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PEDIATRIC ANESTHESIA

Autoantibodies Associated with Volatile Anesthetic Hepatitis Found in the Sera of a Large Cohort of Pediatric Anesthesiologists

Dolores B. Njoku, MD^*, Robert S. Greenberg, MD^*, Mohammed Bourdi, PhD, Craig B. Borkowf, PhD, Elizabeth M. Dake, MS^*, Jackie L. Martin, MD^*, and Lance R. Pohl, Pharm D, PhD

Departments of *Anesthesiology and Critical Care Medicine and Pediatrics, The Johns Hopkins Medical Institutions, Baltimore, Maryland, the Molecular and Cellular Toxicology Section of the Laboratory of Molecular Immunology, and the Division of Epidemiology and Clinical Applications, Office of Biostatistics Research, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland

Address correspondence and reprint requests to Dolores B. Njoku, MD, Department of Anesthesiology and Critical Care Medicine, The Johns Hopkins Hospital, Blalock 906A, 600 N Wolfe St., Baltimore, MD 21287. Address e-mail to dnjoku{at}jhmi.edu

	Abstract

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Anesthetic-induced hepatitis is thought to have an immune-mediated basis, in part because many patients who develop hepatitis have serum autoantibodies that react with specific hepatic proteins. The present study shows that pediatric anesthesiologists also have these serum autoantibodies. Moreover, levels of these autoantibodies are higher than those of general anesthesiologists. We collected sera from 105 pediatric and 53 general anesthesiologists (including 3 nurse anesthetists), 20 halothane hepatitis patients, and 20 control individuals who were never exposed to inhaled anesthetics. Serum cytochrome P450 2E1 (P450 2E1) and 58-kd hepatic endoplasmic reticulum protein (ERp58) autoantibodies were measured by enzyme-linked immunosorbent assays. Positive values were 2 SD above median control values. Two multiple regression models were constructed. Pediatric anesthesiologists, like halothane hepatitis patients, had higher serum autoantibody levels of ERp58 and P450 2E1 than general anesthesiologists and controls, which was possibly because of their increased occupational exposures to anesthetics. Female anesthesiologists had higher levels of ERp58 autoantibodies than male anesthesiologists, whereas female pediatric anesthesiologists had higher levels of P450 2E1 autoantibodies than all other anesthesiologists. One female pediatric anesthesiologist had symptoms of hepatic injury. Because most anesthesiologists do not develop volatile anesthetic-induced hepatic injury, the findings suggest that pathogenic ERp58 and P450 2E1 autoantibodies may not directly cause volatile anesthetic hepatitis. Female anesthesiologists have high levels of these autoantibodies; however, the majority of these individuals do not develop hepatitis, suggesting that autoantibodies may not have a pathological role in volatile anesthetic-induced hepatitis.

IMPLICATIONS: Environmental exposure of anesthesiology personnel to certain inhaled anesthetics can induce the formation of autoantibodies that have been associated with anesthetic hepatitis. Female anesthesiologists have high levels of these autoantibodies; however, the majority of these individuals do not develop hepatitis, suggesting that autoantibodies may not have a pathological role in volatile anesthetic-induced hepatitis.

	Introduction

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Hepatic injury after environmental exposure to volatile, halogenated anesthetic gases is a rare event. However, it has been described in operating room personnel, recovery room nurses, and in laboratory workers anesthetizing laboratory animals (1–4). With regards to operating room personnel, most events have been described before the introduction and widespread use of scavenging systems (5). Despite this precaution, pediatric anesthesiologists can still be exposed to significant levels of these anesthetics during frequent mask inductions of general anesthesia and from the practice of using uncuffed endotracheal tubes.

Previous investigations have demonstrated that several specific hepatic proteins become covalently trifluoroacetylated (TFA) by the reactive metabolites of halothane and isoflurane (6). Similar adducts may also be formed at very small levels after exposure to desflurane (6). These TFA neoantigens are important because it is thought that they induce immune responses against either the TFA neoantigens, the native protein components (autoantigens) of the TFA neoantigens, or both of these classes of antigens in individuals that are susceptible to inhaled anesthetic-induced hepatitis (7). For example, studies in rats have shown that after exposure to halothane, a 58-kd hepatic endoplasmic reticulum protein (ERp58) becomes covalently modified by the trifluoroacetyl chloride metabolite of halothane (8). Subsequently, it was found that the majority of halothane hepatitis patients had serum antibodies that reacted with the purified rat TFA-ERp58 neoantigen, native ERp58 autoantigen, or both antigens, and this reactivity was significantly more than that of control patients (9). Moreover, we recently found that 40% of halothane hepatitis patients have serum autoantibodies that react with human liver ERp58 (Martin et al., unpublished results). Similarly, it has been established that cytochrome P450 2E1 (P450 2E1), the primary enzyme responsible for the oxidative metabolism of most volatile anesthetics, also becomes TFA altered when it metabolizes halothane (10). In addition, autoantibodies reacting with P450 2E1 are significantly elevated in the sera of 45%–70% of patients diagnosed with halothane hepatitis, whereas control subjects did not demonstrate increased levels of these autoantibodies (10,11). These findings suggest that pathogenic antibodies directed against ERp58, P450 2E1, or both may have a role in the etiology of volatile anesthetic hepatitis.

