395
Acta, 203 (1991) 395-402 0 1991 Elsevier Science Publishers B.V. All rights reserved 0009-8981/91/$03.50
Clinica Chimica
CCA 05144
Short Communication
Measurement of ornithine carbamyl transferase (OCT) in plasma by means of enzymatic determination of ammonia Henry G. Peltenburg ‘, Mieke A. Janssen 2, Peter B. Soeters 2, J. Guus Flendrig ’ and Wim Th. Hermens 3 Departments
of ’ Internal
Medicine,
’ General Surgery, and 3 Cardiotiascular
Uniuersity of Limburg
Institute,
(Netherlands)
(Received 23 October 1990; revision received 29 August 1991; accepted 16 September 1991) Key words: Ornithine carbamyl transferase; Liver mitochondria; Ammonia assay; Glutamate dehydrogenase; Mitochondrial aspartate aminotransferase
Release of mitochondrial enzymes from the liver is considered to provide strong evidence for hepatic necrosis [1,2], and is also associated with specific forms of liver disease. It has been shown, for instance, that glutamate dehydrogenase (GLDH) correlates well with the presence and extent of necrosis in alcoholic liver disease [3], and the ratio of mitochondrial and total aspartate aminotransferase (mAST) has been proposed as a marker for chronic alcoholism [4]. However, both GLDH and mAST are widely distributed in various organs and lack specificity as a marker of liver injury. Ornithine carbamyl transferase (OCT; EC 2.1.3.3) is located in the mitochondrial matrix of the hepatocyte and plays a role in the urea cycle. Inherited deficiencies are described with impaired ureagenesis and ammonia intoxication [5]. The enzyme was first described by Grisolia [6] and Krebs [7] and further characterized by Reichard [81. Apart from some OCT activity in the small bowel mucosa, the enzyme is liver-specific in man [9]. Plasma OCI activity may rise by a factor of 100-1000 in various forms of liver pathology and thus appears to be a sensitive marker. In view of these properties, the use of OCI as a marker of mitochondrial liver damage was advocated [lo], but its routine use has been hampered by the complicated assay procedure. Measurement of the forward reaction by estimation of citrulline production, has been proposed by Brown and Grisolia [ll], and
Correspondence to: Dr. H.G. Peltenburg M.D. Ph.D, Department of Internal Medicine, University Hospital Maastricht, P.O. Box 5800, 6202 AZ Maastricht, The Netherlands.
396
modified [12]. Reichard originally proposed measurement of the reverse reaction by addition of arsenate instead of phosphate. The carbamyl arsenate thus formed is unstable and decomposes spontaneously, producing ammonia and carbon dioxide. By using radiolabelled citrulline, the measurement of 14C0, provides a sensitive assay [8]. Alternative measurement of ammonia was first performed with the microdiffusion technique using the Nessler reagent [13]. This method was simplified by direct detection of ammonia with the phenol reagent (Berthelot’s reaction [141X Both procedures require a deproteinization step. A method for the enzymatic determination of ammonia without the deproteinization step was first described by Da Fonseca Wollheim [15]. This method was applied in the present study for an improved assay of OCT activity. Materials and methods The following solutions were prepared with deionized water (Millipore Q system). Unless indicated otherwise, reagents were obtained from Sigma Chemical Co. (St. Louis, MO, U.S.A.) and of analytical grade. Arsenate buffer (Sodium arsenate 0.5 mol/l, pH 7.1) 7.8 g Sodium arsenate was dissolved in 40 ml water, pH adjusted to 7.1 and the buffer was further diluted to 50 ml. Citrulline-arsenate buffer (r_-citrulline 0.1 mol/l) 175 mg L-citrulline was dissolved in 10 ml of the sodium arsenate buffer. A fresh solution should be prepared weekly. Standard solutions of ammonia were prepared by dilution of a stock solution containing 330 mg dried (NH,)$O, (Merck 1217) in 1 1 water (5 mmol/l). An OCT calibration curve (cf. Fig. 2) was obtained with an OCT preparation of Streptococcus fuecalis (Sigma 025011, a lyophilized powder containing 40% protein and 835 U of OCT activity per mg of protein. Incubation procedure for the OCT determination
