Clinical Chemistry 43: 686-687, 1997;
(Clinical Chemistry. 1997;43:686-687.)
© 1997 American Association for Clinical Chemistry, Inc.
Characterization of the Aspartate Aminotransferase–Immunoglobulin M Complex in a Japanese Woman,
Koichi Asanuma,
Atsuhito Yagihashi and
Naoki Watanabea
Dept. of Lab. Diagnosis, Sapporo Medical Univ. School of Med., South 1, West 16, chuo-ku, Sapporo, 060, Japan
a author for
correspondence: fax 81-11-622-7502
Alterations in serum aspartate aminotransferase (AST)
concentration usually correlate with the clinical stages of
organ-specific disease. However, persistently increased AST
concentration is sometimes due to the formation of a macrocomplex
between immunoglobulin (Ig) and AST. In most healthy cases, AST-Ig
complexes were detected after detailed studies to determine the cause
of a persistent high concentration of serum AST. If high AST
concentrations are found in the absence of organ-specific disease, we
should suspect the presence of an AST-Ig complex. In eight previously
reported healthy individuals with a high AST concentration
(1)(2)(3)(4)(5)(6), the Ig that was bound to AST was always IgG. Here
we characterize the immunological and biochemical properties of the
AST-IgM complex in an individual without serious disease.
The patient, a 61-year-old woman, was referred to our hospital for
evaluation of persistently high serum AST concentration of 9 years
duration. Her AST concentration was measured six times between February
and July 1992 in our hospital. Each time, AST was high but all other
liver function tests were normal. Laboratory tests at the time of
admission showed (reference range): AST, 294 (5–40) U/L; IgM, 2.83
(0.7–1.70) g/L; C3, 0.53 (0.63–0.99) g/L; antimacrosomal antibody,
x400 (<x100). Results of hepatitis B surface and core antibody
assays were positive. Computed tomography and ultrasonography of the
abdomen revealed no evidence of liver disease such as hepatocellular
carcinoma, liver cirrhosis, or fatty liver. A liver biopsy sample was
normal.
Total AST activities were measured by an Olympus AU-560 automated
analyzer (Olympus, Tokyo, Japan) with a commercially available kit
(Wako Pure Chemical Co., Osaka, Japan). AST isoenzymes were separated
by electrophoresis on a Titan-III lipocellulose acetate membrane
(Helena Labs., Beaumont, TX) in a pH 8.7 buffer containing 25 mmol/L
sodium pyrophosphate and 11 mmol/L barbital, according to the method
described previously (7). To visualize AST activity, the
membrane was stained with an AST isoenzyme reagent staining kit (Wako
Pure Chemical Co.) consisting of 60 mmol/L Tris-HCl, pH 7.4 (1.4 mL),
0.1 mol/L sodium L-asparaginase (0.8 mL), 0.1 mol/L
-ketoglutaric acid (0.2 mL), 1 g/L pyridoxal-5 phosphate (0.05 mL),
and Fast Violet B salt, 3 mg; a principle of this procedure is the
diazo reaction. The patient's serum produced a single band on the
membrane between supernatant AST (s-AST) on the anodal side and
mitochondrial AST (m-AST) on the cathodal side, suggesting that
something was bound to AST. To determine the heavy-chain and
light-chain classes and the type of a suspected Ig complex, we
performed electroimmunosyneresis (without deproteinization)
(8) and immunoturbidimetry
(9)(10) by using antisera against human IgG,
IgA, and IgM, and light-chain 
and 
(Dakopatts, Glostrup,
Denmark). With electroimmunosyneresis, the bands of the patient's AST
activities that precipitated with anti-IgM and anti- antibody were
shifted to the cathodal side (Fig. 1
). Immunoturbidimetry showed that the absorbances in tubes to
which anti-IgM or anti- antibody had been added were higher than
those in tubes containing control serum. Absorbance at 570 nm was
measured by means of a Toshiba TBA-180 analyzer (Toshiba Medical,
Tokyo, Japan). The Ig bound to AST was judged to be IgM of the
type. To dissociate the AST-IgM complex, the patient's serum was
treated with 0.1 mol/L 2-mercaptoethanol and incubated for 60 min at
37 °C. After this treatment, the AST activity remained unchanged,
and the electrophoretic pattern of the s-AST isoenzyme normalized.