Low levels of P450 2E1 autoantibodies have also been found in the sera of a small group of anesthesiology personnel, suggesting that they may have been formed as a result of environmental exposure to halothane, enflurane, isoflurane, or possibly desflurane (10). If this were the case, then it seems reasonable that pediatric anesthesiologists may have higher levels of P450 2E1 autoantibodies than general anesthesiologists as a consequence of direct exposure to inhaled anesthetics from mask inductions and uncuffed endotracheal tubes. To test this idea, and to determine whether anesthesiologists also have ERp58 autoantibodies in their sera, we collected sera from a large group of pediatric anesthesiologists at the 1998 Society of Pediatric Anesthesiology winter meeting and analyzed them for the presence of ERp58 and P450 2E1 autoantibodies. We found that pediatric anesthesiologists had higher levels of both ERp58 and P450 2E1 autoantibodies than did general anesthesiologists. However, because most anesthesiologists do not develop volatile anesthetic-induced liver injury, the results suggest that pathogenic ERp58 and P450 2E1 autoantibodies may not cause volatile anesthetic hepatitis.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Chemicals and Reagents
Affinity purified alkaline phosphatase (AP) conjugated goat antihuman immunoglobulin G was obtained from Life Technologies (Bethesda, MD), AP substrate reaction mixture was from BIO-RAD (Hercules, CA), and the alanine aminotransferase (ALT) test kit was from Sigma Diagnostics (St Louis, MO). Purified human ERp58 (12) and P450 2E1 (10) were prepared as previously described.

Human Sera
The Joint Committee on Clinical Investigation IRB at the Johns Hopkins University School of Medicine approved the studies. After informed consent was obtained, sera from 105 pediatric anesthesiologists and 53 general anesthesiologists (including 3 nurse anesthetists) were collected and stored at -25°C until analyzed. A questionnaire was used to obtain demographic information from the subsets of individuals. Sera previously collected from patients with a clinical diagnosis of halothane hepatitis and control patients without exposure to volatile anesthetics were also analyzed (13). Because we were concerned that degradation of the proteins being tested may have occurred with some of the older sera from halothane hepatitis patients, as well as factitiously low autoantibodies in older control sera, all of the sera were reanalyzed, with the addition of new halothane hepatitis and control patients. We found that older sera results were analogous to new sera results in both groups.

Enzyme Linked Immunosorbent Assay of Human Sera
All assays were performed in triplicate in the wells of Immulon^® 4 microtiter plates (Dynex Incorporated, Chantilly, VA) in a total reaction volume of 100 µL by using a previously described method (14). Washings were performed with an Ultrawash Plus^TM automated microplate washer (Dynatech Technologies). The test antigens were 0.5 µg of ERp58 or P450 2E1 in phosphate-buffered saline. AP product formation was determined at 405 nm after 60 and 90 min, respectively, using a SpectraMax 250 automatic plate reader (Molecular Devices, Sunnyvale, CA).

Demographics
To determine whether differences in the autoantibody levels could be explained in terms of the baseline characteristics of each group, these characteristics were examined for both pediatric and general anesthesiologists. Autoantibody levels, age, work experience, and ALT levels were evaluated using Welch’s two-sided t-test for differences in means with unequal variances (15). Sex, history of autoimmune disease, liver disease, and infectious hepatitis were also examined in both groups using two-sided Fisher’s exact test for proportions (16).

ERp58 and P450 2E1 autoantibody levels for control and halothane hepatitis patients were compared with those levels for pediatric and general anesthesiologists without adjusting for other covariates. Box plots were constructed to compare the means and distributions of these levels among the four groups. The medians of the autoantibody levels of these groups were evaluated using pairwise Wilcoxon’s ranked sum tests (17).

