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On the proposed origin of creatinine from creatine phosphate
ARCHIVES
OF BIOCHEMISTRY
AND
BIOPHYSICS
86,
483-486
(1959)
On the Proposed Origin of Creatinine from Creatine Phosphate’ John F. Van Pilsum and Bruce Hiller From the Department of Physiological Chemistry, University Minnesota Medical School, Minneapolis, Minnesota Received
of
June 11, 1959
INTRODUCTION In 1947 Borsook and Dubnoff (1) calculated that the spontaneous, nonenzymic transformation of creatine phosphate to creatinine plus inorganic phosphate was sufficient to account for the urinary excretion of creatinine. They indicated that the possibility of an enzymic conversion of creatine phosphate to creatinine was not excluded, but no enzyme had been identified. In 1954, Caputto (2) reported the presence of an enzyme in normal rabbit muscle extract which catalyzed the transformation of creatine phosphate into creatinine in the presence of glycogen plus phosphate or glucose l-phosphate. In 1955, Caputto and Carpenter (3) reported that the phosphate of creatine phosphate was transferred to hexose monophosphate with the production of creatinine and hexose diphosphate. Details of their procedures were not published until 1958 (4). Ennor and Morrison (5) have stated in a review, “Biochemistry of the Phosphagens and Related Guanidines,” that no doubts have been cast on these unconfirmed reports of Caputto et al. The experiments of Caputto et al. have been reinvestigated using newer analytical procedures for creatine, creatine phosphate, and creatinine (6). It was found that muscle extracts from normal rabbits catalyzed the conversion of creatine phosphate to creatine, not to creatinine. METHODS Male albino rabbits weighing 1-2 kg. were killed by a blow on the head. A sample of back muscle was immediately removed and ground in a cold mortar and pestle with cold salt solution (4). Then 0.1 ml. of the muscle extract was added to test tubes containing 11 pmoles creatine phosphate, 30 pmoles glucose l-phosphate, and 20 pmoles cysteine. Total volume of the incubation mixture was 2.5 ml. Three control tubes were used: Control No. 1 contained muscle extract, glucose l-phosphate, and r Supported by University of Minnesota Graduate School Grant, No. 387-32012409 and by a grant from the American Cancer Society, Minnesota Division. 483
484
VAN
PILSUM
AND
HILLER
cysteine. The creatine phosphate was added after the mixture had been incubated and the enzyme reaction stopped. Control No. 2 contained muscle extract, creatine phosphate, glucose l-phosphate and cysteine. The reaction was not incubated and was stopped immediately after the addition of the muscle extract. Control No. 3 contained water (instead of muscle extract), creatine phosphate, glucose l-phosphate, and cysteine. The mixture was incubated and stopped. Incubation was for 15 min. at 30°C. without shaking and with an air phase over the solutions. The analytical procedures were as follows:
I. Creatinine Production A. Jaffe (Alkaline Picric Acid) Method. The volumes of incubation mixtures and color reaction were ten times the amount used by Carpenter et al. (4)) so the enzyme reactions were stopped by the addition of 10 ml. alkaline picric acid solution and the volume made up to 30 ml. with distilled water. B. o-Nitrobenzaldehyde Method (6). The large amounts of cysteine and glucose phosphate in the incubation mixture prevented normal color development. The enzyme reactions were stopped by the addition of 0.2 ml. of 2.5 N NaOH and 1 drop of 14% o-nitrobenzaldehyde. The solution was allowed to stand 20 min. at room temperature, 0.5 ml. of HzSOd-phosphate buffer mixture was added, and the mixture was heated for 15 min. in a boiling water bath. The methylguanidine formed by the reaction with o-nitrobenzaldehyde was separated from the interfering cysteine and glucose l-phosphate by allowing the solution to run through a column (1.5 X 5 cm.) containing Amberlite IRA 400 (OH) ion-exchange resin. The column was washed with 5 ml. distilled water, and this wash added to the eluate. Five milliliters of this mixture was withdrawn, and the modified Sakaguchi color reaction developed (6).
II. Creatine Phosphate Utilization The enzyme reactions were stopped by chilling the test tubes in an ice bath and adding 1.0 ml. of ice-cold 3 N perchloric acid. The mixtures were centrifuged in the cold at about 3000 r.p.m. for 10 min., the supernatant was decanted, and cold 30% KOH was added dropwise to the supernatant solution until the pH was 7-8. The solutions were allowed to stand 0.5-l hr. in the cold, and the potassium perchlorate was removed by centrifugation for 3-5 min. at about 3000 r.p.m. The supernatant was decanted and filtered. One milliliter of this filtered supernatant (solution A) was placed in a column (1.5 X 5 cm.) containing Dowex 50, X-16, 100-200 mesh, ion-exchange resin. The column was washed with 10 ml. cold distilled water, and the wash was added to the eluate. This mixture contained the creatine phosphate and was diluted 1:3 with distilled water and was analyzed for creatine by the o-nitrobenzaldehyde procedure (7).
