- Email: [email protected]
The influence of operative trauma on some blood enzyme levels in children
405
SHORTCOMMUNICATIONS
in our laboratories are currently for serum urea determination.
being directedtowarddevelopment
of aurease
Research Division, U&-Tech Chemical Manufacturing Company, Sun Valley, Calif. ad Department of Pathology, Los Angeles Coudy Los Angeles,
Received
method
J. REARDON J. A. FOREMAN R. L. SEARCY
General Hospital (Ulzit 2),
Calif. (U.S.A.)
March r8th,
1966 Cli~z. Chim. Acta, 14 (1966) 403-405
The influence of operative trauma on some blood enzyme levels in children Operative trauma is a strong stimulus acting on the state of homeostasis in the body. An effect on the blood levels of certain enzymes, in particular the transaminases and oxytocinase, was observed in previous studies1-4. On comparing the blood concentrations of the enzymes in relation to the age of the patients, changes in enzymatic adaptation of the body can be observed. In addition, by employing two different substrates decomposed by related enzymatic systems, specificity of enzymes can be studied under the influence of the same operative stimulus. For instance, oxytocinase decomposes L-cystine-di-@aphthylamide as well as L-leucine-/-naphthylamide, the first of which is resistant to the action of leucine aminopeptidase. Estimation of the degree of decomposition of these two substrates therefore allows complex study of two blood aminopeptidase activities, which behave differently during pregnancy and in viral hepatitis6-7. The behavior of well-known enzymes can be utilized also in comparative studies on the specificity and nature of other, less well-known, enzymes, e.g. by means of the Abderhalden reaction8. This reaction is based on the breakdown by blood of specific organ tissues in connection with the appearance in the blood of enzymes in certain pathologic conditions9 and may therefore be clinically applicable. This paper presents the results of a study of this type on the influence of operative trauma in children on levels of the following blood enzymes: glutamicoxalacetic transaminase (GOT), glutamic-pyruvic transaminase (GPT), choline e&erase (ChE), leucine aminopeptidase (LAP), cystine aminopeptidase (CAP-“oxytocinase”) and Abderhalden enzymes, which hypothalamus, hypophysis and liver served as substrates for. MATERIAL
AND
METHODS
The study material consisted of 30 children aged 3 to 14 years (mean 7.1 years), in whom physical examination and laboratory studies before operation revealed no signs of active inflammation. Typical operations used in pediatric surgery were perClin.
Chim.
Acta,
14 (1966) 405-408
406
SHORT
COMMUNICATIONS
formed, according to the type of underlying disease: 20 children with various congenital anatomic anomalies (cleft lip and palate, torticollis, funnel chest), and IO children with inguinal hernia were operated. Enzyme determinations were made before and 2 and IO days after operation. GOT was determined by the method of Cabaud et al. lo, GPT according to Wrbblewski and CabaudI’, LAP according to the method described by Green and modified by Goldbarg et a1.l2, and CAP by the method of Tuppy and Neswadba as modified by Klimek 13. Abderhalden enzymes were estimated calorimetrically by the method described by R. Abderhalden9. GOT and GPT were expressed in terms of units per I y of pyruvic acid/ml/a0 min. ChE is given as percentages of hydrolyzed acetylcholine chloride, and LAP and CAP in pmoles of p-naphthylamine per l/min. Abderhalden’s reaction was graded in four degrees : negative, doubtful, positive and strongly positive. The findings were analyzed statistically with Student’s tests. Curves were plotted according to the standard enzymogram 14.All the methods have been verified and described in our previous communications.
TABLE
I Befove o$wation
Enzyme
Mean GOT GPT ChE
55 :: 17.4 0.65
LAP CAP
S.D.
Range
37 34 8 5.5 0.24
10-146 6-174 50-96 I O-29 0.1-12.8
2nd day after operation
roth day after operation
Mean
S.D.
Mean
S.D.
