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A new colorimetric ultramicromethod for serum glutamicoxalacetic and glutamic-pyruvic transaminase determination
CLINIC.4 CHIMICA ACTA
A NEW COLORTMETRIC OXALACETIC
431
ULTRAMICROMETHOD
AND GT-UTA~IC-~YRU~IC
FOR SERUM
TRA~SA~INASE
GLUTAMICDET~R~INA-
TION
In this paper an ultramicromethod for the determination of the serum glutamicoxalacetic and glutamic-pyruvic transaminase activity is presented; this method is based on the use of glutamate dehydrogenase for the enzymatic estimation of the glutamate formed. The dehydrogenation of the glutamate gives rise to the reduction of a diazonium salt, and it is possible to perform a photometric reading of the colored compound at 520 nm. 20 ~1 of serum and an incubation time of only 45 min at the temperature of 37’ were necessary. The normal values never exceeded 54.5 I.U. for the serum glutamic-oxalacetic transaminase and 52 I.U. for the glutamic-pyruvic transaminase. IJnder conditions of viral hepatitis values of 390 I.U. for glutamic-pyruv~c transaminase and 310 I.U. for serum glutamic-oxalacetic transaminase were obtained.
INTRODUCTION
The most widely used photometric method for the determination of the serum transaminase activity is that proposed by Reitman and Frankell. The method of Babson et al.” can only be used for serum glutamic-oxalacetic transaminase determination. However, there are several points on which the procedure of Reitman and Frankell may be criticized: (I) dinitrophenylhydrazine is a nonspecific reagenP; (2) the blank absorption in normal sera may be stronger than that of the colored sample, so that even small variations in the blank may cause large errors in the measure of the enzymatic activity*; (3) the calibration curve does not comply with the analytical conditions of the whole enzymatic reaction5 and it is linear only over a short range; (4) a disagreement between the dinitrophenylhydrazine method and the spectrophotometric method has been observed many times, so that many falsenormal serum glutamic-oxalacetic transaminase activities have been measured in acute myocardial infarction-‘I. czin.
Chim.
Acta,
28
(1970)
/+3’--1_37
LIPPI,
432 Critical surveys on the phenylhydrazine and by Amador et aL4. The present study concerns the serum transaminase aminases) . MATERIALS
activity
GUInI
method have been published by Fonty12
a new ultramicromethod (glutamic-oxalacetic
for the determination
and glutamic-pyruvic
of
trans-
AND METHODS
The glutamate
derived
from the serum transaminases
is dehydrogenated
by
glutamate dehydrogenase. The NADf-N-methylphenazonium methosulfate system is used as an Hf carrier and a diazonium salt as an H+ acceptor. The formazan so formed is dissolved in 0.35 M HCl and photometric readings are performed at 52.0 nm. The reaction sequence
is as follows:
a-Oxoglutarate+aspartate
SGoT
a-Oxoglutarate+alanine
SGPT
) oxalacetate+glutamate ) glutamate-tpyruvate
COOH
COOH
CH,
CH,
CH,
+ H,O+NAD’-
GDH
(1)
I
, CH, + NH, + NADH
(2)
I CHNH,
c=o
I coo-
I coo-
Glutamate
cc-Oxoglutarate
NADH+INT
PEE
, NAD+ + INT formazan
(3)
SGOT = serum glutamic-oxalacctic transaminase ; SGI”L : strum glutamic-pyruvic transaminasc GDH = glutamate dehydrogenase; INT = r-p-iodophcnyl-3-~~itroph~n~l-5-ph””yl tctrazolium chloride; PZ!IS = X-methyl phenazonium methosulfate.
:
Reagents (I) Glutamic-oxalacetic transaminase buffered substrate : 0.002 M a-oxoglutaric acid and 0.10 M r_(+)-aspartic acid in 0.10 M phosphate buffer (pH 7.4). (2) Glutamic-pyruvic transaminase-buffered substrate : 0.002 lK a-oxoglutaric acid and 0.2 Jt rn-alanine in 0.10 M phosphate buffer (pH 7.4). tetrazo(3) Color reagentI : 0.0039 M z+iodophenyl-3-nitrophenyl-5-phenyl
lium chloride (INT),
0.0075 M NAD+, 0.00162
M n’-methylphenazonium
methosulfate
(PMS). (4) 20 mg/ml glutamate Soehne GmbH, Mannheim). (5) 0.35 M HCl.
