Proc. Natl. Acad. Sci. USA
Vol. 86, pp. 322-326, January 1989
Human anti-endoplasmic reticulum antibodies in sera of patients
with halothane-induced hepatitis are directed against a
H. SATOH*, B. M. MARTINt, A. H. SCHULICK*, D. D. CHRIST*, J. G. KENNA*, AND L. R. POHL*t
*Laboratory of Chemical Pharmacology, National Heart, Lung and Blood Institute, and tClinical Neuroscience Branch, National Institute of Mental Health,
National Institutes of Health, Bethesda, MD 20892
Communicated by Allan H. Conney, September 28, 1988 (receivedfor review July 5, 1988)
tients with halothane-induced hepatitis have serum antibodies
that are directed against novel liver microsomal neoantigens
and have suggested that these neoantigens may play an immu-
nopathological role in development of the patients' liver dam-
age. These investigations have further revealed that the anti-
bodies are directed against distinct polypeptide fractions (100
kDa, 76 kDa, 59 kDa, 57 kDa, 54 kDa) that have been
covalently modified by the reactive trifluoroacetyl halide me-
tabolite of halothane. In this paper, the trifluoroacetylated
(TFA) 59-kDa neoantigen (59-kDa-TFA) recognized by the
patients' antibodies was isolated from liver microsomes of
halothane-treated rats by chromatography on an immunoaf-
finity column ofanti-TFA IgG. Antibodies were raised against
the 59-kDa-TFA protein and were used to purify the native
protein from liver microsomes ofuntreated rats. Based upon its
apparent monomeric molecular mass, NH2-terminal amino
acid sequence, catalytic activity, and other physical properties,
the protein has been identified as a previously characterized
microsomal carboxylesterase (EC 220.127.116.11). A similar strategy
may be used to purify and characterize neoantigens associated
with other drug toxicities that are believed to have an immu-
Previous studies have demonstrated that pa-
It has been estimated that between 3% and 25% of all drug
toxicities, which can include anaphylaxis, serum sickness,
asthma, urticaria, dermatitis, fever, hemolytic anemia,
thrombocytopenia, granulocytopenia, hepatitis, nephritis,
vasculitis, pneumonitis, and lupus-erythematosus-like syn-
drome, are due to hypersensitivity (allergic) reactions (1).
Although most ofthese drug-induced hypersensitivities have
been presumed to be mediated by immunogens formed by the
covalent interaction of a reactive drug metabolite with tissue
carrier macromolecules (2-6), it is only in the case ofhepatitis
caused by the inhalation of anesthetic halothane that this
mechanism has been supported substantially by experimental
Previous studies have demonstrated that the majority of
halothane hepatitis patients have unique serum antibodies
that react with novel neoantigens in livers ofanimals (1, 7, 8)
and humans (9) treated with halothane and have suggested
that these neoantigens may play an immunopathological role
in development of the patients' liver damage. Characteriza-
tion of these neoantigens by immunoblotting with hapten-
specific anti-trifluoroacetyl (TFA) antibodies and sera from
several halothane hepatitis patients has revealed that they
correspond to distinct liver microsomal protein fractions (100
kDa, 76 kDa, 59 kDa, 57 kDa, 54 kDa) (10, 11) that have been
covalently modified by the reactive TFA halide metabolite of
To investigate the role of the halothane-induced neoanti-
gens in the pathogenesis of halothane hepatitis, a general
approach for their purification and characterization has been
developed and utilized to identify one of them.
MATERIALS AND METHODS
Purification of 59-kDa-TFA Protein from Halothane-
Treated Rats by Immunoaffmnity and Anion-Exchange HPLC.
Specific anti-TFA IgG (12) was purified from antisera derived
from rabbits immunized with TFA rabbit serum albumin (13)
as described. Anti-TFA IgG (250 mg) was coupled to Affi-Gel
10 (25 ml) according to the manufacturer's instructions
(Bio-Rad) and packed into a chromatography column (1.6 cm
x 13 cm).
