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Articles |
in Human Liver and Plasma
Department of Gastroenterology, University Hospital St. Radboud, 6500 HB Nijmegen, The Netherlands.
a Address correspondence to this author at: Department of Gastroenterology, University Hospital St. Radboud, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands. Fax 31-243540103; e-mail W.Peters{at}gastro.azn.nl
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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glutathione
S-transferases in human liver, where they are highly
expressed, or in serum.
Methods: Both total class glutathione
S-transferase (GST-, composed of GSTA1-1, GSTA1-2, and
GSTA2-2) as well as GSTA1-1 concentrations were measured by specific
and sensitive ELISA in liver cytosols of 35 organ donors and in
plasma samples of 350 healthy controls.
Results: The mean total GST- and GSTA1-1 in liver cytosols
were 25.1 ± 9.4 and 10.7 ± 5.3 µg/mg protein,
respectively, and did not correlate with activities of aspartate
aminotransferase or alanine aminotransferase. The mean total
GST- in liver was significantly higher in females compared with
males (28.8 ± 10.0 vs 22.0 ± 7.8 µg/mg protein;
P <0.05). In contrast, the median total GST- in
plasma was lower in females compared with males (2.0 and 2.8 µg/L,
respectively; P <0.0001). The median ratios for
GSTA1-1/total GST- in liver and plasma were 0.42 and 0.58,
respectively.
Conclusions: GSTA1-1 constitutes approximately one-half of the
total amount of class GSTs in human plasma and liver. Total GST-
values are higher in female liver but lower in plasma compared with the
respective values in males. © 1999 American
Association for Clinical Chemistry
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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,
µ, 
, and 
, each subdivided into one or more isoenzymes
(1)(2)(3).
Glutathione S-transferase (GST-) is found at high
concentrations in the human liver and is released quickly and in large
quantities into the bloodstream during hepatocellular damage
(4). Because the half-life of GST- in plasma is
~1 h (5), its concentration will follow changes in
hepatocellular damage more rapidly than aspartate aminotransferase
(AST; EC 2.6.1.1) or alanine aminotransferase (ALT; EC 2.6.1.2), which
have plasma half-lives of ~17 and 47 h, respectively
(4).
GSTs are dimeric enzymes, and the
GST- class comprises two immunologically distinct subunits, GSTA1
and GSTA2, which are encoded by separate genes
(6)(7). Two homodimers, GSTA1-1 and
GSTA2-2, and the heterodimer GSTA1-2 have been purified from human
liver (7).
Several immunochemical assays for GST- were published in the 1980s
(8)(9)(10)(11)(12)(13)(14); recently, however, several sensitive and
specific ELISAs have been developed in different laboratories
(15)(16)(17). The introduction of a commercial ELISA kit
[Hepkit; Biotrin International; Ref. (18)]
has facilitated the clinical application of GST- as a marker for
hepatocellular damage. Recent studies demonstrated that measurement of
serum or plasma GST- may improve the monitoring of hepatocellular
integrity in patients with hepatitis C
(19)(20), in anesthetized patients
(21), in liver transplant recipients
(22)(23)(24), and in women with severe preeclampsia
(25)(26).
In several studies, reference values for plasma or serum GST- in
healthy controls were reported
(8)(9)(10)(11)(12)(13)(14)(15)(18); however, Tiainen and Karhi
(16) were the first to notice that males had
significantly higher plasma GST- concentrations than females.
Recently, in a larger study on the plasma GSTA1-1 concentrations of
blood donors, we obtained similar results and also noticed a
significant increase of plasma GSTA1-1 with increasing age in females
but not in males (17).
Little is known about GST- isoenzyme composition in human liver with
respect to gender and age. Corrigall and Kirsch
(27) reported on the concentration of GST- in
livers from nine male subjects. Strange et al. (28)
and Van Ommen et al. (29) measured GSTA1 and GSTA2 in
20 human liver specimens, but no information on gender and age of the
subjects included in these studies was available.
