mar. 28 73.3 Third period of crea-
“ 29 T tine administration.
“ 30 1.42 0.16
‘( 31 1.46 0.27
Air. 1 1.41 0.19
(‘ 2 1.40 0.32
“ 3 1.38 0.36
--
0.97 12.1 1.40 0.26 19.1 Average of 5 days.
--
Apr. 4 73.3 0.97 1.46 0.35 Fourth period of crea-
“ 5 0.97 1.42 0.30 tine administration.
“ 6 0.95 1.48 0.27
“ 7 0.95 1.41 0.40
“ 8 0.95 1.49 0.30
“ 9 0.95 1.43 0.43
“ 10 0.95 1.44 0.34
_-
0.96 11.6 1.45 0.34 19.8 Average of 7 days.
--
Apr. 11 73.5 1.46 0.34 Fifth period of crea-
“ 12 1.49 0.30 tine administration.
“ 13 1.44 0.31
(‘ 14 1.50 0.29
“ 15 1.53 0.30
“ 16 1.55 0.26
“ 17 1.54 0.19
_-
0.95 10.9 1.50 0.28 20.4 Average of 7 days.
-
Apr. 18 73.4 1.52 0.20 Sixth period of crea-
“ 19 Sample lost tine administration.
“ 20 1.47 0.33
“ 21 1.43 0.22
“ 22 1.49 0.21
“ 23,
I‘ 24 I
_- --
0.95; 11.9 1.49 0,24 20.3 Average of 4 days.
-

* Same amount for each day of period.

t Menstruation; urine not collected.
180 Creatine Metabolism
TABLE III-Continued.
-
Urine.
- - - c:reati.
Body nine Remarks.
reight. C reati- ,:005-
‘P
1
1he. ,
1
cient.
-- .- _-
kn. gm. m. m. m.
Apr. 251 73.4 0.95 ISeventh period of
“ 26 0.95 I 1.55 0.37 creatine administra-
“ 27 0.95 3L.53 0.33 tion.
“ 28 0.95 11.43 0.27
“ 29 0.96 I 1.56 0.33
“ 30 0.96 I1.52 I9.50

May 1 0.96 11.46 tD.22

_- -- .- - _-
12.3 91 1.51 9.34
0.95
_- -- .-
I
.- 20.6 --
Average of 6 days.
May 2 73.2 1.53 I0.43 First after period.
Cc 3 1.50 ,0.05
(‘ 4 1.50 ,
0.06
“ 5 1.48 0.03
“ 6 1.56 0
“ 7 1.59 0.06
“ 8 1.54 0
- -_ - -- .- -_ - -
0 11.5 i7 1.53 0.09 Average of 7 days.
-. -_ -- _ - _20.9 --
May 9 ‘73.8 1.52 0.09 Second after period.
(’ 10 1.44 0
‘( 11 1.49 0
“ 12 1.46 0
“ 13 1.57 0
“ 14 1.50 0
“ 15, 1.53 0
- -- _ -_
12.110 1.50 0.01 20.2 I Average of 7 days.
- -. -_ _ -
May 16, 73.8 1.46 Third after period.
“ 17 1.46
“ 1EIt
“ 1E)t
“ 2f )t
‘( 211 1.41
Cc 2:!, 1.33 ,
- - -
11. ii. 1.43 , 0 19.8i- Average of 4 days.
- - - -

t Menstruation; urine not collected.

 Rose, Ellis, and Helming 181
TABLE III-conduded.

Creati
Body ClW&- nine
Remarks.
weight Creati tine 8~ coe5-
nine. crertti- cient.
nine.

ko. 0-L m.
May 23 73.4 1.38 0 Fourth after period.
“ 24 1.40 0
“ 25 1.42 0
“ 26 1.38 0
“ 27 1.37 0
“ 28 1.42 0
“ 29 1.36 0
1.39 0 18.9 Average of 7 days.
May 30 73.4 1.48 0 Fifth after period.
‘< 31 1.40 0
June 1 1.41 0
“ 2 1.35 0
(‘ 3 1.34 0
(‘ 4 1.33 0
“ 5 1.43 0
1.39 0 18.9 Average of 7 days.

its normal level, and after the 1st day of the second period failed to
appear again in the urine. The latter fact is peculiar and unex-
pected in view of the almost constant excretion of small amounts
during the preliminary periods.
The urinary creatinine showed a tendency to rise during the 1st
week of creatine administration, and the increase became unmis-
takable during the 2nd week. The output continued to rise
until the fifth period, and remained fairly constant from that time
until the end of the second after period. As in the male subject,
so in this individual, the maximum daily excretion was secured
during the first after period when on the 6th day it amounted to
1.59 gm., or an increase of 0.32 gm. (25.2 per cent) over the aver-
age output for the preliminary periods. Following the with-
drawal of creatine from the ration, the excretion of creatinine
diminished to the end of the experiment, when it was almost down
to the level of the fore periods.
182 Creatine Metabolism

In accordance with the procedure of Benedict and Osterberg

we have calculated the percentage of the retained creatine which
was transformed into creatinine by the two subjects. Inasmuch
as neither of our subjects manifested decided alterations in body
weight we have thought it advisable to compute the expected
creatinine on the basis of the amounts actually excreted during
the preliminary periods, rather than on the basis of the creatinine
coefhcients. A summary of the calculations in the two experiments

TABLE IV.

