Clin 8iochem, Vol. 23, pp. 139-141, 1990 Printed in Canada. All rights reserved.
0009-9120/90 $3.00 + .00 Copyright © 1990 The Canadian Society of Clinical ChemiSts.
Clearance of Argininosuccinate Synthetase from the Circulation in Acute Liver Disease TAKEYORI SAHEKI, 1 KAZUHIRO KOMORIZONO, 2 TSUTOMU MIURA, 2 HITOSHI ICHIKI, 1 YUKIO YAGI, 1 and SHUJI HASHIMOTO2 1Department of Biochemistry, and 2Second Department of Internal Medicine, Faculty of Medicine, Kagoshima University, Japan Argininosuccinate synthetase is an enzyme which has been found to be a specific marker for liver damage. In patients with acute hepatitis, the concentration in serum increases at the onset of the disease, but later decreases more quickly, so that the time required for normalization is shorter than that of alanine aminotransferase. This is probably caused by rapid clearance of argininosuccinate synthetase from the serum. Rapid clearance was demonstrated in experimental animals given purified enzymes intravenously. Argininosuccinate synthetase disappeared from the serum with a half life of about 15 rain, while the half lives of alanine aminotransferase and aspartate aminotransferase were 4 and 5 h, respectively, under the same conditions.
KEY WORDS: argininosuccinate synthetase; liver diseases; clearance rate; alanine aminotransferase; aspartate aminotransferase; serum enzymes.
Introduction A
rgininosuccinate synthetase (ASS; [EC 6.3.4.5]) is one of the urea cycle enzymes located mainly in the cytosol of hepatocytes, therefore, it may be a potentially useful serum marker specific for liver damage. Previously we have demonstrated t h a t the serum concentration of ASS increased mainly in acute and chronic hepatitis, but only in much fewer cases of non-hepatic diseases (1). We have also found t h a t the ASS levels in acute hepatitis were not much higher t h a n those in chronic hepatitis, whereas, the alanine aminotransferase (ALT; [EC 2.6.1.2]) levels in acute hepatitis are much higher. To investigate the difference between serum levels of these two enzymes, we observed the time course of the serum levels in patients with acute hepatitis, and measured the clearance rate (the rate of disappearance from the serum) of ASS, ALT and aspartate aminotransferase (AST; EC 2.6.1.1) injected intravenously into rats.
Correspondence: Dr. Takeyori Saheki, Department of Biochemistry, Faculty of Medicine, Kagoshima University, Usuki-cho 1208-1, Kagoshima 890, Japan. Manuscript received March 31, 1989; revised July 14, 1989; accepted July 25, 1989.
CLINICAL BIOCHEMISTRY, VOLUME 23, APRIL 1990
Materials a n d m e t h o d s PATIENTS Serum enzymes of patients with acute hepatitis were estimated at least once a day for the first 3 days after admission to the hospital and thereafter, once every 2 days until normalization. Initial values are listed in Table 1. PURIFICATION OF ENZYMES FROM RAT LIVER
ASS was purified to homogeneity by the method of Saheki et al. (2). Purified ASS had a specific activity of 1.6 U per mg of protein at 25 °C and pH 7.5. AST was purified according to Okada and Hirose (3) to apparent homogeneity. The specific activity was 144 U per mg of protein. ALT was partially purified by the method of Matsuzawa and Segal (4) to the anion-exchange column chromatography step (we used Q-Sepharose FF as an anion-exchange resin). The specific activity was 68 U per mg of protein. Protein concentration was determined by the method of Lowry et al. (5). CLEARANCE OF ENZYMES FROM THE SERUM
After being passed through a Sephadex column (PD-10) equilibrated with 0.15 mol/L NaC1, the TABLE 1
List of Patients with Acute Hepatitis Described in This Paper Patient No. Name Sex
Initial Serum Levels Age (y) Diagnosis
ALT (U/L)
ASS (~g/L)
1
EJ
f
44
NANB
2259
204
2 3 4 5
KA IH MM FN
m m f f
26 31 36 47
NANB A B A
1768 2697 6396 2074
146 505 280 210
f, m and y denote female, male and years, respectively. Diagnosis is shown as A, B, and non-A non-B (NANB) types of acute hepatitis. The upper limits of the normal values are 40 ~g/L for ASS and 43 U/L for ALT.