We developed several monoclonal antibodies against GST-: one
antibody recognizes GSTA1-1 and GSTA2-2 as well as GSTA1-2
(30), whereas another antibody is specific for GSTA1-1
(17). Two sandwich ELISAs based on these monoclonal
antibodies were developed, and these assays were used for measuring
both GSTA1-1 and total GST- in liver cytosols from 35 organ donors
and in blood plasma from 350 healthy blood donors.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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purification of gst- and preparation of the
antibodies
GST- was purified from human liver (31) and
contained ~40% GSTA1-1. Highly purified GSTA1-1, GSTA1-2, and
GSTA2-2 were a generous gift from Dr. P.J. van Bladeren, TNO Nutrition,
Zeist, The Netherlands.
Production and characterization of the specific monoclonal
antibodies against GST- and GSTA1-1 were described previously
(17)(30). A polyclonal antiserum was
prepared as follows: rabbits were immunized by intracutaneous injection
of 100 µg of total GST- in complete Freund's adjuvant and boosted
twice by subcutaneous injection of 50 µg of GST- in incomplete
Freund's adjuvant.
IgG was purified using Protein A Sepharose CL-4B, according to the manufacturer's instructions (Pharmacia).
elisa procedures
Assays for total GST- and GSTA1-1 were performed in 96-well
polystyrene plates (Greiner). All incubations were done at room
temperature in 100 µL/well, unless stated otherwise. Between
incubations, plates were washed five times with >200 µL/well
phosphate-buffered saline (PBS) supplemented with 0.5 mL/L Tween 20
detergent (PBS-T). Plates were coated overnight at 4 °C with 10
mg/L purified monoclonal antibody in PBS and blocked for 1
h with 200 µL/well PBS-T supplemented with 10 g/L bovine serum
albumin (BSA). Calibrators (0.04–20 µg/L GSTA1-1 or 0.2–50.0
µg/L GST-), diluted in PBS-T supplemented with 10 mmol/L EDTA and
100 mL/L normal human plasma that had been heated to
60 °C for 30 min, and plasma samples diluted with an equal volume of
PBS-T-EDTA-normal human plasma were then added to the wells. Plates
were incubated overnight, washed, incubated with rabbit GST-
antiserum diluted 1:4000 in PBS-T-BSA for 3 h, washed, and
subsequently incubated for 2 h with peroxidase-labeled swine
anti-rabbit (Dako) diluted 1:2000 in PBS-T-BSA. After a final wash,
plates were stained with
o-phenylenediamine/H2O2.
The absorbance was read at 492 nm with a background subtraction at 620
nm. All calibrators and samples were measured in duplicate. A
four-parameter weighted logistic regression model was used to calculate
calibration curves and unknowns.
Within- and between-assay coefficients of variation (CVs; SD/mean
x 100%) for the total GST- assay were calculated from five
measurements of 16 plasma samples containing between 1.0 and 43.8
µg/L GST-. Analytical recoveries of this assay were determined by
the addition of 0.8, 1.6, 3.1, 6.2, 12.5, and 25.0 µg/L GST- to
plasma samples from 10 healthy controls.
The GSTA1-1 ELISA showed 7% and 0.1% cross-reactivity with GSTA1-2 and GSTA2-2, respectively, and did not cross-react at all with GSTP1-1 and GSTM1-1 (17). Additional characteristics of the GSTA1-1 assay were described previously (17).
sources of human plasma and liver cytosols
Liver cytosols were obtained from the International Institute for
the Advancement of Medicine (Exton, PA). Liver cytosols from subjects
with a history of diabetes or recent corticosteroid use were excluded
from the study.
Thirty-five liver cytosols from organ donors were studied, 19 males
(median age, 36 years; range, 16–66 years) and 16 females (median age,
41 years; range, 18–60 years). Twenty-nine of these organ donors were
eligible with respect to smoking habits, alcohol consumption, and use
of drugs or medication. These data are given in Table 1
.
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Blood from 350 healthy blood donors was collected at the Red Cross Blood Bank, Nijmegen, The Netherlands, in tubes containing K3 EDTA (Becton and Dickinson) and centrifuged at 2000g for 10 min within 2 h after collection. Plasma samples were stored at -20 °C.
miscellaneous
Protein concentrations in liver cytosols were determined according
to Lowry et al. (32). The GST concentrations in
calibrator preparations were quantified using a Bio-Rad protein assay
(Bio-Rad Laboratories) because GSTs tend to give spuriously high
results in the Lowry protein assay. In both protein assays, BSA
calibration curves were used.
