- Email: [email protected]
Creating in urine: Its determination by means of creatine phosphokinase. Observations in normal men and women
CLINICA CHIMICA ACTA
408
CREATINE
IN URINE:
ITS DETERMINATION
BY MEANS OF CREATINE
PHOSPHOKINASE OBSERVATIONS
IN NORMAL
MEN AND WOMEN*
A. C. KIBRICK Institute for Muscle Disease, Inc., New York, N.Y. (Received
(l7.S.A.J
May rSth, 1964)
SUMMARY
A modification of the method of Tanzer and GilvargB is described for the determination of creatine in urine by means of creatine phosphokinase. The reliability of the modified procedure is evidenced by the results of recovery experiments and by comparison with the values obtained by use of the Jaffe reaction in urine of patients with muscular dystrophy. Values obtained in Ig normal men and women on a regular diet are II to 189 mg per 24 h for the men, and xg to 270 for the women. Other data show that the large range of normal values may be due to variation in the amounts of creatine in the diets and to variation in the activity of the subjects. The menstrual cycle may also influence the excretion of creatine.
Two commonly used methods of determining creatine in biological fluids are with the Jaffe reaction after conversion of creatine to creatininel, and with cc-naphthol and diacetyl in alkaline solution 2. By either method the results are questionable in normal urine which contains IO to 20 times as much creatinine as creatine. We have shown the effect of creatinine estimated by the Jaffe reaction in such urine, and in patients with muscular dystrophy the reliability of the method in urine that contains approximately equal amounts of creatine and creatinine3. A large amount of creatinine on a small amount of creatine has the same effect in the determination with a-naphthol and diacetyl since creatinine is converted to creatine in the alkaline medium and then reacts with the reagentst. In view of these difficulties, attempts to separate creatine from creatinine by various means5-7 have not been quite satisfactory. Tanzer and Gilvarg8 have shown that creatine can be determined in blood and urine by means of creatine phosphokinase, coupled with pyruvic kinase and lactic dehydrogenase, in which the change from DPNH to DPN is a direct measure of the * This study was aided by a grant from Muscular Dystrophy Associations of America, Jnc. Clin. Chim. Acta. II (1965) 408-413
URINARY
CREATINE
DETERMINATION
409
concentration of creatine. However, in using this method for urinary determination we had difficulty in obtaining enzyme of sufficient activity for use in the unmodified procedure, partly because of the lability of the enzyme while in the refrigerator, since it seems to be more stable at -zoo, and partly because of difficulty in dissolving the enzyme, which deteriorates rapidly in solution. We later found that the enzyme dissolves quite readily in 0.01 M glycine buffer at pH 9.0. Even with such precautions, crea tine phosphokinase from rabbit skeletal muscle manufactured by Boehringer and purchased from CalBiochem, has never been sufficiently active for the unmodified method, nor does it always yield a water-clear solution. We have, therefore, devised a modified method for urinary determinations which is more convenient for routine work. Instead of following the reaction after addition of creatine phosphokinase with periodic readings, only one reading is made, and the Bausch and Lomb spectrophotometer is used since it is more readily available to clinical laboratories. EXPERIMENTAL
In preliminary work with the reagents of Tanzer and Gilvarg, concentration of creatine was calculated from values of d 0. D. 35 min after addition of creatine phosphokinase, since the reaction was essentially complete after 25 min and reached its maximum at 35 or 40 min. The effect of glycocyamine on the determination is shown in Fig. I ; only when the concentration of this substance is 40 rng% in urine does it affect the determination of creatine. This result supports the conclusions of the previous workers.