139
SAHEKI, KOMORIZONO, MIURA, ET AL.
enzyme preparations (about 0.5 mL) were injected into the jugular vein of anesthetized rats (Wistar strain, 300-350 g body weight). The amounts of protein injected were 38-128 ~Lg, 47--141 ~Lg, and 200-350 ~Lg for ASS, AST, and ALT, respectively. Blood was collected from the jugular vein or tail vein under ether anesthesia at appropriate time intervals. Serum was isolated after standing for 30 min and stored at - 8 0 °C until enzyme concentration and activities were estimated.
500o 500G
2000
ASS concentration in the serum was determined by the ELISA method described by Ichiki et al. (6). ALT and AST activities in the serum were determined with commercial kits supplied by Wako Pure Chemicals (Tokyo, Japan) at 37 °C.
I0OO
IOOC
500
=-D E T E R M I N A T I O N OF ASS CONCENTRATION A N D AST A N D ALT ACTIVITIES
2000
J < E 2
\
500 CO 09
20C
200
< E
I00
co
co
IOO 5o
50
20
20
Results and discussion R A T E OF DECREASE OF S E R U M ASS A N D ALT DURING THE CLINICAL COURSE OF ACUTE HEPATITIS 0
The serum ASS concentration was high in acute hepatitis, but the mean value was not markedly greater t h a n t h a t in chronic hepatitis: mean value ± SD was 318 +_ 273 tLg/L and 137 _+ 155 }xg/L for acute and chronic hepatitis, respectively (1). This was in strong contrast to serum ALT (where the values were 1610 _ 1050 U/L and 161 _ 166 U/L for acute and chronic hepatitis, respectively). To explain the difference between the elevation of ASS and ALT in acute hepatitis, we followed the time course of these enzymes in patients with acute hepatitis. As shown in Figure 1, the serum ASS concentration decreased much more quickly t h a n the serum ALT. The rate of decrease of the serum ALT, expressed as half-life (tlj2) and calculated from a semilogarithmic graph, was 2.0 ± 0.7 days; the half-life for ASS was too short to be determined (Table 2). Table 2 also shows the number of days from the highest serum enzyme levels to when they fell within the range of the normal values. Serum ASS concentration returned to the normal level about nine times faster t h a n ALT activity. The rapid decrease of serum ASS may account for the relatively small increase of serum ASS concentration in acute hepatitis. The rapid decrease of ASS may be caused by its faster clearance from the serum or by selective cessation of its release from the liver. In this study, we tested the former possibility by injecting the enzymes ASS, ALT and AST intravenously into rats and estimating their clearance rates from the serum. C L E A R A N C E OF INTRAVENOUSLY INJECTED ASS, ALT A N D AST
Purified or partially purified enzymes were injected intravenously into rats and the serum levels 140
i
~ii
,
i
I
t
I
I
I
I
I
2
5
4
5
6
7
8
9
I0
~0
Doys of Clinicol Course F i g u r e 1 - - C h a n g e s o f s e r u m ASS c o n c e n t r a t i o n a n d A L T
activity during the clinical course of acute hepatitis. Serum levels of ASS concentration and ALT activity of the five patients with acute hepatitis listed in Table 1 were plotted on a semilogarithmic graph. Open and closed symbols show ALT activity and ASS concentration, respectively. Each symbol represents one patient.
were monitored. In an initial experiment, we measured the decrease of injected AST from the serum. Our data show t h a t the calculated half-life of AST was similar to t h a t of Kakimoto et al. (7). The decreases of ASS and ALT in rat serum are shown in Figure 2; the calculated half-lives of the enzymes are listed in Table 3. These results demonstrate the extremely rapid clearance of serum ASS with a half-life of only about 15 min, in contrast to several hours for AST and ALT. These values were similar, regardless of whether the enzymes were injected separately or simultaneously (data not shown). We also tested purified h u m a n liver ASS as an enzyme
TABLE 2 Rate of Decrease (tlm) of S e r u m A S S Concentration and
A L T Activity,and Number of Days Required for Normalization of the Serum Enzymes of the Five Patients with Acute Hepatitis Listed in Table 1
Enzyme ALT ASS
tl/2 (Days)
D a y s R e q u i r e d for Normalization
2.0 - 0.7 < 1
24.4 -+ 13.8 2.6 --- 1.5
D a t a a r e m e a n - SD. CLINICAL BIOCHEMISTRY, VOLUME 23, APRIL 1990
SERUM
ARGININOSUCCINATE
SYNTHETASE
50O0
5000
"3 2000
2000 J
0009-9120/90 $3.00 + .00 Copyright © 1990 The Canadian Society of Clinical ChemiSts.