105 82 78 17.3 0.8
81 6g
86 89 76 ‘7 0.68
45 47 16
Range 20-336 28-364
13 4.2 0.28
43-95 15-33 0.5-1.62
4.6 0.2
Range 15-194 18-198 34-95 13-21.2 0.5-1.16
RESULTS
The absolute values, means, standard deviations and range of values of GOT, GPT, ChE, LAP and CAP before operation and 2-10 days after operation are given in Table I. Statistical analysis of the findings (Table II) revealed significant increase in the blood levels of both transaminases, 2 as well as IO days after operation, compared with the starting levels. On the other hand, the difference between the values of GOT and GPT, 2 and IO days after operation, is not significant. The changes in concentrations of ChE and LAP are also nonsignificant. The rise in the CAP levels on the second
TABLE
II
Enzyme
Student’s t test before operation and 2nd day after operation
Student’s t test before operation and 10th day after operation
GOT GPT ChE LAP CAP
3.01
0.01
2.32
0.05
2.87 3.73 1.82 0.3 2.03
Clin.
1.42 0.078 2.2 Chim.
Acta,
p
>
0.001
> p p p 0.05 > p
> > > >
0.02 0.05 0.05
14 (1966)
>
405-408
0.02
0.01
>
p
>
p p p p
> > > >
0.001 0.001
0.05 0.05 0.05
Student’s t test 2nd and 10th days after operation I.1
0.45 0.54 0.26 0.48
p p p p p
> > > > >
0.05 0.05 0.05 0.05 0.05
-
SHORT
COMMUNICATIONS
407
day is statistically significant, both in relation to the starting level and the level observed on the tenth day; the two last-mentioned levels were similar. In Table III are summarized the results of parallel determinations of the Abderhalden enzymes. It is worth noting that before operation positive results with hypothalamic substrate (14 times) were twice as frequent as with pituitary (6 times) and liver (7 times) substrates. On the 2nd and 10th days after operation, positive results (56 and 53 times) were observed twice as often as before operation (27 times). The course of the
TABLE
III
Abderhalden’s reaction
Negative Doubtful Positive Strongly
Before operation Hypothalamus
Hypephysis
Liver
‘3
22 2
I7 6
5
3 I3
positive I -____________
I __________
and day after operation
roth day after operation
Hypotha-
Hypotha-
Hypophysis
Livev
I
13
10
6
16
8
5
3 I3
4 II
3 9
:
:
I 16
2
I3
2
8
16
2
5
lamus
_____
lamus
Hypephysis
Lioer
reaction with liver substrate was most characteristic; positive results show twofold increase on the second day after operation, and threefold increase on the 10th day after operation, compared with the starting state. DISCUSSION
AND
CONCLUSIONS
Operative trauma in children produces changes in the blood levels of GOT, GPT and CAP, but has no significant influence on the concentrations of ChE and LAP. The changes in transaminase levels resemble those observed in adults with obstructive jaundice 2+, i.e., a significant rise occurs on the second day after operation, persisting up to the tenth day. In adults without jaundice, this phenomenon has a different course, significant rise in the concentrations occurring on the second day and returning to starting levels on the tenth day 2y4.The behavior of cystine aminopeptidase (“oxytocinase”) in the blood also differs from that in adults, in whom elevated levels are observed on the first and second days after operation and persist until the tenth day32’5. This has not been observed in children. The different response of LAP and CAP levels to the same stimulus suggests differences between the enzymatic systems breaking down the two amide substrates, as confirmed by other writers5T7’18.Operative trauma caused the appearance of the endogenous Abderhalden enzymes in the blood already on the second day after operation, twice as often as before operation. Their specificity was confirmed by the concordance between significant rise in transaminase levels (Table I) and positive results of the Abderhalden reaction with liver substrate (Table II). The experimental findings allow the following conclusions : (I) Operative trauma causes statistically significant elevations of levels of GOT, GPT and CAP in the blood of children. The behavior of concentrations of these enzymes in children differs from Clilz. Chim.