Clin. China. Acta, 28 (1970) 431-437
dehydrogenase
(45 units/mg;
C. I;. Boehringer
and.
COLORIMETRIC
DETERMINATION
OF
SGOT
Glutamic-oxalacetic glutamic-pyruvic
substrate
AND
SGPT
OY transaminase
(ml)
..~___.
433
C&V reagent (flzl) _
0.25
0.2j
0.25
0.25
Procedure The reagents are preheated at 37O, mixed according to the scheme outlined in Table I and then incubated at 37’ in a water bath for 45 min. The reaction is then stopped by adding 5 ml of HCl. The samples are measured at 520 nm against the corresponding blank. Just before use the glutamic-oxalacetic or glutamic-pyruvic transaminase substrate solution and the color reagent are mixed mixture is placed in each test tube. Constriction
in equal parts; then 0.5 ml of this pipettes were used for the addition
of glutamate dehydrogenase. An excess of this latter enzyme does not compromise the course of the reaction. The incubation time is 45 min because at this moment the optical absorption of the reaction product reaches an optimal value. Calibration The
cu~vc calibration
curve was obtained
in the following
way:
samples
were in-
cubated, which contained color reagent together with a known amount of IKT and serum of a very high transaminase activity. The color reagent we used contained 0.09 pmole/ml INT. Several samples containing different amounts of INT were prepared by dilution. Taking into account the amount of serum involved in the reaction and the incubation time, the value we got from the measurements of the absorbance of the undiluted sample, with respect to the blank which lacked serum, corresponds to IOO I.U., where an I.U. is 0.0009 (50000/45) j,6mole. A plot of the absorbance measured at 520 nm of the samples containing different concentrations of INT, against the concentration, gives a straight line passing through zero (Fig. I).
05°C. 040
A-*’
030 020~
./
010 0
./
0
50
100
150
200 I u.
Fig.
I. Calibration
curve
(see text)
Clin. Chim.
Acta,
28 (1970) 431-437
LIPPI,
434 Basal
C;CIDI
glutamate
The amount of glutamate originally present in serum is extremely low, and its value is not modified in normal or pathologic sera. The quantity of the basal glutamate has been determined by performing the same measurements as before with samples in which the substrate solutions were replaced by an equal volume of 0.05 M phosphate buffer (pH 7.4). The difference between the formed glutamate and the basal glutamate lies within such values that it does not effect the enzymatic activity. Kim3tics
of the enzyme
reaction
A pool of normal
sera and some sera with quite a high transaminase
activity
were incubated during periods ranging from 20- to r2o-min duration (Fig. 2). In every case the colored products of the reaction increased linearly with increasing incubation time. between the enzymatic concentration and the progress qf the reaction A pool of normal sera was diluted I : IO-I : 6-1: 3-1: 2 ; 20 ,~l of the solutions
Correlation
were incubated for 45 min and the reaction was stopped by adding 2.5 ml of 0.35 M HCl. The values of the absorbance could be plotted against the concentration in a straight line passing through zero. Another serum with very high transaminase activity was similarly diluted and incubated for 45 min. The reaction was stopped with IO ml of 0.35 M HCl. .4gain the values fitted a straight line, showing zero-order kinetics for the reaction (Fig. 3). A
070
SGPT
060. A
cm-
.
040.
.
. .
a30. cl20 010
.
. .
-
. l
(
0
0
.
010
:
.
l
. .
.
l
l
l
l
l
m
50
70
93
110120
A 070 SGOT 060
t A
040
.
I
.
.
g/::,,“ ox)
30
50 ltwhation
70 time
B 90 (min)
no
120
Fig. 2. Kinetics of the enzymatic reaction at various incubation times at 37O. Data concern sera with quite high (A) and normal (B) glutamic-oxalacetic transaminase (SGOT) and glutamicpyruvic transaminase (SGPT) activities. Clin. Chim. Acta,
28 (1970)
431-437
COLORIIUETRIC DETERMINATION
OF SGOT
AND SGPT
0.40. 044. 040. 036. 032. 028. 024
435
SGOT
Fig. 3. Kinetics of the enzymatic reaction at various serum glutamic-pyruvic transaminase (SGPT) and serum glutamic-oxalacetic transaminase (SGOT) contents. The reaction of a pool of normal sera has been stopped by adding 2.5 ml of 0.35 M HCl. The reaction of a high enzymatic activity serum has been stopped with IO ml of 0.35 M HCl.
