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Clinical value
of the determination of serum guanase activity in patients
with chronic hepatitis type C
Hiroko Matsunaga, Hirohito
Honda, Kenichirou Kubo, Katsutaka Sannomiya,
Xuezhi Cui, Yoshio Toyota, Toshifumi Mori, Naoki Muguruma,
Toshiya Okahisa,
Seisuke Okamura, Ichiro Shimizu and Susumu Ito
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Second Department of Internal
Medicine, The University of Tokushima School of Medicine,
Tokushima, Japan
Abstract: The study examines the clinical
significance of guanase (GU) measurement in patients with
hepatitis C. 688 patients in whom either ALT was abnormal,
or in whom HBsAg or HCVAb was detected in the serum, were
enrolled into this study. The percentage of cases in which
normal ALT while elevated GU was compared among the different
disease groups. Then, the percentage of cases with normal
ALT but elevated GU was compared between HBV and HCV groups.
For the entire population, a significant correlation was observed
between ALT and GU (r=0.872). The overall percentage of cases
with normal ALT but elevated GU activity was 11.4%. In HCV
group, 449 cases had normal ALT. Of these cases, 20.3% had
elevated GU, while ALT was normal. Before 1989, no test to
check donated blood for HCV antibody was available. However,
screening of donated blood for high GU was associated with
a reduced incidence of post-transfusion hepatitis. This is
probably because following the screening, blood donated by
patients with hepatitis C who had normal ALT but elevated
GU was rejected. After the introduction of HCV antibody measurement,
GU measurement is still useful to reveal the pathophysiological
condition in-patients with chronic hepatitis type C. J. Med.
Invest. 50:64-71, 2003
Keywords:chronic hepatitis, HCV, guanase,
ALT
INTRODUCTION
No reliable screening method for donated
blood had been established before 1972. The incidence of post-transfusion
hepatitis was very high (20-40%) before that year (1). HBs
antigen measurement was introduced in 1972 as a means of screening
blood donated for transfusion. Since 1981, alanine aminotransferase
(ALT) activity measurement has been additionally performed
to check donated blood, resulting in a reduction in the incidence
of post-transfusion hepatitis (2). However, non-A, non-B post-transfusion
hepatitis was still seen in about 15% of blood recipients.
Under these circumstances, serum guanase (guanine deaminase;GU)
measurement began to attract attention as a more effective
means of screening donated blood (3-5).
GU is abundantly expressed in the liver, brain and kidney.
It is quite scarce in the skeletal muscles, myocardium, pancreas
and other organs in which aspartate aminotransferase (AST)
and ALT are relatively abundant (6-7). Therefore, it has been
reported that a rise in blood GU activity may be a sign specific
to liver disease, and that measurement of his enzyme may serve
as an excellent means of screening for liver disease (8-10).
It has also been shown that the GU activity in transfused
blood is significantly correlated with the incidence of post-transfusion
hepatitis (3-5), and that discarding donated blood showing
high GU activity can reduce the incidence of post-transfusion
hepatitis (11). Based on these findings, the introduction
of GU measurement as a screening test for blood donated for
transfusion was discussed. However, in 1989, a method for
measuring hepatitis C virus (HCV) antibody was published (12),
which hampered the introduction of GU measurement as a screening
test for donated blood.
In November 1989, checking donated blood for HCV antibody
(C100-3 antibody) and HBc antibody was started. Since then,
a sharp decrease in the incidence of post-transfusion hepatitis
has been reported. Since February 1992, when second-generation
HCV antibody measurement was introduced, the incidence of
post-transfusion hepatitis has decreased to almost zero (13-15).
Data collected from HCV antibody measurements revealed that
most cases of post-transfusion non-A, non-B hepatitis were
cases of hepatitis C. Therefore, it was thought that there
could be some relationship between the serum GU activity and
hepatitis C. To date, however, this relationship has not been
studied. After the introduction of HCV antibody measurement,
the significance of GU measurement has to be revealed.
The present study was undertaken to follow the time-course
of changes in the serum GU activity in patients with hepatitis,
and to examine the clinical significance of serum GU activity
measurement in patients with hepatitis C.
SUBJECTS AND METHODS
Subjects
Of the patients who visited our department between January
1991 and December 1996, 688 patients in whom either serum
ALT activity was abnormal, or in whom HBsAg or HCVAb was detected
in the serum, were enrolled into this study. Fig. 1 shows
the clinical details of these subjects. Elevated serum ALT
activity was seen in 599 cases, HBsAg was positive in 91 cases,
and HCVAb was positive in 436 cases. Thus, many patients had
two or more of the three abnormalities. The subjects underwent
biochemical tests and diagnostic imaging. Biopsy was additionally
performed in some of the subjects. Based on the results, the
subjects were diagnosed as being asymptomatic carriers (ASC),
or as having chronic hepatitis (CH), liver cirrhosis (LC)
or hepatocellular carcinoma (HCC);of all the subjects, 36
were diagnosed as being ASC, 372 as having CH, 130 as having
LC, 53 as having HCC, and 116 as having “other disease".