Clearance of Argininosuccinate Synthetase from the Circulation in Acute Liver Disease TAKEYORI SAHEKI, 1 KAZUHIRO KOMORIZONO, 2 TSUTOMU MIURA, 2 HITOSHI ICHIKI, 1 YUKIO YAGI, 1 and SHUJI HASHIMOTO2 1Department of Biochemistry, and 2Second Department of Internal Medicine, Faculty of Medicine, Kagoshima University, Japan Argininosuccinate synthetase is an enzyme which has been found to be a specific marker for liver damage. In patients with acute hepatitis, the concentration in serum increases at the onset of the disease, but later decreases more quickly, so that the time required for normalization is shorter than that of alanine aminotransferase. This is probably caused by rapid clearance of argininosuccinate synthetase from the serum. Rapid clearance was demonstrated in experimental animals given purified enzymes intravenously. Argininosuccinate synthetase disappeared from the serum with a half life of about 15 rain, while the half lives of alanine aminotransferase and aspartate aminotransferase were 4 and 5 h, respectively, under the same conditions.
KEY WORDS: argininosuccinate synthetase; liver diseases; clearance rate; alanine aminotransferase; aspartate aminotransferase; serum enzymes.
Introduction A
rgininosuccinate synthetase (ASS; [EC 6.3.4.5]) is one of the urea cycle enzymes located mainly in the cytosol of hepatocytes, therefore, it may be a potentially useful serum marker specific for liver damage. Previously we have demonstrated t h a t the serum concentration of ASS increased mainly in acute and chronic hepatitis, but only in much fewer cases of non-hepatic diseases (1). We have also found t h a t the ASS levels in acute hepatitis were not much higher t h a n those in chronic hepatitis, whereas, the alanine aminotransferase (ALT; [EC 2.6.1.2]) levels in acute hepatitis are much higher. To investigate the difference between serum levels of these two enzymes, we observed the time course of the serum levels in patients with acute hepatitis, and measured the clearance rate (the rate of disappearance from the serum) of ASS, ALT and aspartate aminotransferase (AST; EC 2.6.1.1) injected intravenously into rats.
Correspondence: Dr. Takeyori Saheki, Department of Biochemistry, Faculty of Medicine, Kagoshima University, Usuki-cho 1208-1, Kagoshima 890, Japan. Manuscript received March 31, 1989; revised July 14, 1989; accepted July 25, 1989.
CLINICAL BIOCHEMISTRY, VOLUME 23, APRIL 1990
Materials a n d m e t h o d s PATIENTS Serum enzymes of patients with acute hepatitis were estimated at least once a day for the first 3 days after admission to the hospital and thereafter, once every 2 days until normalization. Initial values are listed in Table 1. PURIFICATION OF ENZYMES FROM RAT LIVER
ASS was purified to homogeneity by the method of Saheki et al. (2). Purified ASS had a specific activity of 1.6 U per mg of protein at 25 °C and pH 7.5. AST was purified according to Okada and Hirose (3) to apparent homogeneity. The specific activity was 144 U per mg of protein. ALT was partially purified by the method of Matsuzawa and Segal (4) to the anion-exchange column chromatography step (we used Q-Sepharose FF as an anion-exchange resin). The specific activity was 68 U per mg of protein. Protein concentration was determined by the method of Lowry et al. (5). CLEARANCE OF ENZYMES FROM THE SERUM
After being passed through a Sephadex column (PD-10) equilibrated with 0.15 mol/L NaC1, the TABLE 1
List of Patients with Acute Hepatitis Described in This Paper Patient No. Name Sex
Initial Serum Levels Age (y) Diagnosis
ALT (U/L)
ASS (~g/L)
1
EJ
f
44
NANB
2259
204
2 3 4 5
KA IH MM FN
m m f f
26 31 36 47
NANB A B A
1768 2697 6396 2074
146 505 280 210
f, m and y denote female, male and years, respectively. Diagnosis is shown as A, B, and non-A non-B (NANB) types of acute hepatitis. The upper limits of the normal values are 40 ~g/L for ASS and 43 U/L for ALT.