20
40
60 mg
60 per
100 cent
120
140
160
160
glycocyomine
Fig. 1. The effect of glycocyamine on A 0. D. expressed as creatine 35 min after addition of creatine phosphokinase. Reagents (I) 0.075 M glycine buffer, PH 9.0 75 ml 0.1 iPI glycine (750 mg/Ioo ml) +14 ml 0.1 N sodium hydroxide, mix and adjust to pH 9.0 with either 0.1 N sodium hydroxide or 0.1 N hydrochloric acid and a glass electrode. Finally, dilute to IOO ml and mix. Check pH and readjust if necessary. Stable in refrigerator for 2 weeks. (2) 0.01 M magnesium chloride. Dilute IO ml 0.1 M Mg Cl, (0.952 g/roe ml)
to
ml with water. Stable in refrigerator for 2 weeks. (3) Adenosine-5’-triphosphate, disodium tetrahydrate (ATP) mol. wt. 623, CalBiochem. Dissolve 62.3 mg in 5 ml of water. Stable in freezer for I week. IOO
Clin. Chim. Ada,
II (1965) 408-413
A. C. KIBRICK
410
(4) Diphosphopyridine nucleotide, reduced, disodium (DPNH), mol. wt. 709, CalBiochem. Dissolve 22.6 mg in IO ml of water. Stable in freezer for I week. (5) z-phosphoenolpyruvic acid, tryst., trisodium salt, dihydrate (PEP) mol. wt. 270, CalBiochem. Dissolve 54.0 mg in 5 ml of water. Stable in refrigerator for I week. (6) Prepare the following mixture of reagents: I. Glycine 100 ml 4. DPNH 5.0 ml 2. MgCl, 5.0 ml 5. PEP 2.5 ml 30 ml 3. ATP 2.5 ml 6. water Prepare sufficient for I week. Stable in refrigerator. (7) Lactic dehydrogenase (LDH), 5 mg per ml, CalBiochem. Store in freezer. Pyruvic kinase (PK), IO mg per ml, CalBiochem. Store in freezer. LDH-PK: Mix 0.24 ml LDH and 0.30 ml PK. IO ~1 = 0.022 mg LDH and 0.055 mg PK. Stable in freezer for I week. (8) 0.01 M glycine buffer, PH 9.0. Mix I ml 0.075 A4 glycine (I) with 6.5 ml water. Prepare daily. (9) Creatine phosphokinase (CPK), lyophilized powder (Boehringer), CalBiochem. Store in freezer and dissolve just before using (IO mg/r.o ml reagent 8). (IO) Standard creatine solution. Stock solution: IOO mg in IOO ml water. Place in several small containers and store in freezer. Stable for I month. Working solution (Bopg/ml) : 8 ml dilutedstock (I ml of stock+9 ml of water) +2 ml water. Prepare daily. Procedure
Determinations in series of IO to 15 specimens of urine can be performed conveniently at one time, and two standards containing 80 pugof creatine per ml are included in each series. Place 5 to 20 ml of urine into a mixing cylinder (25 ml long-style, glassstoppered). Use 20 ml for normal adult subjects, 5 ml for dystrophic patients with advanced muscle wasting, and IO ml for those in whom wasting is less advanced. Add water to achieve a concentration, preferably between 50 and IOO ,ug/ml; mix and add 0.5 N sodium hydroxide drop-wise to a PH of 9.0 with a glass electrode electrometer. Do not overtitrate with more than one or two drops, and back-titrate immediately with 0.1 N hydrochloric acid from a PH of 9.2 to 9.3. Rinse the beaker used for the PH readings with small portions of water and add to the mixing cylinder. Finally, add water to a convenient final volume, mix again, and check the PH. After centrifugation, the clear supernatant alkaline solution can be left more than 4 h without change in creatine concentration, but it is preferable to continue the determination within 2 h. Place 0.2 ml of the clear diluted urine of PH 9.0 and 0.2 ml of two standards (80 pg/ml) into rz.6-mm glass cuvettes, and add 3.0 ml of mixed reagent 6, and IO 1~1 of LDH-PK (7). Mix by rotation and take the zero readings against water at IOO’/~ transmission at 340 m,u in a Bausch and Lomb spectrophotometer*. Repeat the readings after IO min. The two readings should be the same within narrow experimental variations; however, if they are not, a third reading may be necessary. The reading when constant is T I. is satisfactory. However, the digital readout * The transistor-regulated Spectronic “20” be used with this instrument since the signal at 340 m,u is too weak. Clin. Chim. Acta,
II
(1965)
408-413
cannot
URINARY CREATINE DETERMINATION
411
Dissolve the CPK (9), in 0.01 M glycine buffer (8) (IO mg/r.o ml of buffer), and use immediately. Add IOO ,ul to the tubes, mix well by rotation, and note the time. After 20 min read the solutions in the tubes again in the same way = T 2. Calcdation
Convert T I and T 2 to optical density = O.D. I and O.D. 2, and correct O.D.2 by the factor s.si 3.31 for dilution with CPK. Then use the differences. 0. D. I - car. 0. D. 2 = d 0. D. d 0. D. St should agree with the value of d 0. D. of the standard curve for 80 rug/ml. Then, ilO.D.Un
x 80 = ,ug per ml in the diluted urine,
d 0. D. St
final volume in cylinder
and this x
X
original volume of urine taken
volume of 24-h specimen 1000
=
= lug/ml of original urine = mg/l,
mg creatine per 24 h.