Plasma and liver cytosolic AST and ALT activities were measured at the local laboratory for clinical chemistry on a Hitachi 747 analyzer.
To evaluate the significance of differences between groups, the Mann–Whitney U-test was used. Correlations between different assays were evaluated using the Pearson linear correlation procedure. Statistical calculations were done using SPSS/PC+, Ver. 5.01, computer software.
This study was approved by the local Medical Ethical Review Committee.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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assay, and the cross-reactivity with GST- and
GST-µ class proteins was <0.1%. The detection limit, corresponding
to 3 SD above the mean signal of five zero calibrators in duplicate,
was 0.2 µg/L. Recoveries in plasma samples supplemented with
GST- ranged from 90% to 118% (mean, 104%), and the mean
within- and between-assay CVs were 3.5% and 5.3%, respectively.
total gst- in plasma from blood donors
Plasma total GST- concentrations in 350 healthy controls
displayed a skewed distribution, which could be nearly normalized by
logarithmic transformation. On the logarithmic scale, the mean value
was 2.4 µg/L and the reference range (mean ± 2 SD) was
0.4–13.7 µg/L GST-. Using the previously published data on
GSTA1-1 plasma concentrations (17), we calculated the
median ratio GSTA1-1/total GST- as 0.58 (0.08–1.63). Males
in the age groups 20–40 years and 40–60 years had significantly
(P <0.0002) higher plasma total GST- concentrations than
females in the same age groups (Table 2
). Plasma total GST- concentrations increased significantly
with age in females (P <0.005) but not in males. Plasma
total GST- values correlated significantly with plasma GSTA1-1
concentrations (r = 0.87; P <0.0001; Fig. 1
).
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total gst- and gsta1-1 in liver cytosols
Total GST- and GSTA1-1 concentrations in 35 liver cytosols
displayed a nearly gaussian distribution, and the mean
concentrations of GSTA1-1 and total GST- were 10.7 ± 5.3 and
25.1 ± 9.4 µg/mg cytosolic protein, respectively. The
concentrations of GSTA1-1 correlated significantly with those of total
GST- (r = 0.82; P <0.0001; Fig. 2
). The mean ratio GSTA1-1/total GST- was 0.42 ± 0.10,
indicating that between 22% and 62% (mean ± 2 SD) of total
GST- in liver is GSTA1-1. The GSTA1-1 and total GST-
concentrations in liver cytosols were not correlated significantly with
AST (r = -0.11, P = 0.51; and
r = -0.27; P = 0.12, respectively) or
ALT activities (r = 0.05; P = 0.78; and
r = 0.06; P = 0.72, respectively).
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The mean total GST- concentration in liver cytosols from females was
significantly higher than that in males (28.8 ± 10.0 µg/mg
protein vs 22.0 ± 7.8 µg/mg protein, respectively;
P = 0.024). The mean hepatic GSTA1-1 concentration was
also higher in females (12.2 ± 5.6 µg/mg protein vs 9.5 ±
4.7 µg/mg protein in males), but the statistical significance of
difference was only borderline (P = 0.055). In
contrast, the mean ALT activity was significantly lower in liver
cytosols from females (324 ± 111 U/mg protein vs 500
± 128 U/mg protein in males; P <0.001). Hepatic AST
activities were similar in females and males (1410 ± 618 U/mg
protein vs 1394 ± 444 U/mg protein; P = 0.39).
The hepatic mean CVs (SD/mean) for total GST- and GSTA1-1 were 0.38
and 0.49, respectively. The mean CVs for AST (1401 ± 522 U/mg
protein) and ALT activities (420 ± 149 U/mg protein) were 0.37
and 0.35, respectively.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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published have been
based on polyclonal antibodies. As pointed out by Beckett and Hayes
(4), antisera from animals immunized with total
GST- may vary in their cross-reactivity toward the isoforms GSTA1-1,
GSTA2-2, and GSTA1-2. Therefore, it could be important to use an
antiserum that recognizes only one of the isoforms or, alternatively,
to use an antiserum that recognizes all isoforms with equal affinity.