0.05 ml Plasma was added to 0.05 ml citrulline arsenate buffer in a microtube (Sample caps blue, Roche no. 10 0678 9). A reference tube containing 0.05 ml plasma and 0.05 ml arsenate buffer was used as a blank. Both tubes were firmly closed, mixed and incubated for 24 h at 37°C. The tubes were either directly assayed for ammonia or stored at -70°C. Ammonia assay
A commercially available test kit (Sigma 170-UV) was used. The assay is based on the method proposed by Da Fonseca-Wollheim [13], the endpoint being the rate of disappearance of NADH, which was measured spectrophotometrically at a wavelength of 340 nm. These measurements were performed in a Cobas Bio
397
centrifugal analyzer (Hoffmann-Laroche) at 25°C and expressed in micromoles of substrate converted per minute per liter of plasma (U/l). Determination of GLDH and AST activities
Glutamate dehydrogenase (GLDH; EC 1.2.1.3) was assayed spectrophotometritally by means of a commercially available kit (Boehringer nr. 124320). Total aspartate aminotransferase CAST, EC 2.6.1.1) activity was measured spectrophotometrically by means of a commercially available kit (Boehringer nr. B258784). Cytosolic and mitochondrial AST were separated on LD gels (Paragon, Beckman) with the method described by Sakakibara [161. The procedure was carried out on a Beckman type P/N 65580 electrophoresis apparatus. Blood sampling
Blood was collected in heparinized tubes kept on ice and centrifuged for 10 min at 1000 X g. Plasma samples were stored at - 70°C if the OCT determination was not performed within 30 min after blood collection. Patients
Patient A, a 42-year-old woman, was admitted to the Intensive Care Unit with a and septicaemia. The patient had severe circulatory failure for approximately 45 min. After that, circulation was restored, and the infection successfully treated. There was no electrocardiographical sign of myocardial infarction, and creatine kinase remained within normal limits. Patient B, a 67-year-old man, suffered from chronic obstructive lung disease. He had an episode of respiratory and circulatory failure after a period of atria1 fibrillation. There was no increase in creatine kinase activity in plasma directly after his transient heart failure. Patient C, a 75-year-old woman, was admitted with cholecystolithiasis and biliary colic. After endogenous retrograde cholangiography and papillotomy, the obstruction was relieved and liver enzymes decreased. There was no sign of cardiac disease and no circulatory failure. Patient D, a 64-year-old man, known with a low output syndrome, had an episode of severe heart failure without ECG signs of myocardial infarction or increased creatine kinase activity. He was admitted to the Intensive Care Unit for ventilatory support.
Staphylococcus aweuS endocarditis
Normal controls
Samples were taken from 13 individuals without a previous history of liver disease. Seven women with a mean age of 29, and 6 men with a mean age of 32 were included.
398
Results Linearity The capacity of the ammonia assay was tested for a series of dilutions of a standard ammonia solution ranging from 0.1 to 1.2 mmol/l. Recoveries not significantly different from 100% were found in a range of ammonia concentrations up to 0.6 mmol/l (Fig. 1). This figure also shows that the assay was not influenced by the presence of citrulline and arsenate in the incubation mixture. This range of ammonia concentrations corresponds to a rate of ammonia production up to 400 nmoi/min/l during the 24-h incubation period, i.e., a range of OCT activity up to 400 mu/l. The linearity of ammonia production during the 24-h incubation period was confirmed using a series of diIutions of the enzyme preparation. As shown in Fig. 2, the production was hnear to more than 5 mmol of ammonia/Z4 h/l, corre-
0
500
Ammonia
standards
1000
(PM)
Fig. 1. Linearity of the ammonia assay. Open circles indicate samples in citrulline arsenate buffer.