RESULTS AND DISCUSSION
Fig. 2 shows that in the modified method as described above the reaction is maximum less than 20 min after addition of the creatine phosphokinase. Fig. 3 is a standard
‘00
pa
20 JJO 0
5
I
I
I
IO
I5
20
T/me
I
25
I
I
50
35
In minuies
Fig. z. Values of n 0. D. for solutions of creatine obtained intervals after addition of creatine phosphokinase.
in the modified
method at varying
curve for creatine obtained under these conditions. Solutions containing as much as 120 ,ug per ml are all on a straight line going through the origin. Repeated standard curves yield the same values of d 0. D. within narrow experimental variations, and daily standards run with the determinations always have values in good agreement with this curve. The recovery of creatine added to urine (4.0 to 12.0 ,ug/ml) in 12 experiments had a range of 89 to IOIO/~ and a mean of 93.6%. Values obtained in urine of dystrophic patients were compared with values of creatine calculated as the CZin. Chim. Acta, II
(1965) 408-413
A. C. KIBRICK
4x2
difference between total and preformed creatinine measured by the Jaffe reaction, since the latter values of creatine are precise in urine containing close to a I to I ratio of creatine to creatinine3. Satisfactory agreement was obtained by the two methods in 36 urines, the average difference being only + 2.2%. 0.320
0.260
0.240
““‘1: 20
40
60
80
100
Creafine,
I20
140
160
I60
200
p/g/ml
Fig. 3. Standard curve for creatine by the modified method.
Urinary creatine was determined by the proposed method in a small series of rg male and Ig female laboratory workers on a regular diet. The values were from II to 189 mg/z4 h with an average of 52.1 in the men, and from rg to 270 mg/24 h with an average of 92.1 in the women. In Fig. 4 are given values of creatine deter32Or
Fig. 4. Excretion of creatine by a young woman over a period of 2 months. M. F. indicates the duration of menstrual flow.
mined in urine samples collected from a normal woman of 25 at intervals over a period of more than 2 months. The values vary from 24 to 300 mg per 24 h. The excretion is low during menstruation when she believes her diet may contain little creatine (no meat) and her activity may be less. Each of the three major maxima of creatine excretion shown in the figure commenced about 5 days after menstruation, a period which corresponds with ovulation in a normal cycle and with the first peak of estrogen excretion@. Urinary creatine was also determined in 3 other young women CliVZ. Chim. Ada,
II
(1965)
408-413
URINARY
CREATINE
DETERMINATION
413
and 2 young men on regular diets and on creatine-free diets lo. In one of the male and one of the female subjects the values were quite constant regardless of diet, whereas in the others the excretion of creatine was reduced when intake was restricted. It is difficult to analyze these results for the effects of diet, exercise, and menstruation. Although the data are insufficient to draw positive conclusions, it seems that both diet and exercise may affect the excretion of creatine in normal men and women and that there may be a pattern of excretion of this substance in the menstrual cycle. These factors would then explain the unusually large differences found in the values of urinary creatine in both men and women. REFERENCES I 0. FOLIN, J. Bid. Chem., 17 (1914) 475. 2 P. EGGLETON, S. R. ELSDEN AND N. GOUGH, Biochem. J., 37 (1943) 526. 3 A. C. KIBRICK AND A. T. MILHORAT, Abstr., 5th Intern. Congr. Clin. Chem., 4 (1963) 489. 4 A. H. ENNOR AND L. A. STOCKEN, Biochem. J., 55 (1953) 310. 5 B. F. MILLER, M. J. ALLINSON AND Z. BAKER, J. Biol. Chem., 130 (1939) 383. 6 H. BORSOOK, J. Biol. Chem., IIO (1935) 481. 7 W. S. ADAMS, F. W. DAVIS AND L. E. HANSEN, Anal. Chcm., 34 (1962) 854. 8 M. L. TANZER AND C. GILVARG, J. Biol. Chem., 234 (1959) 3201. 9 M. BODANSKY AND 0. BODANSKY, Biochemistry of Disease, Macmillan, New York, 1946, p. 451. IO A. T. MILHORAT AND H. G. WOLFF, Arch. Neural. Psvchiat., 38 (1937) 992.