III.
Creatine Production
One milliliter of solution A (in the creatine phosphate procedure ) was diluted with 7 vol. water, and 1 ml. of this dilution was used for the diacetyl color reaction (8). Creatine will react with ol-naphthol and diacetyl in the presence of alkali to yield a color, whereas creatine phosphate will not. .A11 measurements were made with a Bausch and Lomb “Spectronic 20” colorimeter.
485
CREATINE PHOSPHATE TO CREATININE
TABLE I Produclion of Creatine from Creatine Phosphate by Extracts of Normal Rabbit Muscle The reaction tube contained 11 rmoles creatine phosphate, 0.1 ml. extract, 30 pmoles glucose l-phosphate, and 20 pmoles cysteine. Total volume was 2.5 ml., and incubation was for 15 min. at 30°C. without shaking and with an air phase over the solutions. The compositions of the three control tubes were averaged and compared with the incubation mixtures to give net production or utilization. The compositions of the control tubes are explained in the text. Composition
of incubation and (pwles)
Creatine
Creatinine “0-nitro” method
Pk:ixi;;id
Reaction tube Control 1 Control 2 Control 3 Net production utilization
or
control
D&-t!&
0.044 0.035 0.039 0.035 0.008
0.50 0.29 0.21 0.20 0.27
mixtures Creatine “0-nitro”
phosphate method
8.1 10.7 11.2 10.4 2.6
2.3 0 0 0 2.3
RESULTS The results obtained with a normal rabbit muscle extract are shown in Table I. Similar results were obtained with extracts from four additional normal rabbits. The creatinine production, as measured by the alkaline
picric acid method, of 0.27 pmole was 60 % greater than the value of 0.168 pmole reported by Carpenter et al. (4). The creatinine production of 0.008 pmole as measured by the o-nitrobenzaldeyhde procedure was not considered to be a significant production. The creatine phosphate utilization of 2.6 pmoles was 70% greater than the value for “phosphate transfer” of 1.112 pmoles
(12-min.
incubation
period)
reported
by Carpenter
et al. (4).
The 2.6 pmoles creatine phosphate utilized was similar to the production 2.3 pmoles creatine, as measured by the diacetyl color reaction.
of
DISCUSSION
The product formed in the incubation that was responsible for a positive color reaction with picric acid in the presence of alkali is not known. That it is not creatinine was shown by the fact that it could not be measured by the o-nitrobenzaldehyde procedure, and creatinine added to the incubation mixtures
was recovered
completely
by this procedure.
Its color reaction
with picric acid was not the same as the reaction of creatinine with picric acid. For example, after the 20-min. color development the optical density
486
VAN PILSUM AND HILLER
of the solution increased with time, whereas the optical density of the creatinine standard remained constant. Also if water was added (to make the total volume of 30 ml.) prior to the addition of the alkaline picric acid, the color would not form with the incubation mixtures but would develop with the creatinine standard. SUMMARY
1. Solutions containing extracts of normal rabbit muscle, glucose l-phosphate, creatine phosphate, and cysteine were incubated and analyzed for creatine and creatinine formation and creatine phosphate utilization. 2. The muscle extract catalyzed the conversion of creatine phosphate to creatine without any detectable formation of creatinine. REFERENCES 1. BORSOOK, H., AND DUBNOFF, J. W., J. Biol. Chem. 168, 493 (1947). 2. CAPUTTO, R., Arch. Biochem. Biophys. 62, 280 (1964). 3. CAPUTTO, R., AND CARPENTER, M. P., Federation Proc. 14, 608 (1966). 4. CARPENTER, M., MCKAY, P., AND CAPUTTO, R., Proc. Sot. Exptl. Biol. Med. 97, 205 (1958) . 6. ENNOR, A. H., AND MORRISON, J. F., Physiol. Revs. 38, 631 (1958). 6. VAN PILSUM, J. F., MARTIN, R. P., KITO, E., AND HESS, J., J. Biol. Chem. 222, 225 (1956). 7. VAN PILSUM, J. F., J. Biol. Chem. 228, 145 (1957). 8. EGGLETON, P., ELSDEN, S. R., AND GOUGH, N., Biochem. J. 37,526 (1943).