139
SAHEKI, KOMORIZONO, MIURA, ET AL.
enzyme preparations (about 0.5 mL) were injected into the jugular vein of anesthetized rats (Wistar strain, 300-350 g body weight). The amounts of protein injected were 38-128 ~Lg, 47--141 ~Lg, and 200-350 ~Lg for ASS, AST, and ALT, respectively. Blood was collected from the jugular vein or tail vein under ether anesthesia at appropriate time intervals. Serum was isolated after standing for 30 min and stored at - 8 0 °C until enzyme concentration and activities were estimated.
500o 500G
2000
ASS concentration in the serum was determined by the ELISA method described by Ichiki et al. (6). ALT and AST activities in the serum were determined with commercial kits supplied by Wako Pure Chemicals (Tokyo, Japan) at 37 °C.
I0OO
IOOC
500
=-D E T E R M I N A T I O N OF ASS CONCENTRATION A N D AST A N D ALT ACTIVITIES
2000
J < E 2
\
500 CO 09
20C
200
< E
I00
co
co
IOO 5o
50
20
20
Results and discussion R A T E OF DECREASE OF S E R U M ASS A N D ALT DURING THE CLINICAL COURSE OF ACUTE HEPATITIS 0
The serum ASS concentration was high in acute hepatitis, but the mean value was not markedly greater t h a n t h a t in chronic hepatitis: mean value ± SD was 318 +_ 273 tLg/L and 137 _+ 155 }xg/L for acute and chronic hepatitis, respectively (1). This was in strong contrast to serum ALT (where the values were 1610 _ 1050 U/L and 161 _ 166 U/L for acute and chronic hepatitis, respectively). To explain the difference between the elevation of ASS and ALT in acute hepatitis, we followed the time course of these enzymes in patients with acute hepatitis. As shown in Figure 1, the serum ASS concentration decreased much more quickly t h a n the serum ALT. The rate of decrease of the serum ALT, expressed as half-life (tlj2) and calculated from a semilogarithmic graph, was 2.0 ± 0.7 days; the half-life for ASS was too short to be determined (Table 2). Table 2 also shows the number of days from the highest serum enzyme levels to when they fell within the range of the normal values. Serum ASS concentration returned to the normal level about nine times faster t h a n ALT activity. The rapid decrease of serum ASS may account for the relatively small increase of serum ASS concentration in acute hepatitis. The rapid decrease of ASS may be caused by its faster clearance from the serum or by selective cessation of its release from the liver. In this study, we tested the former possibility by injecting the enzymes ASS, ALT and AST intravenously into rats and estimating their clearance rates from the serum. C L E A R A N C E OF INTRAVENOUSLY INJECTED ASS, ALT A N D AST
Purified or partially purified enzymes were injected intravenously into rats and the serum levels 140
i
~ii
,
i
I
t
I
I
I
I
I
2
5
4
5
6
7
8
9
I0
~0
Doys of Clinicol Course F i g u r e 1 - - C h a n g e s o f s e r u m ASS c o n c e n t r a t i o n a n d A L T
activity during the clinical course of acute hepatitis. Serum levels of ASS concentration and ALT activity of the five patients with acute hepatitis listed in Table 1 were plotted on a semilogarithmic graph. Open and closed symbols show ALT activity and ASS concentration, respectively. Each symbol represents one patient.
were monitored. In an initial experiment, we measured the decrease of injected AST from the serum. Our data show t h a t the calculated half-life of AST was similar to t h a t of Kakimoto et al. (7). The decreases of ASS and ALT in rat serum are shown in Figure 2; the calculated half-lives of the enzymes are listed in Table 3. These results demonstrate the extremely rapid clearance of serum ASS with a half-life of only about 15 min, in contrast to several hours for AST and ALT. These values were similar, regardless of whether the enzymes were injected separately or simultaneously (data not shown). We also tested purified h u m a n liver ASS as an enzyme
TABLE 2 Rate of Decrease (tlm) of S e r u m A S S Concentration and
A L T Activity,and Number of Days Required for Normalization of the Serum Enzymes of the Five Patients with Acute Hepatitis Listed in Table 1
Enzyme ALT ASS
tl/2 (Days)
D a y s R e q u i r e d for Normalization
2.0 - 0.7 < 1
24.4 -+ 13.8 2.6 --- 1.5
D a t a a r e m e a n - SD. CLINICAL BIOCHEMISTRY, VOLUME 23, APRIL 1990
SERUM
ARGININOSUCCINATE
SYNTHETASE
50O0
5000
"3 2000
2000 J