In those patients in whom liver biopsy was not performed,
the diagnosis of ASC, CH, LC or HCC was made based on the
following criteria. The diagnosis of ASC was made in cases
where the serum ALT activity had never been abnormal. The
diagnosis of CH was made in cases where the serum ALT activity
had remained abnormal for 6 months or longer, but the criteria
for diagnosis of LC and HCC were not satisfied. The diagnosis
of LC was made in cases where three or more of the following
criteria were satisfied, and diagnostic imaging also indicated
signs of LC:1) serum AST/ALT ratio>1.0, 2) serum cholinesterase
activity (ChE) below 0.50ΔpH, 3) serum albumin level
(Alb) below 3.5 g/dl, 4) blood platelet count (Plt) below
100,000/ml, 5) serum hepaplastin activity (HPT) below 70%,
and 6) ICG R15 over 15%.The diagnosis of HCC was made in cases
where diagnostic imaging (abdominal ultrasonography, dynamic
CT, or angiography) revealed signs suggestive of HCC. The
diagnosis of “other disease" was made in cases
where none of the aforementioned liver conditions were diagnosed.
The subjects were divided into the HBsAg-positive group (HBV
group, excluding patients who were both HBsAg-and HCVAb-positive),
the HCVAb-positive group (HCV group, excluding patients who
were both HBsAg- and HCVAb-positive), the HBsAg + HCVAb-positive
group (comprising patients who were both HBsAg- and HCVAb-positive),
and the HBsAg, HCVAb-negative group (the NBNC group), as shown
in Table 1. Of all the patients, 90 were assigned to the HBV
group, 449 patients to the HCV group, 3 patients to the HBsAg
+ HCVAb-positive group, and 165 patients to the NBNC group.
During long-term follow-up, CH progressed to LC in 7 cases,
CH advanced to HCC in 1 case, and LC progressed to HCC in
11 cases. These 19 cases were counted in duplicate. For each
of the followed up cases, hematological testing was conducted
once every 1-6 months. When reporting the results, the number
of cases was expressed as N, and the number of samples as
n.
Measurement
HBs antigen was measured by the reversed passive hemagglutination
(R-PHA) method;HCV antibody was measured by enzyme immunoassay
(EIA);ChE was measured by the enzyme method;Alb was measured
by bromcresol green (BCG);HPT was measured automatically with
an MDA;AST and ALT were measured by ultraviolet (UV);GU was
measured by the enzyme method, using a Hitachi 736 automated
analyzer.
Methods
1) Correlation between serum ALT and serum GU activity
For all samples (n=3744) obtained from 688 patients (707
cases, including duplicate counts), the correlation between
the serum ALT and serum GU activity was evaluated. For each
disease group, the correlation between the two was analyzed
by further subdividing the group into the HBV group and
HCV group.
2) Analysis of cases not showing a relationship between
the serum ALT and serum GU activity
The percentage of cases in which the serum ALT activity
was high while the serum GU activity was normal was compared
between the HBV group and the HCV group, and also among
the different disease groups. Then, the percentage of cases
with normal serum ALT activity but elevated serum GU activity
was compared between the HBV and the HCV groups. Patients
with chronic hepatitis C in whom a normal ALT activity was
recorded at least once during the follow-up period were
deemed as cases showing normal ALT activity. The incidence
of elevated GU activity during periods of normal ALT activity
was investigated in these patients.
3) Testing the significance of differences
The F-test was used to test the coefficient of correlation.
P<0.05 was regarded as denoting statistical significance.
Fisher's exact test was employed to test the percentage
of cases not showing any correlation between the serum ALT
and serum GU activity, and Student's t-test was used for
other tests. P<0.01 was regarded as denoting significance
in these tests.