OF BIOCHEMISTRY
AND
BIOPHYSICS
86,
483-486
(1959)
On the Proposed Origin of Creatinine from Creatine Phosphate’ John F. Van Pilsum and Bruce Hiller From the Department of Physiological Chemistry, University Minnesota Medical School, Minneapolis, Minnesota Received
of
June 11, 1959
INTRODUCTION In 1947 Borsook and Dubnoff (1) calculated that the spontaneous, nonenzymic transformation of creatine phosphate to creatinine plus inorganic phosphate was sufficient to account for the urinary excretion of creatinine. They indicated that the possibility of an enzymic conversion of creatine phosphate to creatinine was not excluded, but no enzyme had been identified. In 1954, Caputto (2) reported the presence of an enzyme in normal rabbit muscle extract which catalyzed the transformation of creatine phosphate into creatinine in the presence of glycogen plus phosphate or glucose l-phosphate. In 1955, Caputto and Carpenter (3) reported that the phosphate of creatine phosphate was transferred to hexose monophosphate with the production of creatinine and hexose diphosphate. Details of their procedures were not published until 1958 (4). Ennor and Morrison (5) have stated in a review, “Biochemistry of the Phosphagens and Related Guanidines,” that no doubts have been cast on these unconfirmed reports of Caputto et al. The experiments of Caputto et al. have been reinvestigated using newer analytical procedures for creatine, creatine phosphate, and creatinine (6). It was found that muscle extracts from normal rabbits catalyzed the conversion of creatine phosphate to creatine, not to creatinine. METHODS Male albino rabbits weighing 1-2 kg. were killed by a blow on the head. A sample of back muscle was immediately removed and ground in a cold mortar and pestle with cold salt solution (4). Then 0.1 ml. of the muscle extract was added to test tubes containing 11 pmoles creatine phosphate, 30 pmoles glucose l-phosphate, and 20 pmoles cysteine. Total volume of the incubation mixture was 2.5 ml. Three control tubes were used: Control No. 1 contained muscle extract, glucose l-phosphate, and r Supported by University of Minnesota Graduate School Grant, No. 387-32012409 and by a grant from the American Cancer Society, Minnesota Division. 483
484
VAN
PILSUM
AND
HILLER
cysteine. The creatine phosphate was added after the mixture had been incubated and the enzyme reaction stopped. Control No. 2 contained muscle extract, creatine phosphate, glucose l-phosphate and cysteine. The reaction was not incubated and was stopped immediately after the addition of the muscle extract. Control No. 3 contained water (instead of muscle extract), creatine phosphate, glucose l-phosphate, and cysteine. The mixture was incubated and stopped. Incubation was for 15 min. at 30°C. without shaking and with an air phase over the solutions. The analytical procedures were as follows:
I. Creatinine Production A. Jaffe (Alkaline Picric Acid) Method. The volumes of incubation mixtures and color reaction were ten times the amount used by Carpenter et al. (4)) so the enzyme reactions were stopped by the addition of 10 ml. alkaline picric acid solution and the volume made up to 30 ml. with distilled water. B. o-Nitrobenzaldehyde Method (6). The large amounts of cysteine and glucose phosphate in the incubation mixture prevented normal color development. The enzyme reactions were stopped by the addition of 0.2 ml. of 2.5 N NaOH and 1 drop of 14% o-nitrobenzaldehyde. The solution was allowed to stand 20 min. at room temperature, 0.5 ml. of HzSOd-phosphate buffer mixture was added, and the mixture was heated for 15 min. in a boiling water bath. The methylguanidine formed by the reaction with o-nitrobenzaldehyde was separated from the interfering cysteine and glucose l-phosphate by allowing the solution to run through a column (1.5 X 5 cm.) containing Amberlite IRA 400 (OH) ion-exchange resin. The column was washed with 5 ml. distilled water, and this wash added to the eluate. Five milliliters of this mixture was withdrawn, and the modified Sakaguchi color reaction developed (6).
II. Creatine Phosphate Utilization The enzyme reactions were stopped by chilling the test tubes in an ice bath and adding 1.0 ml. of ice-cold 3 N perchloric acid. The mixtures were centrifuged in the cold at about 3000 r.p.m. for 10 min., the supernatant was decanted, and cold 30% KOH was added dropwise to the supernatant solution until the pH was 7-8. The solutions were allowed to stand 0.5-l hr. in the cold, and the potassium perchlorate was removed by centrifugation for 3-5 min. at about 3000 r.p.m. The supernatant was decanted and filtered. One milliliter of this filtered supernatant (solution A) was placed in a column (1.5 X 5 cm.) containing Dowex 50, X-16, 100-200 mesh, ion-exchange resin. The column was washed with 10 ml. cold distilled water, and the wash was added to the eluate. This mixture contained the creatine phosphate and was diluted 1:3 with distilled water and was analyzed for creatine by the o-nitrobenzaldehyde procedure (7).