Percentage Recovery of Administered Creatine, and Percentage Transforma-

tion of the “Retained Creatine”* into Creatinine as Calcu-
lated from Tables II and III.
Figures for creatine are expressed as creatinine.
-

Subject.

-
Lt 1
1, male.
2, female.
-
* In accordance with the procedure of Benedict and Osterberg (1923)
we have used the term “retained oreatine” to denote the creatine not ex-
creted as such.
t In calculating the total excretion of creatine and creatinine allowance
was made for the days in which, because of accidental loss or the menstrual
cycle, the urines were not collected. In each instance of this sort the figure
representing the average output for the other days of the same’period was
employed in the calculation.

is presented in Table IV. As will be observed, 33.0 and 42.2 per

cent of the retained creatine were transformed into creatinine in
the male and female subjects respectively. These figures are
rather close to those secured by Benedict and Osterberg in dogs,
which manifested transformations of 29.1 to 34.2 per cent. The
dosage of creatine in our experiments was relatively much smaller
than that employed by Benedict and Osterberg. The latter ad-
ministered 37.5 to 43.0 mg. (expressed as creatinine) per kilo of
body weight per day. In our subjects, the daily dosage amounted
 Rose, Ellis, and Helming 183

to 11.4 mg. per kilo. Thus it is rather striking that the two series
of investigations involving such widely different dosages should
show such closely agreeing values for creatine dehydration. Bene-
dict and Osterberg suggest that creatine is metabolized in two or
more ways, and that from 3 molecules of creatine only 1 mole-
cule of creatinine results. The present investigation appears to
ocnfirm this opinion.
Our data indicate quite clearly that the creatinuria of females
is not associated with an inability to transform creat.ine into
creatinine. Perhaps the fact that the female excreted a some-
what larger amount of extra creatinine than the male is not signifi-
cant; but at least she was just as effective in accomplishing the
transformation as was the subject of opposite sex. On the other
hand, the female eliminated 6.0 gm. of unchanged creatine in
excess of the expected amount, while the male excreted none. The
total recovery of administered creatine amounted to 50.8 per cent
in the female as compared with 33.0 per cent in the male. This
fact suggests that females may be less efficient than males in stor-
ing or metabolizing that portion of the creatine which does not
yield creatinine. If this conception is correct, one might expect
to observe the excretion of creatine in women under circumstances
which do not induce creatinuria in men. Perhaps the intermit-
tent excretion of creatine by females and the creatinuria of high
protein diets may be explained upon this basis.
The body weights of our subjects do not indicate that creatine
ingestion exerts an influence upon nitrogen retention in the human
organism such as was observed by Benedict and Osterberg in dogs.
The weight of the male was quite constant throughout the experi-
ment. The female showed a slight gain, but scarcely enough to
justify the assumption that-the administered creatine was in-
strumental in the change. It must be recalled, however, that the
dosage employed by Benedict and Osterberg was approximately
3.5 times as large as that used by us. This difference may account
for the greater influence upon nitrogen retention observed by the
former investigators.
SUMMARY.

1. Long continued feeding experiments have shown that male

and female human subjects possess the power of transforming crea-
184 Creatine Metabolism

tine into creatinine to approximately the same degree. This finding

indicates that the creatinuria of women is not to be attributed to
a failure in the conversion of creatinine into its anhydride.
2. Approximately one-third of the retained creatine was re-
covered as creatinine. This would seem to indicate, as Benedict
and Osterberg suggest, that creatine is metabolized by two or
more methods only one of which yields creatinine.
3. In the female, creatine reappears unchanged in larger
amounts following its oral administration than in the male sub-
jected to the same dosage. This suggests that the powers of re-
tention and storage of creatine, or the ability to catabolize it by
methods which do not yield creatinine, may be less effective in
women than in men. Obviously, our data upon a single subject
of each sex are not sufficient to justify one in making a positive
assertion in this regard.

BIBLIOGRAPHY.

Benedict, S. R., and Osterberg, E., J. Biol. Chem., 1923, lvi, 229.
Folin, O., Hammarsten Festschrift, Upsala, 1906,l.
Folin, O., J. Biol. Chem., 1914, xvii, 469.
Hunter, A., Physiol. Rev., 1922, ii, 586.
Rose, W. C., and Dimmitt, F. W., J. Biol. Chem., 1916, xxvi, 345.