SGPT 08 07
1
,’ ,/
,,-’
Fig. 4. “Scalen” quantities of a serum with a very high serum glutamic-pyruvic transaminase (SGPT) activity have been incubated under standard conditions. The enzymatic reaction has been stopped with 20 ml of 0.35 M HCl.
In another experiment, different quantities of a serum with very high transaminase activity (0.010, 0.020, 0.040, 0.050, 0.060, 0.080 ml) were incubated under standard conditions. The absorbance remained constant for the samples containing 0.050 ml of serum (Fig. 4). Important is the observation that an excess of NAD+ in the reaction mixture slows down the enzymatic reaction. We performed simultaneously 20 determinations using a pool of normal sera and some sera with very high enzymatic activity. The absorbance values of the colored samples were always reproducible within every group of sera. Particularly, it may be interesting and very useful to point out that the 1:2 dilution of the colored solution with 0.35 M HCl gives rise to absorbance readings of half values. Normal values and pathological values 92 determinations of the transaminase activity on sera of healthy individuals (blood donors and young soldiers) were performed. The values obtained never exceeded 54.5 I.U. for serum glutamic-oxalacetic transaminase and 52 I.U. for serum glutamic-pyruvic transaminase, with a great incidence of 43-48 I.U. and of 43.5-48 Clin. Chim.
Acta,
28 (1970)
431-437
LIPPI, GCIDI
SGPT
356
SGOT
IU.
(
L
133 %
IU.
8.8 %
l-
I
37-40 41-44 45-4c
1
4.4 %
Fig. 5. Xormal distribution of serum glutamic-pyruvic oxalacetic transminase (SGOT) values.
4452 53-545 transaminase (SGPT) and serum glutamic-
In some pathological cases (viral hepatitis) serunl glutamic-oxalacetic values of up to 310 and 390 I.ll., respectively, were found.
and -pyruvic
transaminase
Srra
Sormal
Pathological
(viral hepatitis)
I.U., respectively (Fig. 5). Table II lists the values of serum glutamic-oxalacetic transaminase and serum glutamic-pyruvic transaminase in normal and pathological sera. REFERENCES I S. KE1Tnl.w AND 5. FRAKKEL, z4wz. ,I. Clin. Pathol., 28 (1957) 56. L A. L. BABSON, P. 0. SHAPIRO, I’. A. R. WILLIAMS ASD G. IX. PHILLIPS, Clix. Chim. (1962) 199. 3 H. SAIKAWA, Okayama Igakkai Zasshi, 76 (1964) 559. 4 E. AMADOR, M. F. Rbssoo AND R. J, FRA;UEY, Am. ,I. Clin. Pathol., 47 (1967) 479. Clin.
Ckim.
Acta,
2X (1970) 431-437
Acta,
7
COLORIMIETRIC
DETERMINATION
OF
SGOT
AND
SGPT
437
E. ARIADOR, H. HFISSTEIX ASD N. BENOTTI, Am. J. Clis. Pathnl., .++ (1965) 62. Ii. F. W'ITTER AND L. 31. GRUBRS, Cli?z. Chinz. Acta, 13 (1966) 524. J. BURSTEIN ASD A. HARJANNE, Acta Md. Stand., 163 (1959) 175. H. I\. DEWAR, ?;. K. ROWELL ASD .4. J. SMITH, Hvit. Xrd. J., 2 (1958) 1121. 9 P. Y. GRIFFITHS, Bait. Hcavt J., 28 (1966) Igg. ICI T. 1%‘. STEWART AXD F. G. WARBURTON, lint. Heavt J., ~3 (1961) 236. II J. STUART, T. C. CRAWFORD, J. FORSHALL ASD J. A. OWEN, Brit. JIrd. ,f,, I (1965) 4~3. I2 P. L'OKTU, _!Tn~ymZo~i~, nlonographie .\IInuclle de la Sm. I;ran$. dc Rid. Clin., Paris, 1964. 5 6 7 Y
‘3
A.
I<. 13\~son-
ASD
G.