RESULTS
1) Correlation between the serum ALT and serum GU activity
Table 2 shows the coefficient of correlation between the serum
ALT and serum GU activity and the p value for all samples
(n=3744) obtained from 688 patients (707 cases if some patients
were counted in duplicate). For the entire population, a significant
correlation was observed between the ALT and GU activity (r=0.872,
p<0.0001). When analyzed by disease, a significant (p<0.0001)
correlation between the serum ALT and GU activity was observed
in the CH group (r=0.756), the LC group (r=0.662), the HCC
group (r=0.944) and the “other disease" group
(r=0.918). Favorable correlation were observed among the HCC
group and the “other disease" group. When analyzed
by the type of virus, the correlation between the ALT and
GU activity was significantly stronger (p<0.05) in the
HCV group (r=0.891) than in the HBV group (r=0.756). Among
patients with CH, the correlation was less strong in the HCV
group (r=0.747) than in the HBV group (r=0.854), while among
patients with HCC, the correlation tended to be stronger in
the HCV group (r=0.946) than in the HBV group (r=0.816). However,
none of these differences between the HBV and the HCV groups
was statistically significant.
2) Analysis of cases not showing any correlation between the
serum ALT and serum GU activity
Fig. 2 shows the percentage of cases not showing any correlation
between the serum ALT and serum GU activity in each virus-type
group. The overall percentage of cases with normal serum ALT
activity but elevated serum GU activity was 11.4%. This percentage
was higher in the HCV group (12.2%) than in the HBV group
(4.4%), although this difference was not significant. The
overall percentage of cases with elevated serum ALT activity
but normal serum GU activity was 30.4%. This percentage was
higher in the HBV group (34.4%) than in the HCV group (29.2%),
although the difference was not statistically significant.
In both the HBV and the HCV groups, the percentage of cases
with normal serum ALT activity but elevated serum GU activity
was significantly lower than the percentage of cases with
elevated serum ALT activity and normal serum GU activity.
We then conducted a disease-wise analysis of the percentage
of cases with elevated serum ALT activity but normal serum
GU activity (Table 3). There was no significant difference
in this percentage between the HBV and the HCV groups. In
the HCV group, this percentage was significantly higher in
patients with CH (38.4%) than in those with LC or HCC. In
the HBV group also, this percentage was lower in patients
with LC or HCC than in patients with CH, similar to the relationship
observed in the HCV group;the difference between the two groups
was not statistically significant.
The percentage of cases with normal serum ALT activity but
elevated serum GU activity was also analyzed disease-wise
(Table 4). Among patients with CH, this percentage was significantly
higher in the HCV group (9.7%) than in the HBV group (0.0%).
In both the HBV and the HCV groups, this percentage tended
to increase as the underlying disease advanced from CH to
LC or from LC to HCC, but the change was not statistically
significant.
3) Analysis of cases with normal serum ALT activity
In the HCV group, we analyzed the percentage of cases with
normal serum ALT activity and the percentage of cases with
normal ALT but elevated serum GU activity (Table 4). Of the
449 HCV-positive cases, 60.4% had normal ALT activity. Of
these cases, 20.3% had elevated serum GU activity, while the
serum ALT activity was normal. This percentage tended to increase
as the disease advanced, however, the change was not statistically
significant.
DISCUSSION
Guanase is an enzyme that was first detected
in rabbit liver homogenates by Schmidt in 1932. In 1963, Passananti
found that the serum guanase activity rose in patients with
liver disease. Since then, guanase has been utilized in liver
function testing, and its relationship to post-transfusion
hepatitis has recently attracted close attention. In 1989,
HCV antibody was detected (12), and some relationship between
hepatitis C and serum GU activity was suggested. Thereafter,
however, no report on this relationship has been published.
Under these circumstances, the present study was undertaken
to examine the relationship between the serum GU and serum
ALT activity in patients with hepatitis, and to examine the
clinical significance of measuring the serum GU activity when
dealing with hepatitis C. On the whole, a good correlation
was found between the serum ALT and serum GU activity, but
no correlation was seen in some cases. When analyzed by disease,
the correlation was more marked in patients with HCC or “other
disease" than in patients with CH or LC. When analyzed
by virus type, the correlation tended to be higher in the
HCV group than in the HBV group. Among patients with LC, the
degree of correlation differed little between the HBV and
the HCV groups. Among patients with CH, the correlation was
more marked in the HBV group, while among patients with HCC,
the correlation was more marked in the HCV group. In the cases
of “other disease" and NBNC group, they include
several different etiologies. So, the meanings of the differences
concerned them are unknown. Thus, no definitive tendency was
observed in this analysis. This suggests that the serum ALT
and serum GU activities are not directly related to the underlying
pathology or the pathogenic virus type, but rather to differences
in the quantum of release of these enzymes from the liver
depending on the underlying pathophysiological condition.
Takino et al. (16) and Noma et al. (17) have published some
reports previously regarding the correlation between the serum
ALT and serum GU activity in patients with liver disease.