Acta, 14 (1966) 405-408
SHORT COMMUNICATIONS
408
that observed in adults. LAP and ChE are not significantly affected. (2) Operative trauma in children can liberate endogenous Abderhalden enzymes into the blood. Ilnd
Surgical Clinic*, IInd Clinic of Internal Medicine**,
J.
and the Ist Clinic of Obstetrics and Gynaecology***, Medical Academy,
2 3 4
5 6
R. H.
Cracow (Polalzd)
I J, GROCHOWSKI, in R. KLIMEK AND W. KR~L (Eds.),
GROCHOWSKI
Oxytocin
and
its Analogues,
KLIMEK D~oiBi
Cracow,
1964, P. 75. J. GROCHOWSKI AND R. KLIMEK, Pd. Przegl. Chir., 23 (1961) 527. R. KLIMEK. M. PIETRZYCKA AND J. GROCHOWSKI, Clin. Chim. Ada, 6 (1961) 326. J. OSZACKI, J. GROCHOWSKI AND R. KLIMEK, Pd. Przegl. Chir., 35 (1963) 523. H. DRO~D~, Serum Aminopeptidase Activity in Infectious Hepatitis, Thesis, Cracow, 1965. M. B. GLENDENING, M. A. TITUS, S. A. SCHROEDER, G. MOHUN AND E. W. PAGE, Am. J, Obstet.
Gynscol., 92 (1965) 814. 7 E. W. PAGE, M. A. TITUS, G. MOHUN AND M. B. GLENDENING, Am. J. Obstet. Gynecol., 82 (1961) 1090. E. ABDERHALDEN, Hoppe-Seylers 2. Physiol. Chem., 61 (1909) 200. R. ABDERHALDEN, in Ergeb. Enzymforsch., II (1950) I. P. CABAUD, R. LEEPER AND F. WR~BLE~SKI, Am. J. Clin. Pathol., 26 (1956) 1101. F. WR~BLEWSKI AND P. CABAUD, Am. J. Clin. Pathol., 27 (1957) 235. J. A. GOLDBARG AND A. M. RUTENBURG, Cancer, II (1958) 283. R. KLIMEK, PTL, 19 (1964) ZOOI. R. KLIRIEK, PTL, in press (1966). J. OSZACKI AND J. GROCHOWSKI, in R. KLIMEK AND W. KR~L (Eds.), Oxytocin and ifs Analogues, Cracow, 1964, p. 71. 16 H. TUPPY, Biochem. Pharmacol., 12, Suppl. 141 (1963). 17 D. VINCENT AND G. SEGONZAC, Ann. Biol. Clin., 3/4 (1958) 227.
8 9 IO II 12 13 14 15
Received
January
* Director: ** Director: *** Director:
6th, 1966
Prof. Dr. J. Oszacki. Assoc. Prof. Dr. S. Kirchmayer. Prof. Dr. S. Schwarz.
CZilz. Chim. Actn, 14 (1966) 4055408
Adenine nucleotide content of erythrocytes patients
of multiple
sclerosis
In multiple sclerosis, the multifocal disintegration of the myelin sheath is a very characteristic symptom. Cummings’ attributed the process to the disturbance of the lipid metabolism, Adamus to that of the metabolism of albumins. Recent biochemical investigations of multiple sclerosis suspect disturbances of the carbohydrate metabolism, which is the fundamental metabolic process of erythrocytes. The purpose of this work was to ascertain whether the appearance of changes in neural tissues in multiple sclerosis is reflected also in the carbohydrate metabolism of erythrocytes. To appreciate the changes which occur in erythrocytes, chromatographic analysis of the adenine compounds in the erythrocytes was carried out using Dowex I x 8 chloride 20-50 mesh according to Bartlett 3. Samples of blood were taken from a group of IO fasting normal individuals. Samples were also taken from the group of 7 fasting multiple sclerosis patients, and again 60 min after consumption of 50 g of glucose. The normal persons were not investigated after intake of glucose taking into consideration the results obtained by Gin. Chim. Acta, 14 (1966) 408-409
SHORTCOMMUNICATIONS
in our laboratories are currently for serum urea determination.
being directedtowarddevelopment
of aurease
Research Division, U&-Tech Chemical Manufacturing Company, Sun Valley, Calif. ad Department of Pathology, Los Angeles Coudy Los Angeles,
Received
method
J. REARDON J. A. FOREMAN R. L. SEARCY
General Hospital (Ulzit 2),
Calif. (U.S.A.)