R&&e
concentration of OCT
Fig. 2. Linearity of the production of ammonia during the 24-h incubation procedure.
399
TABLE I Precision of the OCT assay Mean blU/l)
E
13 14 16
55 1188 10268
2.1 2.9 1.9
3 3
220 2052
4.4
n
Ifma assay variation Normal plasma Plasma from patients Liver homogenate Interassay variation OCT (Sir. faec.1 1:
100 CXT (Str. faec.) 1: 10
1.7
spending to an OCT activity of 3500 mU/l. The concentration of citrulline in the incubation mixture hardly changed during the production of this amount of ammonia (from 200 to 195 mmol/l) and remained well above the value of K, = 34 mmoI/I estimated for citrulline l-171. From these data it is apparent that the linearity of the overall OCI assay was limited by the enzymatic measurement of ammonia, In order to preserve a single closed vessel procedure, we advocate the use of reduced sample volumes instead of dilution. It was verified that reduction of the sample volume to 5 mcl did not result in a significant reduction of accuracy. This reduction of sample volume was applied as soon as the limiting value of 400 mU/I was exceeded. Only for samples with OCT activities exceeding 4000 mU/l was manual dilution applied.
Both day-to-day and within-day precision was assessed by analyzing plasma samples, liver homogenates and the Streptococcusfaecalis preparation. The results are shown in Table I.
Plasma samples were assayed with the standard Berthelot reaction using the phenol reagent [14], and with the enzymatic method. The best fitting straight line was calculated by means of linear regression. A correlation coefficient of 0.987 was obtained with a slope of 1.01 and an intercept at the ordinate of -0.11 (Fig. 31. Limitation of incubation period There was a linear relationship between incubation time and ammonia production (Fig. 4). An incubation period of at least 4 h is advocated to minimize” errors introdu~d by thawing.
400
10-
Split-sample
comparison
Enzymatic
VI Berthslot’s
method
method
E
0
10 Enzymat?
method
Fig. 3. Comparison of the enzymatic procedure with Berthelot’s phenol reaction.
1
0
60
120
160
Incubation
240
300
360
time (min)
4. Limitation of the incubation period as an alternative for sample dilution.
600
? 3 x .% .>
0-O mAST A--rGLDH n --BOCT xl 00
400
2 E b
200
15
0 0
12
24
36
48
60
72
84
96
Time (h)
Fig. 5. Time-activity curves of three mitochondrial enzymes in four patients after acute liver injury. Mean values + SEM are shown.
401
Time-activitycurves of mitochondrial enzymes Figure 5 shows the mean time activity curves of three mitochondrial liver enzymes (OCT, mAST, GLDH) for the four patients described in the Methods section. Discussion The properties of OCT have been studied by Kalousek 1171.The enzyme is quite stable over a temperature range from - 50°C to 60°C and over a pH range from 5.8 to 8.2. An apparent K, for ornithine of 0.4 mmol/l and for carbamyl phosphate of 0.16 mmol/l has been estimated. The value of K, for citrulline in the reverse reaction is 34 mmol/l for the arsenate system [181. The maximal catalytic rate for the reverse reaction is about ten times lower than for the forward reaction [19]. Because of its much higher velocity, the forward reaction allows determination of OCT activities without the lengthy incubation period. This approach has, however, some disadvantages. First, an incubation step with urease is necessary to remove urea from the plasma to avoid formation of ammonia. Second, the instability of carbamyl phosphate requires fresh substrate solutions every two h [12]. Further, the procedures with ‘a deproteinization step (the microdiffusion technique and the Berthelot’s reaction) also