Clin. Chim. Acta, II (1965) 408-413
408
CREATINE
IN URINE:
ITS DETERMINATION
BY MEANS OF CREATINE
PHOSPHOKINASE OBSERVATIONS
IN NORMAL
MEN AND WOMEN*
A. C. KIBRICK Institute for Muscle Disease, Inc., New York, N.Y. (Received
(l7.S.A.J
May rSth, 1964)
SUMMARY
A modification of the method of Tanzer and GilvargB is described for the determination of creatine in urine by means of creatine phosphokinase. The reliability of the modified procedure is evidenced by the results of recovery experiments and by comparison with the values obtained by use of the Jaffe reaction in urine of patients with muscular dystrophy. Values obtained in Ig normal men and women on a regular diet are II to 189 mg per 24 h for the men, and xg to 270 for the women. Other data show that the large range of normal values may be due to variation in the amounts of creatine in the diets and to variation in the activity of the subjects. The menstrual cycle may also influence the excretion of creatine.
Two commonly used methods of determining creatine in biological fluids are with the Jaffe reaction after conversion of creatine to creatininel, and with cc-naphthol and diacetyl in alkaline solution 2. By either method the results are questionable in normal urine which contains IO to 20 times as much creatinine as creatine. We have shown the effect of creatinine estimated by the Jaffe reaction in such urine, and in patients with muscular dystrophy the reliability of the method in urine that contains approximately equal amounts of creatine and creatinine3. A large amount of creatinine on a small amount of creatine has the same effect in the determination with a-naphthol and diacetyl since creatinine is converted to creatine in the alkaline medium and then reacts with the reagentst. In view of these difficulties, attempts to separate creatine from creatinine by various means5-7 have not been quite satisfactory. Tanzer and Gilvarg8 have shown that creatine can be determined in blood and urine by means of creatine phosphokinase, coupled with pyruvic kinase and lactic dehydrogenase, in which the change from DPNH to DPN is a direct measure of the * This study was aided by a grant from Muscular Dystrophy Associations of America, Jnc. Clin. Chim. Acta. II (1965) 408-413
URINARY
CREATINE
DETERMINATION
409
concentration of creatine. However, in using this method for urinary determination we had difficulty in obtaining enzyme of sufficient activity for use in the unmodified procedure, partly because of the lability of the enzyme while in the refrigerator, since it seems to be more stable at -zoo, and partly because of difficulty in dissolving the enzyme, which deteriorates rapidly in solution. We later found that the enzyme dissolves quite readily in 0.01 M glycine buffer at pH 9.0. Even with such precautions, crea tine phosphokinase from rabbit skeletal muscle manufactured by Boehringer and purchased from CalBiochem, has never been sufficiently active for the unmodified method, nor does it always yield a water-clear solution. We have, therefore, devised a modified method for urinary determinations which is more convenient for routine work. Instead of following the reaction after addition of creatine phosphokinase with periodic readings, only one reading is made, and the Bausch and Lomb spectrophotometer is used since it is more readily available to clinical laboratories. EXPERIMENTAL
In preliminary work with the reagents of Tanzer and Gilvarg, concentration of creatine was calculated from values of d 0. D. 35 min after addition of creatine phosphokinase, since the reaction was essentially complete after 25 min and reached its maximum at 35 or 40 min. The effect of glycocyamine on the determination is shown in Fig. I ; only when the concentration of this substance is 40 rng% in urine does it affect the determination of creatine. This result supports the conclusions of the previous workers.