Regression Models
To determine whether differences in the autoantibody levels of pediatric and general anesthesiology groups were statistically different because of predictor variables, two separate multiple regression models were constructed (17). The two response variables, Y₁ and Y₂, were the ERp58 and P450 2E1 autoantibody levels, respectively. In both groups, autoantibody levels were skewed to the right. Therefore, these variables were transformed by natural logarithms in the regression models. Thus, log(Y₁) and log(Y₂) are linear functions of the predictor variables in such models. The candidate predictor variables included I₁, type of anesthesiologist (0 for general, 1 for pediatric), I₂, sex (0 for men, 1 for women), I₃, history of autoimmune disease (0 for no, 1 for yes), I₄, history of liver disease (0 for no, 1 for yes), and I₅, history of infectious hepatitis (0 for no, 1 for yes). Other variables also included functions of age (yr), work experience (yr), and ALT levels (U/L), which were transformed by natural logarithms because they were skewed to the right. Additionally, because age and work experience were highly correlated, only one of these predictor variables was included in each model. Thus, the variables used in the model were X₁ = log(age) (log-yr) or X₂ = log(work experience) (log-yr) and X₃ = log(ALT levels + 1) (log-IU/L).

Other characteristics obtained from the questionnaire included ethnicity, chronic medications, and alcohol consumption. None of these correlated with the autoantibody levels and were excluded from the final analysis. Additionally, the reported number of general anesthetics per day was also considered too unreliable to be included in the analysis. Interactions between anesthesiologist type (I₁) and some of the other covariates were also considered. However, to avoid over-fitting the model, and because these covariates were balanced between both pediatric and general anesthesiologist groups, interactions between anesthesiologist type (I₁) and the medical history indictor variables of autoimmune diseases (I₃), liver disease (I₄), and infectious hepatitis (I₅) were excluded from the final models. One of the general anesthesiologists was deleted from the analysis because of missing questionnaire data.

All subset regression was used to select good candidate models. The criteria used to select the best model for each protein were Mallow’s Cp statistic, parsimony (i.e., models with the fewest number of variables), and interpretive value (18). The F test was used to determine whether the complete set of variables used in the model significantly explained the variability of the antibody levels. All variables included in the model were required to be significant at the 5% level. The statistical analyses were performed in the SAS 6.12 programming language (SAS Inc, Cary, NC).

	Results

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Box-Plots Analyses
Absorbance readings were linear with respect to antibody concentrations in the observed range of values. The pairwise Wilcoxon’s ranked sum test showed that the median of the ERp58 autoantibody levels for the control patients was significantly lower than the median levels for pediatric anesthesiologists and halothane hepatitis patients (Fig. 1). The median for the general anesthesiologist autoantibody levels was significantly lower than that for the pediatric anesthesiologists and halothane hepatitis patients, but it was not different from control patients. In contrast, the median autoantibody levels for the pediatric and halothane hepatitis patients could not be distinguished. Similar analysis showed that the median of the P450 2E1 autoantibody levels for control patients was significantly lower than the median levels for general anesthesiologists, pediatric anesthesiologists, and halothane hepatitis patients (Fig. 2). Furthermore, there was a highly significant increasing trend for the median levels of general anesthesiologists, pediatric anesthesiologists, and halothane hepatitis patients.

View larger version (18K): [in this window] [in a new window]   Figure 1. Box plots of the 58-kd endoplasmic reticulum protein (ERp58) autoantibody levels. The box plots were derived from the ELISA determinations of ERp58 serum autoantibody levels as expressed as absorbance at 405 nm on a logarithmic scale for controls (n = 20), general anesthesiologists (n = 52), pediatric anesthesiologists (n = 105), and halothane hepatitis patients (n = 20). The thick bar denotes the median, the lower and upper edges of the box denote the inter-quartile range (IQR), the whiskers extend to all observations within 1.5 times the IQR of the median, and the outliers are designated by asterisks. The shaded region denotes a 95% confidence interval for the median. The median of the ERp58 autoantibody levels for the control patients was significantly lower than the median level for pediatric anesthesiologists (P < 0.0001) and halothane hepatitis patients (P < 0.001). The median for the general anesthesiologists was significantly lower than the median for pediatric anesthesiologists and halothane hepatitis patients (P < 0.0001). In contrast, the median for the pediatric anesthesiologists and halothane hepatitis patients could not be distinguished (P = 0.19).