have the risk of erroneously increasing ammonia [15]. In order to optimize the ammonia determination a closed vessel procedure should be preserved with reduced sample volumes instead of manual dilution. This sample volume reduction may be applied above.OCT activities of 400 mu/l. For values above 4000 mU/l samples should be diluted manually, or, alternatively, the incubation time may be limited to appro~mately 6 h. A shorter incubation time may also be used if a stat analysis is wanted. In Fig. 5, apparently, the enzymes reach peak activity simultaneously. The disappearance rate from plasma for OCT and GLDH is by comparison slower than for mAST. In conclusion, the combination of the shorter incubation procedure with the enzymatic ammonia assay, without a urease or deproteinization step, as proposed in the present study, facilitates the OCT determination and makes it more suitable for use in automatic analyzers. References 1 Schmidt E, Schmidt FW. Aspekte der Enzym-Diagnostik. Med Welt 1970;21:805-816. 2 Frederiks WM, Vogels IMC, Fronik GM. Plasma omithine carbamyl transferase level as an indicator of ischaemic injury of rat liver. Cell Biochem Funct 1984;2:217-220. 3 Van Waes L, Lieber CS. Glutamate dehydrogenase: a reliable marker of liver cell necrosis in the alcoholic. Br Med J 1977;2:1508-1510. 4 Nalpas B, Vassault A, Charpin S, Lacour B, Berthetot P. Serum mitochondrial aspartate aminotransferase as a marker of chronic alcoholism: diagnostic value and interpretation in a liver unit. Hepatology 1986;6:608-614.
402 5 Striver CR, Rosenberg LE. Amino acid metabolism and its disorders. Philadelphia, WB Saunders, 1973:234-249. 6 Grisolia S, Cohen P. Catalytic role of carbamyl glutamate in citrulline synthesis. J Biol Chem 1952;198:561-565. 7 Krebs HA, Eggleston LV, Knivett VA. Arsenolysis and phosphorolysis of citrulline in mammalian liver. Biochem J 1955;59:185-193. 8 Reichard H, Reichard P. Determination of ornithine carbamyl transferase in serum. J Lab Clin Med 1958;52:709-717. 9 Reichard H. Ornithine carbamyl transferase activity in human serum in diseases of the liver and the biliary system. J Lab Clin Med 1961;57:78-87. IO Sato T, Tanaka J, Kono Y, Jones RT, Cowley RA, Trump BF. Hepatic cellular injury following lethal E. coli bacteremia in rats. Lab Invest 1982;47:304-310. I1 Brown RW, Grisolia S. Ornithine transcarbamylase activity in serum. J Lab Clin Med 1959;54:617620. I2 Ceriotti G. Ornithine carbamyl transferase. In: Bergmeyer HU, Methoden der Enzymatischen Analyse. Weinheim/Bergstr: Verlag Chemie, 1974;732-739. I3 Reichard H. Determination of ornithine carbamyl transferase with microdiffusion technique. Stand J Clin Lab Invest 1957;9:311-312. I4 Kontinnen A. Simple method for the determination of ornithine transcarbamylase activity in serum. Clin Chim Acta 1967;18:147-150. I5 Da Fonseca-Wollheim F. Direkte Plasmaammoniakbestimmung ohne Enteiweissung. Z Klin Chem Klin Biochem 1973;10:426-431. I6 Sakakibara S, Shiomi S, Kobayashi S, lkeda T, lrrai S, Kagamiyama H. A convenient and sensitive method for the determination of serum aspartate aminotransferase isozymes after electropheresis. Clin Chim Acta 1983;133:119-123. I7 Kalousek F, Francois B, Rosenberg LE. Isolation and characterization of ornithine transcarbamylase from normal human liver. J Biol Chem 1978;253:3939-3944. I8 Korzenovsky M, Werkman C. Conversion of citrulline to ornithine by cell-free extracts of Str. lactis. Arch Biochem Biophys 1953;46:174-185. I9 Schimke RT. Ornithine Carbamyltransferase (Mycoplasma). In: Colowick SP, Kaplan NO, eds. Methods in Enzymology, Vol. XXVlIA. New York: AC. Press, 1970:295-297.