Male Sprague-Dawley rats were treated with halothane
and after 12 hr liver microsomes were prepared as described
elsewhere (12). The microsomes (-3 g) from 20 rats were
solubilized by stirring gently for 1 hr at 40C in 200 ml of10mM
potassium phosphate (pH 7.4) containing 0.2 mM EDTA,
0.5% (wt/vol) sodium cholate, 0.2% (vol/vol) Emulgen 911,
20% (vol/vol) glycerol, and a mixture ofproteinase inhibitors
(aprotinin, 87 ,g/ml; leupeptin, 0.7 ,ug/ml; pepstatin A, 0.7
,ug/ml; and trypsin inhibitor, 50 ,ug/ml) (buffer A). After
centrifugation at 105,000 x g for 90 min, the supernatant was
applied (10 ml/hr) at 4°C to the anti-TFA IgG affinity column,
which had been equilibrated with buffer A. The column was
washed (1 ml/min) with 3 column volumes of 100 mM
potassium borate (pH 8.4) containing 1 M KCI, 0.2 mM
EDTA, and the proteinase inhibitors (buffer B). The TFA
proteins were eluted (1 ml/min) from the column with 200 ml
of 20 mM NM-TFA-L-lysine (TFA-Lys) in 10 mM potassium
phosphate (pH 7.4) containing 0.1 mM EDTA, 20% (vol/vol)
glycerol, and 0.5% (wt/vol) sodium cholate. The eluent was
concentrated, dialyzed against 20 mM Tris acetate (pH 7.5)
containing 0.2% (vol/vol) Lubrol PX (buffer C), and injected
onto a Bio-Gel TSK DEAE-5-PW (7.5 mm x 7.5 cm) HPLC
column (Bio-Rad). Elution, monitored at 280 nm, was at a
flow rate of 1 ml/min with a 90-min solvent program con-
sisting of an initial 60-min linear gradient of buffer C to 35%
bufferD (bufferC containing 0.8M sodium acetate), followed
by a 15-min linear gradient to 100% buffer D, and an
additional 15 min at 100%Wo buffer D. The 59-kDa-TFA protein
was isolated in the eluent from the HPLC column in a yield
of 1.3 mg.
Purification of 59-kDa Native Protein from Rats by Immu-
noaffiity and Anion-Exchange HPLC. The purification pro-
cedure was similar to that described for the purification ofthe
Abbreviation: TFA, trifluoroacetyl.
tTo whom reprint requests should be addressed.
The publication costs ofthis article were defrayed in part by page charge
payment. This article must therefore be hereby marked "advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Proc. Natl. Acad. Sci. USA 86 (1989)
59-kDa-TFA protein. In short, antisera were raised against
the purified 59-kDa-TFA protein by injecting a female New
Zealand White rabbit with 100pugof the protein in an equal
volume of Freund's complete adjuvant i.m. and s.c. at
several sites. After 4 weeks, a booster of100,gofthe protein
was administered by i.v. injection and antisera were collected
weekly for 5 weeks. Anti-59-kDa-TFA IgG (462 mg) was
coupled to Affi-Gel 10 and packed into a column. Liver
microsomes from 20 untreated male rats were solubilized and
loaded onto the affinity column. The column was washed
with 1 column volume of buffer A and 3 column volumes of
buffer B containing 0.5% (wt/vol) sodium cholate, and the
59-kDa protein was eluted with 3 column volumes of 2 M
KSCN (pH 7.5). After dialysis and concentration, the 59-kDa
protein was further purified by HPLC on the Bio-Gel TSK
DEAE-5-PW column. A 20-min solvent elution program was
used consisting ofan initial 9 min with buffer C, followed by
a 10-min linear gradient to 12% buffer D. The 59-kDa protein
was isolated in the eluent from the HPLC column in a yield
of 2.7 mg.
NH2-Terminal Amino Acid Sequence Analysis. Samples of
the 59-kDa protein were transblotted to polyvinylidene di-
fluoride membranes and sequenced as described by Mat-
sudaira (14) with modifications as reported by Martin et al.
(15). Automated Edman degradation was conducted employ-
ing an Applied Biosystems model 470 A gas-phase sequencer
with an on-line model 120 A phenylthiohydantoin (PTH)
amino acid analyzer. Normal program 03R PTH was em-
ployed as provided by Applied Biosystems.
NaDodSO4/PAGE and Immunoblotting with Anti-TFA IgG
and Human Sera. Procedures for polypeptide electrophoretic
separation, staining, transfer, and immunoperoxidase detec-
tion with anti-TFA IgG and human sera from halothane
hepatitis patients as well as conditions for antibody blocking
with the hapten derivatives TFA-Lys and N6-acetyl-L-lysine
(Ac-Lys) have been described in detail elsewhere (10-12, 16).
Comparative TFA Labeling of Liver Microsomal Proteins
After the Administration ofHalothane, Ethyl Trifluoroacetate,
Trifluoroethanol, or Sodium Trifluoroacetate. Halothane (10
mmol/kg), ethyl trifluoroacetate (5 mmol/kg), or trifluoro-
ethanol (5 mmollkg), dissolved in sesame oil, was adminis-
tered i.p. to rats and sodium trifluoroacetate (1.6 mmol/kg,
dissolved in water) was given by gavage. After 12 hr, rats
were killed and TFA proteins in liver microsomes were
detected by immunoblotting as described (12).