Ji. PHILLIPS,
Clin.
Chim.
4cta.
I2
(1965)
210.
A NEW COLORTMETRIC OXALACETIC
431
ULTRAMICROMETHOD
AND GT-UTA~IC-~YRU~IC
FOR SERUM
TRA~SA~INASE
GLUTAMICDET~R~INA-
TION
In this paper an ultramicromethod for the determination of the serum glutamicoxalacetic and glutamic-pyruvic transaminase activity is presented; this method is based on the use of glutamate dehydrogenase for the enzymatic estimation of the glutamate formed. The dehydrogenation of the glutamate gives rise to the reduction of a diazonium salt, and it is possible to perform a photometric reading of the colored compound at 520 nm. 20 ~1 of serum and an incubation time of only 45 min at the temperature of 37’ were necessary. The normal values never exceeded 54.5 I.U. for the serum glutamic-oxalacetic transaminase and 52 I.U. for the glutamic-pyruvic transaminase. IJnder conditions of viral hepatitis values of 390 I.U. for glutamic-pyruv~c transaminase and 310 I.U. for serum glutamic-oxalacetic transaminase were obtained.
INTRODUCTION
The most widely used photometric method for the determination of the serum transaminase activity is that proposed by Reitman and Frankell. The method of Babson et al.” can only be used for serum glutamic-oxalacetic transaminase determination. However, there are several points on which the procedure of Reitman and Frankell may be criticized: (I) dinitrophenylhydrazine is a nonspecific reagenP; (2) the blank absorption in normal sera may be stronger than that of the colored sample, so that even small variations in the blank may cause large errors in the measure of the enzymatic activity*; (3) the calibration curve does not comply with the analytical conditions of the whole enzymatic reaction5 and it is linear only over a short range; (4) a disagreement between the dinitrophenylhydrazine method and the spectrophotometric method has been observed many times, so that many falsenormal serum glutamic-oxalacetic transaminase activities have been measured in acute myocardial infarction-‘I. czin.
Chim.
Acta,
28
(1970)
/+3’--1_37
LIPPI,
432 Critical surveys on the phenylhydrazine and by Amador et aL4. The present study concerns the serum transaminase aminases) . MATERIALS
activity
GUInI
method have been published by Fonty12
a new ultramicromethod (glutamic-oxalacetic
for the determination
and glutamic-pyruvic
of
trans-
AND METHODS
The glutamate
derived
from the serum transaminases
is dehydrogenated
by
glutamate dehydrogenase. The NADf-N-methylphenazonium methosulfate system is used as an Hf carrier and a diazonium salt as an H+ acceptor. The formazan so formed is dissolved in 0.35 M HCl and photometric readings are performed at 52.0 nm. The reaction sequence
is as follows:
a-Oxoglutarate+aspartate
SGoT
a-Oxoglutarate+alanine
SGPT
) oxalacetate+glutamate ) glutamate-tpyruvate
COOH
COOH
CH,
CH,
CH,
+ H,O+NAD’-
GDH
(1)
I
, CH, + NH, + NADH
(2)
I CHNH,
c=o
I coo-
I coo-
Glutamate
cc-Oxoglutarate
NADH+INT
PEE
, NAD+ + INT formazan
(3)
SGOT = serum glutamic-oxalacctic transaminase ; SGI”L : strum glutamic-pyruvic transaminasc GDH = glutamate dehydrogenase; INT = r-p-iodophcnyl-3-~~itroph~n~l-5-ph””yl tctrazolium chloride; PZ!IS = X-methyl phenazonium methosulfate.
:
Reagents (I) Glutamic-oxalacetic transaminase buffered substrate : 0.002 M a-oxoglutaric acid and 0.10 M r_(+)-aspartic acid in 0.10 M phosphate buffer (pH 7.4). (2) Glutamic-pyruvic transaminase-buffered substrate : 0.002 lK a-oxoglutaric acid and 0.2 Jt rn-alanine in 0.10 M phosphate buffer (pH 7.4). tetrazo(3) Color reagentI : 0.0039 M z+iodophenyl-3-nitrophenyl-5-phenyl
lium chloride (INT),
0.0075 M NAD+, 0.00162
M n’-methylphenazonium
methosulfate
(PMS). (4) 20 mg/ml glutamate Soehne GmbH, Mannheim). (5) 0.35 M HCl.