Acta, 203 (1991) 395-402 0 1991 Elsevier Science Publishers B.V. All rights reserved 0009-8981/91/$03.50
Clinica Chimica
CCA 05144
Short Communication
Measurement of ornithine carbamyl transferase (OCT) in plasma by means of enzymatic determination of ammonia Henry G. Peltenburg ‘, Mieke A. Janssen 2, Peter B. Soeters 2, J. Guus Flendrig ’ and Wim Th. Hermens 3 Departments
of ’ Internal
Medicine,
’ General Surgery, and 3 Cardiotiascular
Uniuersity of Limburg
Institute,
(Netherlands)
(Received 23 October 1990; revision received 29 August 1991; accepted 16 September 1991) Key words: Ornithine carbamyl transferase; Liver mitochondria; Ammonia assay; Glutamate dehydrogenase; Mitochondrial aspartate aminotransferase
Release of mitochondrial enzymes from the liver is considered to provide strong evidence for hepatic necrosis [1,2], and is also associated with specific forms of liver disease. It has been shown, for instance, that glutamate dehydrogenase (GLDH) correlates well with the presence and extent of necrosis in alcoholic liver disease [3], and the ratio of mitochondrial and total aspartate aminotransferase (mAST) has been proposed as a marker for chronic alcoholism [4]. However, both GLDH and mAST are widely distributed in various organs and lack specificity as a marker of liver injury. Ornithine carbamyl transferase (OCT; EC 2.1.3.3) is located in the mitochondrial matrix of the hepatocyte and plays a role in the urea cycle. Inherited deficiencies are described with impaired ureagenesis and ammonia intoxication [5]. The enzyme was first described by Grisolia [6] and Krebs [7] and further characterized by Reichard [81. Apart from some OCT activity in the small bowel mucosa, the enzyme is liver-specific in man [9]. Plasma OCI activity may rise by a factor of 100-1000 in various forms of liver pathology and thus appears to be a sensitive marker. In view of these properties, the use of OCI as a marker of mitochondrial liver damage was advocated [lo], but its routine use has been hampered by the complicated assay procedure. Measurement of the forward reaction by estimation of citrulline production, has been proposed by Brown and Grisolia [ll], and
Correspondence to: Dr. H.G. Peltenburg M.D. Ph.D, Department of Internal Medicine, University Hospital Maastricht, P.O. Box 5800, 6202 AZ Maastricht, The Netherlands.
396
modified [12]. Reichard originally proposed measurement of the reverse reaction by addition of arsenate instead of phosphate. The carbamyl arsenate thus formed is unstable and decomposes spontaneously, producing ammonia and carbon dioxide. By using radiolabelled citrulline, the measurement of 14C0, provides a sensitive assay [8]. Alternative measurement of ammonia was first performed with the microdiffusion technique using the Nessler reagent [13]. This method was simplified by direct detection of ammonia with the phenol reagent (Berthelot’s reaction [141X Both procedures require a deproteinization step. A method for the enzymatic determination of ammonia without the deproteinization step was first described by Da Fonseca Wollheim [15]. This method was applied in the present study for an improved assay of OCT activity. Materials and methods The following solutions were prepared with deionized water (Millipore Q system). Unless indicated otherwise, reagents were obtained from Sigma Chemical Co. (St. Louis, MO, U.S.A.) and of analytical grade. Arsenate buffer (Sodium arsenate 0.5 mol/l, pH 7.1) 7.8 g Sodium arsenate was dissolved in 40 ml water, pH adjusted to 7.1 and the buffer was further diluted to 50 ml. Citrulline-arsenate buffer (r_-citrulline 0.1 mol/l) 175 mg L-citrulline was dissolved in 10 ml of the sodium arsenate buffer. A fresh solution should be prepared weekly. Standard solutions of ammonia were prepared by dilution of a stock solution containing 330 mg dried (NH,)$O, (Merck 1217) in 1 1 water (5 mmol/l). An OCT calibration curve (cf. Fig. 2) was obtained with an OCT preparation of Streptococcus fuecalis (Sigma 025011, a lyophilized powder containing 40% protein and 835 U of OCT activity per mg of protein. Incubation procedure for the OCT determination