20
40
60 mg
60 per
100 cent
120
140
160
160
glycocyomine
Fig. 1. The effect of glycocyamine on A 0. D. expressed as creatine 35 min after addition of creatine phosphokinase. Reagents (I) 0.075 M glycine buffer, PH 9.0 75 ml 0.1 iPI glycine (750 mg/Ioo ml) +14 ml 0.1 N sodium hydroxide, mix and adjust to pH 9.0 with either 0.1 N sodium hydroxide or 0.1 N hydrochloric acid and a glass electrode. Finally, dilute to IOO ml and mix. Check pH and readjust if necessary. Stable in refrigerator for 2 weeks. (2) 0.01 M magnesium chloride. Dilute IO ml 0.1 M Mg Cl, (0.952 g/roe ml)
to
ml with water. Stable in refrigerator for 2 weeks. (3) Adenosine-5’-triphosphate, disodium tetrahydrate (ATP) mol. wt. 623, CalBiochem. Dissolve 62.3 mg in 5 ml of water. Stable in freezer for I week. IOO
Clin. Chim. Ada,
II (1965) 408-413
A. C. KIBRICK
410
(4) Diphosphopyridine nucleotide, reduced, disodium (DPNH), mol. wt. 709, CalBiochem. Dissolve 22.6 mg in IO ml of water. Stable in freezer for I week. (5) z-phosphoenolpyruvic acid, tryst., trisodium salt, dihydrate (PEP) mol. wt. 270, CalBiochem. Dissolve 54.0 mg in 5 ml of water. Stable in refrigerator for I week. (6) Prepare the following mixture of reagents: I. Glycine 100 ml 4. DPNH 5.0 ml 2. MgCl, 5.0 ml 5. PEP 2.5 ml 30 ml 3. ATP 2.5 ml 6. water Prepare sufficient for I week. Stable in refrigerator. (7) Lactic dehydrogenase (LDH), 5 mg per ml, CalBiochem. Store in freezer. Pyruvic kinase (PK), IO mg per ml, CalBiochem. Store in freezer. LDH-PK: Mix 0.24 ml LDH and 0.30 ml PK. IO ~1 = 0.022 mg LDH and 0.055 mg PK. Stable in freezer for I week. (8) 0.01 M glycine buffer, PH 9.0. Mix I ml 0.075 A4 glycine (I) with 6.5 ml water. Prepare daily. (9) Creatine phosphokinase (CPK), lyophilized powder (Boehringer), CalBiochem. Store in freezer and dissolve just before using (IO mg/r.o ml reagent 8). (IO) Standard creatine solution. Stock solution: IOO mg in IOO ml water. Place in several small containers and store in freezer. Stable for I month. Working solution (Bopg/ml) : 8 ml dilutedstock (I ml of stock+9 ml of water) +2 ml water. Prepare daily. Procedure
Determinations in series of IO to 15 specimens of urine can be performed conveniently at one time, and two standards containing 80 pugof creatine per ml are included in each series. Place 5 to 20 ml of urine into a mixing cylinder (25 ml long-style, glassstoppered). Use 20 ml for normal adult subjects, 5 ml for dystrophic patients with advanced muscle wasting, and IO ml for those in whom wasting is less advanced. Add water to achieve a concentration, preferably between 50 and IOO ,ug/ml; mix and add 0.5 N sodium hydroxide drop-wise to a PH of 9.0 with a glass electrode electrometer. Do not overtitrate with more than one or two drops, and back-titrate immediately with 0.1 N hydrochloric acid from a PH of 9.2 to 9.3. Rinse the beaker used for the PH readings with small portions of water and add to the mixing cylinder. Finally, add water to a convenient final volume, mix again, and check the PH. After centrifugation, the clear supernatant alkaline solution can be left more than 4 h without change in creatine concentration, but it is preferable to continue the determination within 2 h. Place 0.2 ml of the clear diluted urine of PH 9.0 and 0.2 ml of two standards (80 pg/ml) into rz.6-mm glass cuvettes, and add 3.0 ml of mixed reagent 6, and IO 1~1 of LDH-PK (7). Mix by rotation and take the zero readings against water at IOO’/~ transmission at 340 m,u in a Bausch and Lomb spectrophotometer*. Repeat the readings after IO min. The two readings should be the same within narrow experimental variations; however, if they are not, a third reading may be necessary. The reading when constant is T I. is satisfactory. However, the digital readout * The transistor-regulated Spectronic “20” be used with this instrument since the signal at 340 m,u is too weak. Clin. Chim. Acta,
II
(1965)
408-413
cannot
URINARY CREATINE DETERMINATION
411
Dissolve the CPK (9), in 0.01 M glycine buffer (8) (IO mg/r.o ml of buffer), and use immediately. Add IOO ,ul to the tubes, mix well by rotation, and note the time. After 20 min read the solutions in the tubes again in the same way = T 2. Calcdation
Convert T I and T 2 to optical density = O.D. I and O.D. 2, and correct O.D.2 by the factor s.si 3.31 for dilution with CPK. Then use the differences. 0. D. I - car. 0. D. 2 = d 0. D. d 0. D. St should agree with the value of d 0. D. of the standard curve for 80 rug/ml. Then, ilO.D.Un
x 80 = ,ug per ml in the diluted urine,
d 0. D. St
final volume in cylinder
and this x
X
original volume of urine taken
volume of 24-h specimen 1000
=
= lug/ml of original urine = mg/l,
mg creatine per 24 h.