Other Methods. Carboxylesterase (EC 18.104.22.168) enzyme
activity was determined spectrophotometrically with p-
nitrophenyl acetate as substrate according to the method of
McLean et al. (17). Deglycosylation with endoglycosidase H
was performed as described by Harano et al. (18). Protein
was determined according to the method ofLowry et al. (19)
with bovine serum albumin as a standard.
Purification ofthe 59-kDa-TFA and Native 59-kDa Proteins.
As previously shown (11), 12 hr after the administration of
halothane to rats, protein fractions of 100 kDa, 76 kDa, 59
kDa, 57 kDa, and 54 kDa are among the major TFA labeled
constituents in liver microsomes, with the 59-kDa fraction
being the most prominent TFA component (Fig. 1A, lane 1).
The TFA proteins were separated from other microsomal
proteins (Fig. 1A, lane 2) by binding them selectively to an
affinity column of anti-TFA IgG and then, after thorough
washing of the column, eluting them selectively with the
hapten derivative TFA-Lys (Fig. 1A, lane 3). Further sepa-
ration by HPLC anion-exchange chromatography (Fig. 1B)
resulted in the purification of the 59-kDa-TFA protein (Fig.
RETENTION TIME (Min)
ofhalothane to rats. (A) Lane 1, immunoperoxidase staining with anti-TFA IgG ofa NaDodSO4/polyacrylamide gel blot ofthe liver microsomes
before purification. Lanes 2-4, NaDodSO4/polyacrylamide gels stained with Coomassie blue. Lane 2, liver microsomes beforepurification; lane
3, mixture ofTFA proteins that were isolated from livermicrosomes by chromatography on an anti-TFA IgG affinity column; lane 4, 59-kDa-TFA
(59-TFA) that was purified from otherTFA proteins by HPLC anion-exchange column as indicated in B. (B) Further purification of59-kDa-TFA
by anion-exchange HPLC. The 59-kDa-TFA (59-TFA) protein eluted from the column between 23 and 28 min. Preliminary results indicate that
the 100-kDa-TFA and 76-kDa-TFA proteins eluted from the column in the broad fraction between 70 and 90 min.
Purification of59-kDa-TFA protein from liver microsomes by immunoaffinity and anion-exchange HPLC 12 hr after administration
Medical Sciences: Satoh et al.
Medical Sciences: Satoh et al.
1A, lane 4). The numberofTFA moieties bound to the 59-kDa
protein remains to be determined.
Polyclonal antibodies raised against the 59-kDa-TFA pro-
tein selectively recognized not only the 59-kDa-TFA protein
in liver microsomes of halothane-treated rats and a
halothane-treated patient but also the native 59-kDa protein
in liver microsomes of untreated rats, as determined by
immunoblotting (results not shown). Based upon these find-
ings, the native 59-kDa protein was isolated from control rat
liver microsomes by affinity chromatography on an anti-59-
kDa-TFA column (Fig. 2A, lane 1). It was further purified by
HPLC anion-exchange chromatography (Fig. 2B) to apparent
homogeneity (Fig. 2A, lane 2).
Characterization of the 59-kDa Protein as a Carboxylester-
ase. The NH2-terminal amino acid sequence of the 59-kDa
protein was identical to a previously characterized rat micro-
somal serine-type carboxylesterase of apparent monomeric
molecular mass of59 kDa (18) and highly similar to a reported
60-kDa rabbit liver microsomal serine-type carboxylesterase
(20) (Table 1). Further analysis of our 59-kDa protein re-
vealed other similarities to those reported for the rat car-
boxylesterase. It is an apparent trimer and is a high-mannose
type of glycoprotein that can be deglycosylated by endogly-
cosidase H to a peptide ofapparent monomeric size of57 kDa
(18, 21) (results not shown). Moreover, the 59-kDa-TFA
protein hydrolyzed p-nitrophenyl acetate at a rate of 63.6
,umol/min per mg, which was comparable to the rates
reported for the rat 59-kDa carboxylesterase (44.4 ,umoUmin
per mg) (21). This activity was abolished by the serine
esterase and peptidase inhibitor phenylmethylsulfonyl fluo-
ride, as observed with the rat 59-kDa carboxylesterase (18).