Clin. China. Acta, 28 (1970) 431-437
dehydrogenase
(45 units/mg;
C. I;. Boehringer
and.
COLORIMETRIC
DETERMINATION
OF
SGOT
Glutamic-oxalacetic glutamic-pyruvic
substrate
AND
SGPT
OY transaminase
(ml)
..~___.
433
C&V reagent (flzl) _
0.25
0.2j
0.25
0.25
Procedure The reagents are preheated at 37O, mixed according to the scheme outlined in Table I and then incubated at 37’ in a water bath for 45 min. The reaction is then stopped by adding 5 ml of HCl. The samples are measured at 520 nm against the corresponding blank. Just before use the glutamic-oxalacetic or glutamic-pyruvic transaminase substrate solution and the color reagent are mixed mixture is placed in each test tube. Constriction
in equal parts; then 0.5 ml of this pipettes were used for the addition
of glutamate dehydrogenase. An excess of this latter enzyme does not compromise the course of the reaction. The incubation time is 45 min because at this moment the optical absorption of the reaction product reaches an optimal value. Calibration The
cu~vc calibration
curve was obtained
in the following
way:
samples
were in-
cubated, which contained color reagent together with a known amount of IKT and serum of a very high transaminase activity. The color reagent we used contained 0.09 pmole/ml INT. Several samples containing different amounts of INT were prepared by dilution. Taking into account the amount of serum involved in the reaction and the incubation time, the value we got from the measurements of the absorbance of the undiluted sample, with respect to the blank which lacked serum, corresponds to IOO I.U., where an I.U. is 0.0009 (50000/45) j,6mole. A plot of the absorbance measured at 520 nm of the samples containing different concentrations of INT, against the concentration, gives a straight line passing through zero (Fig. I).
05°C. 040
A-*’
030 020~
./
010 0
./
0
50
100
150
200 I u.
Fig.
I. Calibration
curve
(see text)
Clin. Chim.
Acta,
28 (1970) 431-437
LIPPI,
434 Basal
C;CIDI
glutamate
The amount of glutamate originally present in serum is extremely low, and its value is not modified in normal or pathologic sera. The quantity of the basal glutamate has been determined by performing the same measurements as before with samples in which the substrate solutions were replaced by an equal volume of 0.05 M phosphate buffer (pH 7.4). The difference between the formed glutamate and the basal glutamate lies within such values that it does not effect the enzymatic activity. Kim3tics
of the enzyme
reaction
A pool of normal
sera and some sera with quite a high transaminase
activity
were incubated during periods ranging from 20- to r2o-min duration (Fig. 2). In every case the colored products of the reaction increased linearly with increasing incubation time. between the enzymatic concentration and the progress qf the reaction A pool of normal sera was diluted I : IO-I : 6-1: 3-1: 2 ; 20 ,~l of the solutions
Correlation
were incubated for 45 min and the reaction was stopped by adding 2.5 ml of 0.35 M HCl. The values of the absorbance could be plotted against the concentration in a straight line passing through zero. Another serum with very high transaminase activity was similarly diluted and incubated for 45 min. The reaction was stopped with IO ml of 0.35 M HCl. .4gain the values fitted a straight line, showing zero-order kinetics for the reaction (Fig. 3). A
070
SGPT
060. A
cm-
.
040.
.
. .
a30. cl20 010
.
. .
-
. l
(
0
0
.
010
:
.
l
. .
.
l
l
l
l
l
m
50
70
93
110120
A 070 SGOT 060
t A
040
.
I
.
.
g/::,,“ ox)
30
50 ltwhation
70 time
B 90 (min)
no
120
Fig. 2. Kinetics of the enzymatic reaction at various incubation times at 37O. Data concern sera with quite high (A) and normal (B) glutamic-oxalacetic transaminase (SGOT) and glutamicpyruvic transaminase (SGPT) activities. Clin. Chim. Acta,
28 (1970)
431-437
COLORIIUETRIC DETERMINATION
OF SGOT
AND SGPT
0.40. 044. 040. 036. 032. 028. 024
435
SGOT
Fig. 3. Kinetics of the enzymatic reaction at various serum glutamic-pyruvic transaminase (SGPT) and serum glutamic-oxalacetic transaminase (SGOT) contents. The reaction of a pool of normal sera has been stopped by adding 2.5 ml of 0.35 M HCl. The reaction of a high enzymatic activity serum has been stopped with IO ml of 0.35 M HCl.