March r8th,
1966 Cli~z. Chim. Acta, 14 (1966) 403-405
The influence of operative trauma on some blood enzyme levels in children Operative trauma is a strong stimulus acting on the state of homeostasis in the body. An effect on the blood levels of certain enzymes, in particular the transaminases and oxytocinase, was observed in previous studies1-4. On comparing the blood concentrations of the enzymes in relation to the age of the patients, changes in enzymatic adaptation of the body can be observed. In addition, by employing two different substrates decomposed by related enzymatic systems, specificity of enzymes can be studied under the influence of the same operative stimulus. For instance, oxytocinase decomposes L-cystine-di-@aphthylamide as well as L-leucine-/-naphthylamide, the first of which is resistant to the action of leucine aminopeptidase. Estimation of the degree of decomposition of these two substrates therefore allows complex study of two blood aminopeptidase activities, which behave differently during pregnancy and in viral hepatitis6-7. The behavior of well-known enzymes can be utilized also in comparative studies on the specificity and nature of other, less well-known, enzymes, e.g. by means of the Abderhalden reaction8. This reaction is based on the breakdown by blood of specific organ tissues in connection with the appearance in the blood of enzymes in certain pathologic conditions9 and may therefore be clinically applicable. This paper presents the results of a study of this type on the influence of operative trauma in children on levels of the following blood enzymes: glutamicoxalacetic transaminase (GOT), glutamic-pyruvic transaminase (GPT), choline e&erase (ChE), leucine aminopeptidase (LAP), cystine aminopeptidase (CAP-“oxytocinase”) and Abderhalden enzymes, which hypothalamus, hypophysis and liver served as substrates for. MATERIAL
AND
METHODS
The study material consisted of 30 children aged 3 to 14 years (mean 7.1 years), in whom physical examination and laboratory studies before operation revealed no signs of active inflammation. Typical operations used in pediatric surgery were perClin.
Chim.
Acta,
14 (1966) 405-408
406
SHORT
COMMUNICATIONS
formed, according to the type of underlying disease: 20 children with various congenital anatomic anomalies (cleft lip and palate, torticollis, funnel chest), and IO children with inguinal hernia were operated. Enzyme determinations were made before and 2 and IO days after operation. GOT was determined by the method of Cabaud et al. lo, GPT according to Wrbblewski and CabaudI’, LAP according to the method described by Green and modified by Goldbarg et a1.l2, and CAP by the method of Tuppy and Neswadba as modified by Klimek 13. Abderhalden enzymes were estimated calorimetrically by the method described by R. Abderhalden9. GOT and GPT were expressed in terms of units per I y of pyruvic acid/ml/a0 min. ChE is given as percentages of hydrolyzed acetylcholine chloride, and LAP and CAP in pmoles of p-naphthylamine per l/min. Abderhalden’s reaction was graded in four degrees : negative, doubtful, positive and strongly positive. The findings were analyzed statistically with Student’s tests. Curves were plotted according to the standard enzymogram 14.All the methods have been verified and described in our previous communications.
TABLE
I Befove o$wation
Enzyme
Mean GOT GPT ChE
55 :: 17.4 0.65
LAP CAP
S.D.
Range
37 34 8 5.5 0.24
10-146 6-174 50-96 I O-29 0.1-12.8
2nd day after operation
roth day after operation
Mean
S.D.
Mean
S.D.