Immunoblotting the 59-kDa-TFA and Native 59-kDa Pro-
teins with Sera from Halothane Hepatitis Patients. The sera of
five halothane hepatitis patients were previously reported to
contain antibodies that reacted with a 59-kDa-TFA polypep-
tide fraction in liver microsomes from halothane-treated rats
(11). This is illustrated by the immunoblot in Fig. 3A (serum
from patient 1, lane HAL MS). As found earlier (11), this
patient's serum antibodies also recognized additional TFA
protein with that of rat and rabbit carboxylesterases
Comparison of NH2-terminal sequences of the 59-kDa
*Data taken from Harano et al. (18).
tData taken from Korza and Ozols (20).
polypeptide fractions of 100 kDa, 76 kDa, and 54 kDa and,
when control microsomes were tested by immunoblotting,
staining ofthe 100-kDa, 76-kDa, 59-kDa, or 54-kDa polypep-
tide fractions was negligible (Fig. 3A, serum from patient 1,
lane SES MS).
Antibodies in the serum from patient 1 reacted strongly
with purified 59-kDa-TFA (Fig. 3A, lane 59-TFA) but negli-
gibly with native 59 kDa (Fig. 3A, lane 59). Sera from three
additional halothane hepatitis patients, previously found to
react with a 59-kDa-TFA fraction in liver microsomes from
halothane-treated rats (11), also reacted with the 59-kDa-
TFA protein, but negligibly if at all with the native 59-kDa
protein (Fig. 3A, sera from patients 2-4). The immunoblot in
Fig. 3A, stained with TFA hapten-specific rabbit IgG instead
ofhuman sera, confirms that59-kDa-TFA contained theTFA
RETENTION TIME (Min)
1 and 2, NaDodSO4/polyacrylamide gels stained with Coomassie blue. Lane 1, partially purified 59-kDa protein that was isolated from liver
microsomes by chromatography on an anti-59-kDa-TFA IgG affinity column; lane 2, 59-kDa protein that was purified furtherby anion-exchange
HPLC as indicated in B. (B) Further purification of the 59-kDa protein by anion-exchange HPLC. The protein eluted from the column between
8 and 11 min.
Purification of 59-kDa protein from liver microsomes of untreated rats by immunoaffinity and anion-exchange HPLC. (A) Lanes
Proc. Natl. Acad. Sci. USA 86(1989)
Proc. Natl. Acad. Sci. USA 86 (1989)
Serum from halothane
halothane hepatitis patients are not directed against the TFA
hapten but instead against new epitopes that have been
produced as a result of the covalent attachment of the TFA
moiety to the specific carrier proteins (1, 11)
Mechanism of TFA Labeling of 59-kDa Carboxylesterase
andOtherMicrosomal Proteins. Previous studies in vitro have
clearly indicated that the microsomal proteins become la-
beled with the TFA group by their direct interaction with the
reactive TFA halide metabolite of halothane (11). To elimi-
nate the possibility that the TFA labeling of the microsomal
proteins might be produced instead from trifluoroacetic acid,
the hydrolytic product ofTFA halide (22), after its possible
metabolic activation into reactive acyl glucuronide (23) or
acyl coenzyme A derivatives (24), rats were treated with
sodium trifluoroacetate andethyltrifluoroacetate or trifluo-
roethanol, which would be expected to be metabolized to
trifluoroacetic acid in the liver by enzymatic hydrolysis and
oxidations, respectively (21, 25). None of these compounds,
however, produced any detectable labeling of liver microso-
mal proteins with the TFA moiety (data not shown).
of patients with halothane hepatitis after NaDodSO4/PAGE and
immunoblotting. (A) Liver microsomes (20 ,ug per lane) from halo-
thane-treated (HAL MS) or control (sesame oil, SES MS)-treated
rats or purified 59-kDa-TFA (59-TFA) or native 59-kDa protein (59)
(2,ugper lane) were probed forimmunoreactivity with serafrom four
patients with halothane hepatitis or with hapten-specific anti-TFA
IgG. Sera from three of these patients were previously found by
NaDodSO4/PAGE and immunoblotting to contain antibodies recog-
nizing the following liver microsomal TFA neoantigenic fractions in
addition to the 59-kDa-TFA protein: 100, 76, and 54 kDa (patient 1);
100 kDa (patient 2); 100 and 76 kDa (patient 3) (11). (B) Recognition
of the 59-kDa-TFA protein by serum antibodies from patient 1
(Control lane) was partially inhibited by preincubation of the serum
for90 min with 1 mM hapten derivative TFA-Lys (TFA-Lys lane) but
not by 1 mM Ac-Lys (AC-Lys lane). (C) In contrast, 1 mM TFA-Lys
did not affect the recognition of the 59-kDa-TFA protein by serum
antibodies from patient 2. Results similar to these were found with
the sera from patients 3 and 4 (data not shown).