0.05 ml Plasma was added to 0.05 ml citrulline arsenate buffer in a microtube (Sample caps blue, Roche no. 10 0678 9). A reference tube containing 0.05 ml plasma and 0.05 ml arsenate buffer was used as a blank. Both tubes were firmly closed, mixed and incubated for 24 h at 37°C. The tubes were either directly assayed for ammonia or stored at -70°C. Ammonia assay
A commercially available test kit (Sigma 170-UV) was used. The assay is based on the method proposed by Da Fonseca-Wollheim [13], the endpoint being the rate of disappearance of NADH, which was measured spectrophotometrically at a wavelength of 340 nm. These measurements were performed in a Cobas Bio
397
centrifugal analyzer (Hoffmann-Laroche) at 25°C and expressed in micromoles of substrate converted per minute per liter of plasma (U/l). Determination of GLDH and AST activities
Glutamate dehydrogenase (GLDH; EC 1.2.1.3) was assayed spectrophotometritally by means of a commercially available kit (Boehringer nr. 124320). Total aspartate aminotransferase CAST, EC 2.6.1.1) activity was measured spectrophotometrically by means of a commercially available kit (Boehringer nr. B258784). Cytosolic and mitochondrial AST were separated on LD gels (Paragon, Beckman) with the method described by Sakakibara [161. The procedure was carried out on a Beckman type P/N 65580 electrophoresis apparatus. Blood sampling
Blood was collected in heparinized tubes kept on ice and centrifuged for 10 min at 1000 X g. Plasma samples were stored at - 70°C if the OCT determination was not performed within 30 min after blood collection. Patients
Patient A, a 42-year-old woman, was admitted to the Intensive Care Unit with a and septicaemia. The patient had severe circulatory failure for approximately 45 min. After that, circulation was restored, and the infection successfully treated. There was no electrocardiographical sign of myocardial infarction, and creatine kinase remained within normal limits. Patient B, a 67-year-old man, suffered from chronic obstructive lung disease. He had an episode of respiratory and circulatory failure after a period of atria1 fibrillation. There was no increase in creatine kinase activity in plasma directly after his transient heart failure. Patient C, a 75-year-old woman, was admitted with cholecystolithiasis and biliary colic. After endogenous retrograde cholangiography and papillotomy, the obstruction was relieved and liver enzymes decreased. There was no sign of cardiac disease and no circulatory failure. Patient D, a 64-year-old man, known with a low output syndrome, had an episode of severe heart failure without ECG signs of myocardial infarction or increased creatine kinase activity. He was admitted to the Intensive Care Unit for ventilatory support.
Staphylococcus aweuS endocarditis
Normal controls
Samples were taken from 13 individuals without a previous history of liver disease. Seven women with a mean age of 29, and 6 men with a mean age of 32 were included.
398
Results Linearity The capacity of the ammonia assay was tested for a series of dilutions of a standard ammonia solution ranging from 0.1 to 1.2 mmol/l. Recoveries not significantly different from 100% were found in a range of ammonia concentrations up to 0.6 mmol/l (Fig. 1). This figure also shows that the assay was not influenced by the presence of citrulline and arsenate in the incubation mixture. This range of ammonia concentrations corresponds to a rate of ammonia production up to 400 nmoi/min/l during the 24-h incubation period, i.e., a range of OCT activity up to 400 mu/l. The linearity of ammonia production during the 24-h incubation period was confirmed using a series of diIutions of the enzyme preparation. As shown in Fig. 2, the production was hnear to more than 5 mmol of ammonia/Z4 h/l, corre-
0
500
Ammonia
standards
1000
(PM)
Fig. 1. Linearity of the ammonia assay. Open circles indicate samples in citrulline arsenate buffer.