III.
Creatine Production
One milliliter of solution A (in the creatine phosphate procedure ) was diluted with 7 vol. water, and 1 ml. of this dilution was used for the diacetyl color reaction (8). Creatine will react with ol-naphthol and diacetyl in the presence of alkali to yield a color, whereas creatine phosphate will not. .A11 measurements were made with a Bausch and Lomb “Spectronic 20” colorimeter.
485
CREATINE PHOSPHATE TO CREATININE
TABLE I Produclion of Creatine from Creatine Phosphate by Extracts of Normal Rabbit Muscle The reaction tube contained 11 rmoles creatine phosphate, 0.1 ml. extract, 30 pmoles glucose l-phosphate, and 20 pmoles cysteine. Total volume was 2.5 ml., and incubation was for 15 min. at 30°C. without shaking and with an air phase over the solutions. The compositions of the three control tubes were averaged and compared with the incubation mixtures to give net production or utilization. The compositions of the control tubes are explained in the text. Composition
of incubation and (pwles)
Creatine
Creatinine “0-nitro” method
Pk:ixi;;id
Reaction tube Control 1 Control 2 Control 3 Net production utilization
or
control
D&-t!&
0.044 0.035 0.039 0.035 0.008
0.50 0.29 0.21 0.20 0.27
mixtures Creatine “0-nitro”
phosphate method
8.1 10.7 11.2 10.4 2.6
2.3 0 0 0 2.3
RESULTS The results obtained with a normal rabbit muscle extract are shown in Table I. Similar results were obtained with extracts from four additional normal rabbits. The creatinine production, as measured by the alkaline
picric acid method, of 0.27 pmole was 60 % greater than the value of 0.168 pmole reported by Carpenter et al. (4). The creatinine production of 0.008 pmole as measured by the o-nitrobenzaldeyhde procedure was not considered to be a significant production. The creatine phosphate utilization of 2.6 pmoles was 70% greater than the value for “phosphate transfer” of 1.112 pmoles
(12-min.
incubation
period)
reported
by Carpenter
et al. (4).
The 2.6 pmoles creatine phosphate utilized was similar to the production 2.3 pmoles creatine, as measured by the diacetyl color reaction.
of
DISCUSSION
The product formed in the incubation that was responsible for a positive color reaction with picric acid in the presence of alkali is not known. That it is not creatinine was shown by the fact that it could not be measured by the o-nitrobenzaldehyde procedure, and creatinine added to the incubation mixtures
was recovered
completely
by this procedure.
Its color reaction
with picric acid was not the same as the reaction of creatinine with picric acid. For example, after the 20-min. color development the optical density
486
VAN PILSUM AND HILLER
of the solution increased with time, whereas the optical density of the creatinine standard remained constant. Also if water was added (to make the total volume of 30 ml.) prior to the addition of the alkaline picric acid, the color would not form with the incubation mixtures but would develop with the creatinine standard. SUMMARY
1. Solutions containing extracts of normal rabbit muscle, glucose l-phosphate, creatine phosphate, and cysteine were incubated and analyzed for creatine and creatinine formation and creatine phosphate utilization. 2. The muscle extract catalyzed the conversion of creatine phosphate to creatine without any detectable formation of creatinine. REFERENCES 1. BORSOOK, H., AND DUBNOFF, J. W., J. Biol. Chem. 168, 493 (1947). 2. CAPUTTO, R., Arch. Biochem. Biophys. 62, 280 (1964). 3. CAPUTTO, R., AND CARPENTER, M. P., Federation Proc. 14, 608 (1966). 4. CARPENTER, M., MCKAY, P., AND CAPUTTO, R., Proc. Sot. Exptl. Biol. Med. 97, 205 (1958) . 6. ENNOR, A. H., AND MORRISON, J. F., Physiol. Revs. 38, 631 (1958). 6. VAN PILSUM, J. F., MARTIN, R. P., KITO, E., AND HESS, J., J. Biol. Chem. 222, 225 (1956). 7. VAN PILSUM, J. F., J. Biol. Chem. 228, 145 (1957). 8. EGGLETON, P., ELSDEN, S. R., AND GOUGH, N., Biochem. J. 37,526 (1943).