SGPT 08 07
1
,’ ,/
,,-’
Fig. 4. “Scalen” quantities of a serum with a very high serum glutamic-pyruvic transaminase (SGPT) activity have been incubated under standard conditions. The enzymatic reaction has been stopped with 20 ml of 0.35 M HCl.
In another experiment, different quantities of a serum with very high transaminase activity (0.010, 0.020, 0.040, 0.050, 0.060, 0.080 ml) were incubated under standard conditions. The absorbance remained constant for the samples containing 0.050 ml of serum (Fig. 4). Important is the observation that an excess of NAD+ in the reaction mixture slows down the enzymatic reaction. We performed simultaneously 20 determinations using a pool of normal sera and some sera with very high enzymatic activity. The absorbance values of the colored samples were always reproducible within every group of sera. Particularly, it may be interesting and very useful to point out that the 1:2 dilution of the colored solution with 0.35 M HCl gives rise to absorbance readings of half values. Normal values and pathological values 92 determinations of the transaminase activity on sera of healthy individuals (blood donors and young soldiers) were performed. The values obtained never exceeded 54.5 I.U. for serum glutamic-oxalacetic transaminase and 52 I.U. for serum glutamic-pyruvic transaminase, with a great incidence of 43-48 I.U. and of 43.5-48 Clin. Chim.
Acta,
28 (1970)
431-437
LIPPI, GCIDI
SGPT
356
SGOT
IU.
(
L
133 %
IU.
8.8 %
l-
I
37-40 41-44 45-4c
1
4.4 %
Fig. 5. Xormal distribution of serum glutamic-pyruvic oxalacetic transminase (SGOT) values.
4452 53-545 transaminase (SGPT) and serum glutamic-
In some pathological cases (viral hepatitis) serunl glutamic-oxalacetic values of up to 310 and 390 I.ll., respectively, were found.
and -pyruvic
transaminase
Srra
Sormal
Pathological
(viral hepatitis)
I.U., respectively (Fig. 5). Table II lists the values of serum glutamic-oxalacetic transaminase and serum glutamic-pyruvic transaminase in normal and pathological sera. REFERENCES I S. KE1Tnl.w AND 5. FRAKKEL, z4wz. ,I. Clin. Pathol., 28 (1957) 56. L A. L. BABSON, P. 0. SHAPIRO, I’. A. R. WILLIAMS ASD G. IX. PHILLIPS, Clix. Chim. (1962) 199. 3 H. SAIKAWA, Okayama Igakkai Zasshi, 76 (1964) 559. 4 E. AMADOR, M. F. Rbssoo AND R. J, FRA;UEY, Am. ,I. Clin. Pathol., 47 (1967) 479. Clin.
Ckim.
Acta,
2X (1970) 431-437
Acta,
7
COLORIMIETRIC
DETERMINATION
OF
SGOT
AND
SGPT
437
E. ARIADOR, H. HFISSTEIX ASD N. BENOTTI, Am. J. Clis. Pathnl., .++ (1965) 62. Ii. F. W'ITTER AND L. 31. GRUBRS, Cli?z. Chinz. Acta, 13 (1966) 524. J. BURSTEIN ASD A. HARJANNE, Acta Md. Stand., 163 (1959) 175. H. I\. DEWAR, ?;. K. ROWELL ASD .4. J. SMITH, Hvit. Xrd. J., 2 (1958) 1121. 9 P. Y. GRIFFITHS, Bait. Hcavt J., 28 (1966) Igg. ICI T. 1%‘. STEWART AXD F. G. WARBURTON, lint. Heavt J., ~3 (1961) 236. II J. STUART, T. C. CRAWFORD, J. FORSHALL ASD J. A. OWEN, Brit. JIrd. ,f,, I (1965) 4~3. I2 P. L'OKTU, _!Tn~ymZo~i~, nlonographie .\IInuclle de la Sm. I;ran$. dc Rid. Clin., Paris, 1964. 5 6 7 Y
‘3
A.
I<. 13\~son-
ASD
G.
Ji. PHILLIPS,
Clin.
Chim.
4cta.
I2
(1965)
210.