The coefficient of correlation reported by them is considerably
lower than that obtained in our study. In the study conducted
by Takino et al., a correlation between the serum ALT and
GU activities was observed in patients with acute hepatitis
and CH, but they noted no correlation between the two in the
LC group (r=0.290). In the study conducted by Noma et al.,
a correlation was seen in patients with acute hepatitis, but
not in patients with CH or LC. The low coefficient of correlation
noted in the studies of Takino et al. and Noma et al. could
probably be attributed to the conventional method with low
sensitivity that they employed in these studies to determine
the enzyme activities in the serum. In our study, the coefficient
of correlation was higher in the HCC group and the “other
disease" group than in the CH and the LC groups. These
findings suggest that whether or not the serum ALT activity
is correlated with the serum GU activity depends on the underlying
pathophysiological condition in each patient.
In our analysis of the cases in whom no correlation was detected
between the serum ALT and serum GU activity, the percentage
of cases with normal serum ALT activity but abnormally elevated
serum GU activity was significantly lower than the percentage
of cases in whom the reverse was true. This finding could
be associated with the differences in tissue distribution
between ALT and GU, differences in the half-lives in the blood
of the two enzymes, or differences in their release from the
hepatocytes depending on the underlying pathophysiological
condition. The percentage of cases with normal serum ALT activity
but elevated serum GU activity was significantly higher in
HCV-positive patients with CH. In both the HBV and the HCV
groups, this percentage tended to increase as CH advanced
to LC or LC advanced to HCC. One factor that could be responsible
for this result is that as liver disease advances, the serum
ALT activity returns towards normal more frequently than serum
GU activity. In the HCV group, the percentage of cases with
abnormally high serum ALT activity but normal serum GU activity
was significantly higher in patients with CH than in patients
with LC or HCC. This could be because the release of ALT and
GU into the blood varies depending on the underlying liver
disease.
Of the HCV-positive patients with normal serum ALT activity,
20.3% had abnormally high GU activity. Patients with hepatitis
C are often asymptomatic and their serum levels are rarely
elevated. It is highly probable that asymptomatic HCV-positive
patients who were ASC, or had CH or LC, donated blood before
the HCV antibody was discovered in this study. The serum ALT
activity was probably normal in many of these patients, and
it would seem that 20% of these patients could have been detected
by serum GU activity measurement. Therefore, the results obtained
by us endorses the view that serum GU measurement could serve
as a useful means for screening blood donated for transfusion.
Overall, a significant correlation was observed between the
serum GU and serum ALT activity in patients with liver disease.
However, because the release of these enzymes into the blood
varies depending on the pathogenic virus type and the underlying
liver condition, there were cases in which the serum ALT activity
was normal while the serum GU activity was elevated. The percentage
of these cases was high among patients with chronic hepatitis
C. Patients with chronic hepatitis C are often asymptomatic,
and their condition is difficult to diagnose unless a test
for HCV antibody is performed. Before 1989, no method to check
for HCV antibody in the serum was available. It is highly
probable that blood donated by HCV-positive individuals with
normal serum ALT activity were used for transfusion before
1989. Serum GU activity measurement thus can probably serve
as a very useful means for the screening of blood donated
by HCV-positive individuals with normal ALT activity. The
fact that the incidence of post-transfusion hepatitis decreased
following the introduction of screening of donated blood for
high GU activity implies that this screening test made it
possible to reject blood donated by patients of hepatitis
C with normal serum ALT activity but elevated serum GU activity.
Conclusion
Serum ALT and GU activities were analyzed in relation to the
pathogenic virus type and the underlying liver disease. A
significant correlation was observed in all the groups examined
between the serum ALT and serum GU activity. However, the
degree of this correlation varied, probably because the release
of these enzymes into the blood varies depending on the type
of the pathogenic virus and the underlying liver disease.
The percentage of cases with normal serum ALT activity but
elevated serum GU activity was high in the HCV group. Before
1989, no test to check donated blood for HCV antibody was
available. However, screening of donated blood for high GU
activity was associated with a reduced incidence of post-transfusion
hepatitis. This is probably because following the screening,
blood donated by patients with hepatitis C who had normal
serum ALT activity but elevated serum GU activity was rejected.
After the introduction of HCV antibody measurement, GU measurement
is still useful to reveal the pathophysiological condition
in patients with chronic hepatitis type C.
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Received for publication November 22, 2002;accepted January
7, 2003.
Address correspondence and reprint requests to Prof. Susumu
Ito, Second Department of Internal Medicine, The University
of Tokushima School of Medicine, Kuramoto-cho, Tokushima770-8503,
Japan and Fax:+81-88-633-9235. |
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