We developed a specific assay for GSTA1-1 as well as an assay measuring
all GST- isoforms. Each assay is based on a specific monoclonal
antibody as coating (catching) antibody, but both assays use the same
polyclonal detecting antibody that recognizes all GST- isoforms with
similar affinity.
The great variety in GSTA1-1/total GST- ratios demonstrated a large
interindividual variation both in liver cytosols and in plasma samples;
however, within a group of controls, GSTA1-1 and GST- displayed a
very significant linear correlation. This highly linear correlation
indicates that variations in cross-reactivity of polyclonal antisera
toward the different GST- isoforms may be of little relevance when
groups of patients or controls are compared, at least as long as the
assay is based upon the same batch of antibodies. However, when
different batches of polyclonal antibodies are used within the same
assay format, variations in the cross-reactivity to GST- isoforms
may have a significant effect upon the GST- concentrations measured
in plasma or tissue samples.
On the basis of theoretical considerations (very high hepatic
concentration, uniform hepatic distribution, and short plasma
half-life), the measurement of plasma GST- may provide a more
sensitive and specific indicator of acute hepatocellular damage than
the aminotransferases AST and ALT (4). Many studies
evaluating the merits of assaying serum or plasma GST- have been
published (8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18). However, data on total GST-
concentrations in human liver are scarce. Corrigall and Kirsch
(27) reported a mean total GST- of 23.8 ± 6.1
µg/mg cytosolic protein in livers from nine male subjects. These data
are in excellent agreement with the value of 22.0 ± 7.8 µg/mg
protein for males reported here. Strange et al. (28)
reported mean GSTA1 and GSTA2 of 12.8 ± 5.6 and 3.7 ± 2.1
µg/mg cytosolic protein, respectively, for 20 human liver specimens.
Van Ommen et al. (29) also studied 20 human liver specimens,
using an HPLC method and reported mean GSTA1 and GSTA2 concentrations
of 20.3 ± 9.0 and 10.7 ± 8.2 µg/mg cytosolic protein,
respectively. The concentrations of GSTA1-1 (10.7 ± 5.3 µg/mg
cytosolic protein) we measured by ELISA are lower, but the total
GST- values (25.1 ± 9.4 µg/mg protein) are comparable to
these earlier data. The studies by Strange et al. (28)
and van Ommen et al. (29) provided no information on
gender and age of the organ donors, whereas we now demonstrate that
livers from females contain significantly higher concentrations of
total GST- than livers from males. These higher hepatic
concentrations in females cannot be explained by induction of GSTs by
smoking or the use of medicaments because intake in females was lower
than in males.
Several studies on GST- in human blood plasma, including those using
the commercial Hepkit, did not mention an influence of gender on plasma
GST- (8)(9)(10)(11)(12)(13)(14)(15)(18). Tiainen and Karhi
(16) reported that males had significantly higher plasma
GST- concentrations than females, which was confirmed by us for
GSTA1-1 previously (17) and in this study for total
GST-. We hypothesized that the higher plasma concentrations found in
males were associated with higher concentrations of GST- in male
livers (17), based on the assumption that GST- in
plasma originates mainly from normal hepatocellular turnover. However,
the present data show the opposite. One possible explanation for the
apparent paradox of low hepatic and high plasma GST- concentrations
could be that males have a higher hepatocellular turnover than females.
Estimation of hepatic contents of ALT, however, did not support this
hypothesis because we found significantly lower ALT activity in the
liver cytosols of females, whereas the plasma ALT activity is not
different between sexes (33). The combination of high
hepatocellular and low plasma concentrations of GST- in women may
therefore be explained by a more rapid plasma clearance of GST- in
females, but no information on this aspect is currently available.
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Acknowledgments |
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Footnotes |
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, glutathione S-transferase ; AST, aspartate aminotransferase; ALT, alanine aminotransferase; PBS, phosphate-buffered saline; PBS-T, phosphate-buffered saline supplemented with Tween 20; and BSA, bovine serum albumin. 
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, males; 
, females. Linear correlation: r =
0.87; P <0.0001.