These results suggest that the binding of IgM to s-AST did not affect
s-AST activity. Further testing with in vitro recombination was used to
study the binding affinity of Ig in the enzyme-Ig complex for human AST
isoenzymes. To dissociate the Ig from the complex, the patient's serum
was dialyzed for 20 h against 50 mmol/L glycine-HCl buffer (pH
3.4). The dissociated IgM was purified by Sephadex S-300 (Pharmacia
Fine Chemicals, Uppsala, Sweden) gel filtration with the same buffer
used as eluant (11). To purifiy s-AST and m-AST, liver
tissue obtained at autopsy was homogenized, then layered on 25 mmol/L
sucrose–20 g/L human albumin, and centrifuged at 1000 rpm for 5 min.
The supernatant was further centrifuged at 13 000 rpm for 3 min. This
supernatant and sediment were used as the starting materials for the
purification. s-AST and m-AST were purified by DEAE-Sephadex A-50
ion-exchange chromatography (Pharmacia Fine Chemicals) by using a
0–0.2 mol/L NaCl gradient in a stepwise elution with 15 mmol/L
phosphate buffer, pH 7.2, at a flow rate of 10 mL/h. The dissociated Ig
was incubated with the purified AST isoenzymes (s-AST 386 U/L, m-AST
986 U/L) in a ratio of 4:1 by volume at 25 °C for 60 min, and the
recombined enzyme-Ig complex was detected by immunoturbidimetry
(11). Immunoturbidimetry showed that the absorbances in
tubes to which anti-IgM and the patient's IgM and s-AST had been added
were higher than in tubes containing m-AST (Table 1
). The results showed that the purified IgM was able to
recombine with s-AST. These results show that the patient's increased
serum AST was related to the presence of an s-AST-IgM--type complex.
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Figure 1. Electroimmunosyneresis (without deproteinization) pattern
of the patient's serum identified as IgM- type.
AST activities on precipitin lines were observed in lanes 4
and 6. Lane 1, patient's serum; lane
2, patient's serum, anti-IgG serum; lane 3, patient's
serum, anti-IgA serum; lane 4, patient's serum, anti-IgM
serum; lane 5, patient's serum, anti- light-chain serum;
lane 6, patient's serum, anti- light-chain serum;
lane 7, control serum from a patient with acute myocardial
infarction.
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We found 53 published cases of high serum AST concentrations that were
due to the formation of an Ig-AST complex (12). In 28
cases, the macro form of AST was found to be an IgA-AST complex; in 18
cases, an IgG-AST complex
(1)(3)(5)(6)(9)(12)(13)(14)(15)(16)(17)(18);
in 1 case, an IgM-AST complex (19); and in 6 cases, AST
was found to be complexed with IgG and IgA
(12)(20). The reported cases with Ig-AST
complexes can be divided into three groups, according to the clinical
characteristics of the patients: (a) 40 patients with
various liver diseases, malignancies, and diseases involving immune
disorders; (b) 8 apparently healthy individuals
(1)(2)(3)(4)(5)(6); and (c) 5 patients whose disease was
not described. However, three of the healthy individuals with IgG-AST
complex had received medication (one, an oral contraceptive
(1); one, a griseofulvin (5); and one, a
weight-reducing drug (5)), and one healthy individual with
IgG-AST complex had hepatitis B vaccination before the increased serum
AST activity was detected. Our patient showed a high antimicrosomal
antibody concentration, suggesting an autoimmune disorder. These
reports suggest that not only the patients with serious liver disease
or immune disorder, but also most of the healthy individuals had
experienced possibly hepatotoxic or host immune response-modifying
episodes before the increased serum AST activity was detected
(15), and these episodes or diseases may be involved in
the production of Ig-AST complexes.
Our patient was discharged with no treatment. After 3 years, the
patient is still clinically well, and her AST serum concentrations are
still high. We are following her enzyme activity to determine the
duration of the high AST activity and whether the IgM bound to AST will
switch to another class of Ig. When high concentrations of serum AST
are observed in the absence of organ-specific disease, the presence of
an Ig-enzyme complex should be investigated to avoid unnecessary or
invasive examinations.
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