Recognition ofpurified 59-kDa-TFA by antibodies in sera
The reactions of the antibodies in the sera of patients 2-4
with 59-kDa-TFA (Fig. 3C, Control) were not inhibited by
preincubation ofthe sera with the hapten derivative TFA-Lys
(Fig. 3C, TFA-Lys) or Ac-Lys (Fig. 3C, AC-Lys). The same
concentration of TFA-Lys, but not Ac-Lys, partially inhib-
ited recognition of 59-kDa-TFA by antibodies in serum from
patient 1 (Fig. 3B). In contrast, we have demonstrated
previously that recognition of TFA microsomal proteins by
hapten-specific anti-TFA antibodies is inhibited nearly com-
pletely by TFA-Lys but not by Ac-Lys (11, 12). These results
confirm the idea that the antibodies in the sera of most
One of the major reasons why relatively little progress has
been made in the understanding of how drugs produce
allergic reactions is that no drug-induced immunogen has
previously been identified (1). In the present study, it has
been shown how immunogens involved in drug hypersensi-
tivities may be purified and identified when the structure of
a covalently bound drug metabolite is known and serum
antibodies directed against drug-induced neoantigens are
A neoantigen recognized by antibodies in the sera of
several halothane hepatitis patients was isolated from liver
microsomes ofhalothane-treated rats by chromatography on
an immunoaffinity column ofanti-TFA IgG and identified as
a 59-kDa-TFA carboxylesterase. The immunogen responsi-
ble for the induction ofthe anti-59-kDa-TFA antibodies in the
patients' sera is presumably a human 59-kDa-TFA carboxyl-
esterase. This conclusion is based upon the fact that the
apparent monomeric molecular masses of the halothane-
induced neoantigens detected in liver microsomes of rats
(11), rabbits (10), and humans (9), by immunoblotting with
sera from halothane hepatitis patients, are very comparable
in size and the fact that a 59-kDa protein has been detected
in liver microsomes of a patient by immunoblotting with
rabbit antibodies directed against the rat 59-kDa-TFA pro-
tein. A similar approach could be used to purify and char-
acterize the neoantigens present in livers ofhalothane-treated
humans, when sufficient amounts oftissue become available.
The purified halothane-induced neoantigens will have sev-
eral important applications. It will be possible to test directly
the relationship between sensitization to the neoantigens and
the pathogenesis of halothane hepatitis by immunizing ani-
mals with the neoantigens prior to challenging them with
halothane (1, 8, 11). Ifthis approach is successful, then it will
be possible to explore whether the hepatocellular damage
caused by halothane is mediated by specific antibodies or
sensitized T cells (1).
A related problem that can be addressed with the purified
neoantigens concerns the reasons for them becoming immu-
nogens in the first place. One contributing factor may be the
number ofcovalently bound TFA moieties. Epitope mapping
ofthe neoantigens with antibodies and T cells from halothane
hepatitis patients may help define other structural features
that make the TFA proteins immunogenic.
Another factor that could possibly determine whether a
TFA protein becomes an immunogen is its rate of degrada-
tion. Those altered proteins that are degraded very rapidly
may not become exposed to the immune system in sufficient
Medical Sciences: Satoh et al.
326 Download full-text
Medical Sciences: Satoh et al.
concentrations to be effective immunogens. It is believed that
this may be a contributing factor as towhy cytochrome P450,
which can become labeled with the TFA moiety (26) and may
correspond to the 54-kDa-TFA neoantigen (11), is, at most,
only a minor immunogen.
Probably one ofthe most important factors in determining
the immunogenic potential of a TFA-altered protein, assum-
ing that it can accumulate to adequate concentrations and be
recognized by the immune system, will be its efficiency of
contacting the immune system. This will be important not
only for the initial induction ofan immune response but also
forthe subsequent development ofimmune-mediated cellular
damage (1). Although TFA adducts have been detected
immunochemically on the outer surface of isolated hepato-
cytes (13), where they could conceivably come in contact
with the immune system, their composition and the mecha-
nisms by which they reached this site remain to be deter-
A practical application of the purified halothane-induced
neoantigens will be for the development of ELISA methods
for the detection of individuals sensitized to halothane that
are more specific and sensitive than procedures currently
available (7, 8, 10, 11, 27). This could prevent patients
sensitized to halothane from being reexposed to this agent or
to otherdrugs that might elicit cross-sensitization, such as the
structurally related inhalation anesthetic enflurane (12, 16).
We thank Dr. James R. Gillette for critically reviewing the
manuscript. D.D.C. was supported by a National Research Service
Award (ES05368) from the National Institute of Environmental
Health Sciences, and J.G.K. was supported in part by a grant from
the Wellcome Trust, U.K.
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