R&&e
concentration of OCT
Fig. 2. Linearity of the production of ammonia during the 24-h incubation procedure.
399
TABLE I Precision of the OCT assay Mean blU/l)
E
13 14 16
55 1188 10268
2.1 2.9 1.9
3 3
220 2052
4.4
n
Ifma assay variation Normal plasma Plasma from patients Liver homogenate Interassay variation OCT (Sir. faec.1 1:
100 CXT (Str. faec.) 1: 10
1.7
spending to an OCT activity of 3500 mU/l. The concentration of citrulline in the incubation mixture hardly changed during the production of this amount of ammonia (from 200 to 195 mmol/l) and remained well above the value of K, = 34 mmoI/I estimated for citrulline l-171. From these data it is apparent that the linearity of the overall OCI assay was limited by the enzymatic measurement of ammonia, In order to preserve a single closed vessel procedure, we advocate the use of reduced sample volumes instead of dilution. It was verified that reduction of the sample volume to 5 mcl did not result in a significant reduction of accuracy. This reduction of sample volume was applied as soon as the limiting value of 400 mU/I was exceeded. Only for samples with OCT activities exceeding 4000 mU/l was manual dilution applied.
Both day-to-day and within-day precision was assessed by analyzing plasma samples, liver homogenates and the Streptococcusfaecalis preparation. The results are shown in Table I.
Plasma samples were assayed with the standard Berthelot reaction using the phenol reagent [14], and with the enzymatic method. The best fitting straight line was calculated by means of linear regression. A correlation coefficient of 0.987 was obtained with a slope of 1.01 and an intercept at the ordinate of -0.11 (Fig. 31. Limitation of incubation period There was a linear relationship between incubation time and ammonia production (Fig. 4). An incubation period of at least 4 h is advocated to minimize” errors introdu~d by thawing.
400
10-
Split-sample
comparison
Enzymatic
VI Berthslot’s
method
method
E
0
10 Enzymat?
method
Fig. 3. Comparison of the enzymatic procedure with Berthelot’s phenol reaction.
1
0
60
120
160
Incubation
240
300
360
time (min)
4. Limitation of the incubation period as an alternative for sample dilution.
600
? 3 x .% .>
0-O mAST A--rGLDH n --BOCT xl 00
400
2 E b
200
15
0 0
12
24
36
48
60
72
84
96
Time (h)
Fig. 5. Time-activity curves of three mitochondrial enzymes in four patients after acute liver injury. Mean values + SEM are shown.
401
Time-activitycurves of mitochondrial enzymes Figure 5 shows the mean time activity curves of three mitochondrial liver enzymes (OCT, mAST, GLDH) for the four patients described in the Methods section. Discussion The properties of OCT have been studied by Kalousek 1171.The enzyme is quite stable over a temperature range from - 50°C to 60°C and over a pH range from 5.8 to 8.2. An apparent K, for ornithine of 0.4 mmol/l and for carbamyl phosphate of 0.16 mmol/l has been estimated. The value of K, for citrulline in the reverse reaction is 34 mmol/l for the arsenate system [181. The maximal catalytic rate for the reverse reaction is about ten times lower than for the forward reaction [19]. Because of its much higher velocity, the forward reaction allows determination of OCT activities without the lengthy incubation period. This approach has, however, some disadvantages. First, an incubation step with urease is necessary to remove urea from the plasma to avoid formation of ammonia. Second, the instability of carbamyl phosphate requires fresh substrate solutions every two h [12]. Further, the procedures with ‘a deproteinization step (the microdiffusion technique and the Berthelot’s reaction) also