105 82 78 17.3 0.8
81 6g
86 89 76 ‘7 0.68
45 47 16
Range 20-336 28-364
13 4.2 0.28
43-95 15-33 0.5-1.62
4.6 0.2
Range 15-194 18-198 34-95 13-21.2 0.5-1.16
RESULTS
The absolute values, means, standard deviations and range of values of GOT, GPT, ChE, LAP and CAP before operation and 2-10 days after operation are given in Table I. Statistical analysis of the findings (Table II) revealed significant increase in the blood levels of both transaminases, 2 as well as IO days after operation, compared with the starting levels. On the other hand, the difference between the values of GOT and GPT, 2 and IO days after operation, is not significant. The changes in concentrations of ChE and LAP are also nonsignificant. The rise in the CAP levels on the second
TABLE
II
Enzyme
Student’s t test before operation and 2nd day after operation
Student’s t test before operation and 10th day after operation
GOT GPT ChE LAP CAP
3.01
0.01
2.32
0.05
2.87 3.73 1.82 0.3 2.03
Clin.
1.42 0.078 2.2 Chim.
Acta,
p
>
0.001
> p p p 0.05 > p
> > > >
0.02 0.05 0.05
14 (1966)
>
405-408
0.02
0.01
>
p
>
p p p p
> > > >
0.001 0.001
0.05 0.05 0.05
Student’s t test 2nd and 10th days after operation I.1
0.45 0.54 0.26 0.48
p p p p p
> > > > >
0.05 0.05 0.05 0.05 0.05
-
SHORT
COMMUNICATIONS
407
day is statistically significant, both in relation to the starting level and the level observed on the tenth day; the two last-mentioned levels were similar. In Table III are summarized the results of parallel determinations of the Abderhalden enzymes. It is worth noting that before operation positive results with hypothalamic substrate (14 times) were twice as frequent as with pituitary (6 times) and liver (7 times) substrates. On the 2nd and 10th days after operation, positive results (56 and 53 times) were observed twice as often as before operation (27 times). The course of the
TABLE
III
Abderhalden’s reaction
Negative Doubtful Positive Strongly
Before operation Hypothalamus
Hypephysis
Liver
‘3
22 2
I7 6
5
3 I3
positive I -____________
I __________
and day after operation
roth day after operation
Hypotha-
Hypotha-
Hypophysis
Livev
I
13
10
6
16
8
5
3 I3
4 II
3 9
:
:
I 16
2
I3
2
8
16
2
5
lamus
_____
lamus
Hypephysis
Lioer
reaction with liver substrate was most characteristic; positive results show twofold increase on the second day after operation, and threefold increase on the 10th day after operation, compared with the starting state. DISCUSSION
AND
CONCLUSIONS
Operative trauma in children produces changes in the blood levels of GOT, GPT and CAP, but has no significant influence on the concentrations of ChE and LAP. The changes in transaminase levels resemble those observed in adults with obstructive jaundice 2+, i.e., a significant rise occurs on the second day after operation, persisting up to the tenth day. In adults without jaundice, this phenomenon has a different course, significant rise in the concentrations occurring on the second day and returning to starting levels on the tenth day 2y4.The behavior of cystine aminopeptidase (“oxytocinase”) in the blood also differs from that in adults, in whom elevated levels are observed on the first and second days after operation and persist until the tenth day32’5. This has not been observed in children. The different response of LAP and CAP levels to the same stimulus suggests differences between the enzymatic systems breaking down the two amide substrates, as confirmed by other writers5T7’18.Operative trauma caused the appearance of the endogenous Abderhalden enzymes in the blood already on the second day after operation, twice as often as before operation. Their specificity was confirmed by the concordance between significant rise in transaminase levels (Table I) and positive results of the Abderhalden reaction with liver substrate (Table II). The experimental findings allow the following conclusions : (I) Operative trauma causes statistically significant elevations of levels of GOT, GPT and CAP in the blood of children. The behavior of concentrations of these enzymes in children differs from Clilz. Chim.