RESULTS AND DISCUSSION
Fig. 2 shows that in the modified method as described above the reaction is maximum less than 20 min after addition of the creatine phosphokinase. Fig. 3 is a standard
‘00
pa
20 JJO 0
5
I
I
I
IO
I5
20
T/me
I
25
I
I
50
35
In minuies
Fig. z. Values of n 0. D. for solutions of creatine obtained intervals after addition of creatine phosphokinase.
in the modified
method at varying
curve for creatine obtained under these conditions. Solutions containing as much as 120 ,ug per ml are all on a straight line going through the origin. Repeated standard curves yield the same values of d 0. D. within narrow experimental variations, and daily standards run with the determinations always have values in good agreement with this curve. The recovery of creatine added to urine (4.0 to 12.0 ,ug/ml) in 12 experiments had a range of 89 to IOIO/~ and a mean of 93.6%. Values obtained in urine of dystrophic patients were compared with values of creatine calculated as the CZin. Chim. Acta, II
(1965) 408-413
A. C. KIBRICK
4x2
difference between total and preformed creatinine measured by the Jaffe reaction, since the latter values of creatine are precise in urine containing close to a I to I ratio of creatine to creatinine3. Satisfactory agreement was obtained by the two methods in 36 urines, the average difference being only + 2.2%. 0.320
0.260
0.240
““‘1: 20
40
60
80
100
Creafine,
I20
140
160
I60
200
p/g/ml
Fig. 3. Standard curve for creatine by the modified method.
Urinary creatine was determined by the proposed method in a small series of rg male and Ig female laboratory workers on a regular diet. The values were from II to 189 mg/z4 h with an average of 52.1 in the men, and from rg to 270 mg/24 h with an average of 92.1 in the women. In Fig. 4 are given values of creatine deter32Or
Fig. 4. Excretion of creatine by a young woman over a period of 2 months. M. F. indicates the duration of menstrual flow.
mined in urine samples collected from a normal woman of 25 at intervals over a period of more than 2 months. The values vary from 24 to 300 mg per 24 h. The excretion is low during menstruation when she believes her diet may contain little creatine (no meat) and her activity may be less. Each of the three major maxima of creatine excretion shown in the figure commenced about 5 days after menstruation, a period which corresponds with ovulation in a normal cycle and with the first peak of estrogen excretion@. Urinary creatine was also determined in 3 other young women CliVZ. Chim. Ada,
II
(1965)
408-413
URINARY
CREATINE
DETERMINATION
413
and 2 young men on regular diets and on creatine-free diets lo. In one of the male and one of the female subjects the values were quite constant regardless of diet, whereas in the others the excretion of creatine was reduced when intake was restricted. It is difficult to analyze these results for the effects of diet, exercise, and menstruation. Although the data are insufficient to draw positive conclusions, it seems that both diet and exercise may affect the excretion of creatine in normal men and women and that there may be a pattern of excretion of this substance in the menstrual cycle. These factors would then explain the unusually large differences found in the values of urinary creatine in both men and women. REFERENCES I 0. FOLIN, J. Bid. Chem., 17 (1914) 475. 2 P. EGGLETON, S. R. ELSDEN AND N. GOUGH, Biochem. J., 37 (1943) 526. 3 A. C. KIBRICK AND A. T. MILHORAT, Abstr., 5th Intern. Congr. Clin. Chem., 4 (1963) 489. 4 A. H. ENNOR AND L. A. STOCKEN, Biochem. J., 55 (1953) 310. 5 B. F. MILLER, M. J. ALLINSON AND Z. BAKER, J. Biol. Chem., 130 (1939) 383. 6 H. BORSOOK, J. Biol. Chem., IIO (1935) 481. 7 W. S. ADAMS, F. W. DAVIS AND L. E. HANSEN, Anal. Chcm., 34 (1962) 854. 8 M. L. TANZER AND C. GILVARG, J. Biol. Chem., 234 (1959) 3201. 9 M. BODANSKY AND 0. BODANSKY, Biochemistry of Disease, Macmillan, New York, 1946, p. 451. IO A. T. MILHORAT AND H. G. WOLFF, Arch. Neural. Psvchiat., 38 (1937) 992.
Clin. Chim. Acta, II (1965) 408-413