have the risk of erroneously increasing ammonia [15]. In order to optimize the ammonia determination a closed vessel procedure should be preserved with reduced sample volumes instead of manual dilution. This sample volume reduction may be applied above.OCT activities of 400 mu/l. For values above 4000 mU/l samples should be diluted manually, or, alternatively, the incubation time may be limited to appro~mately 6 h. A shorter incubation time may also be used if a stat analysis is wanted. In Fig. 5, apparently, the enzymes reach peak activity simultaneously. The disappearance rate from plasma for OCT and GLDH is by comparison slower than for mAST. In conclusion, the combination of the shorter incubation procedure with the enzymatic ammonia assay, without a urease or deproteinization step, as proposed in the present study, facilitates the OCT determination and makes it more suitable for use in automatic analyzers. References 1 Schmidt E, Schmidt FW. Aspekte der Enzym-Diagnostik. Med Welt 1970;21:805-816. 2 Frederiks WM, Vogels IMC, Fronik GM. Plasma omithine carbamyl transferase level as an indicator of ischaemic injury of rat liver. Cell Biochem Funct 1984;2:217-220. 3 Van Waes L, Lieber CS. Glutamate dehydrogenase: a reliable marker of liver cell necrosis in the alcoholic. Br Med J 1977;2:1508-1510. 4 Nalpas B, Vassault A, Charpin S, Lacour B, Berthetot P. Serum mitochondrial aspartate aminotransferase as a marker of chronic alcoholism: diagnostic value and interpretation in a liver unit. Hepatology 1986;6:608-614.
402 5 Striver CR, Rosenberg LE. Amino acid metabolism and its disorders. Philadelphia, WB Saunders, 1973:234-249. 6 Grisolia S, Cohen P. Catalytic role of carbamyl glutamate in citrulline synthesis. J Biol Chem 1952;198:561-565. 7 Krebs HA, Eggleston LV, Knivett VA. Arsenolysis and phosphorolysis of citrulline in mammalian liver. Biochem J 1955;59:185-193. 8 Reichard H, Reichard P. Determination of ornithine carbamyl transferase in serum. J Lab Clin Med 1958;52:709-717. 9 Reichard H. Ornithine carbamyl transferase activity in human serum in diseases of the liver and the biliary system. J Lab Clin Med 1961;57:78-87. IO Sato T, Tanaka J, Kono Y, Jones RT, Cowley RA, Trump BF. Hepatic cellular injury following lethal E. coli bacteremia in rats. Lab Invest 1982;47:304-310. I1 Brown RW, Grisolia S. Ornithine transcarbamylase activity in serum. J Lab Clin Med 1959;54:617620. I2 Ceriotti G. Ornithine carbamyl transferase. In: Bergmeyer HU, Methoden der Enzymatischen Analyse. Weinheim/Bergstr: Verlag Chemie, 1974;732-739. I3 Reichard H. Determination of ornithine carbamyl transferase with microdiffusion technique. Stand J Clin Lab Invest 1957;9:311-312. I4 Kontinnen A. Simple method for the determination of ornithine transcarbamylase activity in serum. Clin Chim Acta 1967;18:147-150. I5 Da Fonseca-Wollheim F. Direkte Plasmaammoniakbestimmung ohne Enteiweissung. Z Klin Chem Klin Biochem 1973;10:426-431. I6 Sakakibara S, Shiomi S, Kobayashi S, lkeda T, lrrai S, Kagamiyama H. A convenient and sensitive method for the determination of serum aspartate aminotransferase isozymes after electropheresis. Clin Chim Acta 1983;133:119-123. I7 Kalousek F, Francois B, Rosenberg LE. Isolation and characterization of ornithine transcarbamylase from normal human liver. J Biol Chem 1978;253:3939-3944. I8 Korzenovsky M, Werkman C. Conversion of citrulline to ornithine by cell-free extracts of Str. lactis. Arch Biochem Biophys 1953;46:174-185. I9 Schimke RT. Ornithine Carbamyltransferase (Mycoplasma). In: Colowick SP, Kaplan NO, eds. Methods in Enzymology, Vol. XXVlIA. New York: AC. Press, 1970:295-297.