Acta, 14 (1966) 405-408
SHORT COMMUNICATIONS
408
that observed in adults. LAP and ChE are not significantly affected. (2) Operative trauma in children can liberate endogenous Abderhalden enzymes into the blood. Ilnd
Surgical Clinic*, IInd Clinic of Internal Medicine**,
J.
and the Ist Clinic of Obstetrics and Gynaecology***, Medical Academy,
2 3 4
5 6
R. H.
Cracow (Polalzd)
I J, GROCHOWSKI, in R. KLIMEK AND W. KR~L (Eds.),
GROCHOWSKI
Oxytocin
and
its Analogues,
KLIMEK D~oiBi
Cracow,
1964, P. 75. J. GROCHOWSKI AND R. KLIMEK, Pd. Przegl. Chir., 23 (1961) 527. R. KLIMEK. M. PIETRZYCKA AND J. GROCHOWSKI, Clin. Chim. Ada, 6 (1961) 326. J. OSZACKI, J. GROCHOWSKI AND R. KLIMEK, Pd. Przegl. Chir., 35 (1963) 523. H. DRO~D~, Serum Aminopeptidase Activity in Infectious Hepatitis, Thesis, Cracow, 1965. M. B. GLENDENING, M. A. TITUS, S. A. SCHROEDER, G. MOHUN AND E. W. PAGE, Am. J, Obstet.
Gynscol., 92 (1965) 814. 7 E. W. PAGE, M. A. TITUS, G. MOHUN AND M. B. GLENDENING, Am. J. Obstet. Gynecol., 82 (1961) 1090. E. ABDERHALDEN, Hoppe-Seylers 2. Physiol. Chem., 61 (1909) 200. R. ABDERHALDEN, in Ergeb. Enzymforsch., II (1950) I. P. CABAUD, R. LEEPER AND F. WR~BLE~SKI, Am. J. Clin. Pathol., 26 (1956) 1101. F. WR~BLEWSKI AND P. CABAUD, Am. J. Clin. Pathol., 27 (1957) 235. J. A. GOLDBARG AND A. M. RUTENBURG, Cancer, II (1958) 283. R. KLIMEK, PTL, 19 (1964) ZOOI. R. KLIRIEK, PTL, in press (1966). J. OSZACKI AND J. GROCHOWSKI, in R. KLIMEK AND W. KR~L (Eds.), Oxytocin and ifs Analogues, Cracow, 1964, p. 71. 16 H. TUPPY, Biochem. Pharmacol., 12, Suppl. 141 (1963). 17 D. VINCENT AND G. SEGONZAC, Ann. Biol. Clin., 3/4 (1958) 227.
8 9 IO II 12 13 14 15
Received
January
* Director: ** Director: *** Director:
6th, 1966
Prof. Dr. J. Oszacki. Assoc. Prof. Dr. S. Kirchmayer. Prof. Dr. S. Schwarz.
CZilz. Chim. Actn, 14 (1966) 4055408
Adenine nucleotide content of erythrocytes patients
of multiple
sclerosis
In multiple sclerosis, the multifocal disintegration of the myelin sheath is a very characteristic symptom. Cummings’ attributed the process to the disturbance of the lipid metabolism, Adamus to that of the metabolism of albumins. Recent biochemical investigations of multiple sclerosis suspect disturbances of the carbohydrate metabolism, which is the fundamental metabolic process of erythrocytes. The purpose of this work was to ascertain whether the appearance of changes in neural tissues in multiple sclerosis is reflected also in the carbohydrate metabolism of erythrocytes. To appreciate the changes which occur in erythrocytes, chromatographic analysis of the adenine compounds in the erythrocytes was carried out using Dowex I x 8 chloride 20-50 mesh according to Bartlett 3. Samples of blood were taken from a group of IO fasting normal individuals. Samples were also taken from the group of 7 fasting multiple sclerosis patients, and again 60 min after consumption of 50 g of glucose. The normal persons were not investigated after intake of glucose taking into consideration the results obtained by Gin. Chim. Acta, 14 (1966) 408-409