View larger version (19K): [in this window] [in a new window]   Figure 2. Box plots of cytochrome P450 2E1 (P450 2E1) autoantibody levels. The box plots were derived from the ELISA determinations of P450 2E1 serum autoantibody levels as expressed as absorbance at 405 nm on a logarithmic scale for controls (n = 20), general anesthesiologists (n = 52), pediatric anesthesiologists (n = 105), and halothane hepatitis patients (n = 20). The thick bar denotes the median, the lower and upper edges of the box denote the inter-quartile range (IQR), the whiskers extend to all observations within 1.5 times the IQR of the median, and outliers are designated by asterisks. The shaded region denotes a 95% confidence interval for the median. The median of the P450 2E1 autoantibody levels for control patients was significantly lower than the median level for general anesthesiologists (P = 0.0009), pediatric anesthesiologists, and halothane hepatitis patients (P < 0.0001). Furthermore, there was a highly significant increasing trend for the median levels of general anesthesiologists, pediatric anesthesiologists, and halothane hepatitis patients (general < pediatric < halothane hepatitis patients, P < 0.0001).

View larger version (31K): [in this window] [in a new window]   Figure 3. Plot of the 58-kd endoplasmic reticulum protein (ERp58) against cytochrome P450 2E1 (P450 2E1) autoantibody levels. The scatter plot was derived from the ELISA determination of ERp58 and P450 2E1 serum autoantibody levels as expressed as absorbance at 405 nm on a logarithmic scale. Solid symbols denote general anesthesiologists, open symbols denote pediatric anesthesiologists, squares denote men, and circles denote women. The dashed lines show the means of the autoantibody levels for the controls, whereas the dotted lines show the means ± 2 SD of the autoantibody levels for the controls.

The best multiple regression model for ERp58 autoantibody levels with the four outliers removed (n = 153 observations) was

equation

where expdenotes the natural exponential function, with an adjusted R² score of 54.7%. The F test shows that the variables pediatric anesthesiologists and female sex explain why ERp58 autoantibody levels were higher among pediatric anesthesiologists than general anesthesiologists (Table 2). Specifically, pediatric anesthesiologists have higher ERp58 autoantibody levels than general anesthesiologists, and female anesthesiologists tend to have higher levels of autoantibodies to this protein than do male anesthesiologists with the same covariates. In the same way, the variable age of general anesthesiologists explains why some of their ERp58 autoantibody levels can be higher than those of other general anesthesiologists. Specifically, as age increases, general anesthesiologists tend to have higher levels of autoantibodies to ERp58. The other predictor variables did not contribute significantly to this model in the presence of the above covariates.

equation

with an adjusted R² score of 70.0%. The F test shows that the variables pediatric anesthesiologists and female pediatric anesthesiologists explain why P450 2E1 autoantibody levels are higher in pediatric anesthesiologists than general anesthesiologists (Table 3). Specifically, pediatric anesthesiologists have higher levels of P450 2E1 autoantibodies than general anesthesiologists with the same covariates, and female pediatric anesthesiologists have higher levels of P450 2E1 autoantibodies than male pediatric anesthesiologists with the same covariates. Moreover, in an alternative model with simply sex (I₂), rather than an interaction between sex and anesthesiologist type (I₁I₂), the coefficient for sex has a P value of 0.08. In the same way, ALT levels of general anesthesiologists explain why the P450 2E1 autoantibody levels of some general anesthesiologists can be higher than those of other general anesthesiologists. Specifically, those general anesthesiologists with higher ALT levels had higher levels of P450 2E1 autoantibodies. The other predictor variables did not contribute significantly to this model in the presence of the above covariates.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The present study has shown that pediatric anesthesiologists have significantly higher serum levels of anesthetic hepatitis-associated ERp58 and P450 2E1 autoantibodies than general anesthesiologists and control patients, whereas general anesthesiologists only have increased serum levels of P450 2E1 autoantibodies when compared with the control patients. These results suggest that chronic occupational exposure to volatile anesthetics can lead to continuous hepatic metabolism of volatile anesthetics and formation of immunogenic TFA protein adducts, including TFA-ERp58 and TFA-P450 2E1, which can induce formation of the ERp58 and P450 2E1 autoantibodies associated with volatile anesthetic hepatitis (7). The results have also shown that female anesthesiologists had higher levels of ERp58 autoantibodies than male anesthesiologists, whereas female pediatric anesthesiologists had higher levels of P450 2E1 autoantibodies than all other anesthesiologists. Antibody responses after immunization are often greater in women than in men and are believed to be caused either by estrogens or pituitary hormones (19). Whether these hormones have a similar effect in the present study is not known.

Only one female pediatric anesthesiologist developed liver injury, even when all of the pediatric anesthesiologists as a group had increased serum levels of anesthetic hepatitis-associated ERp58 and P450 2E1 autoantibodies that were not significantly different from those of halothane hepatitis patients. Thus, our findings suggest that ERp58 and P450 2E1 autoantibodies may not have a role in the development of inhaled anesthetic hepatitis. Alternatively, it is possible that antigen-specific cytotoxic T cells, instead of autoantibodies, which are directed against peptides derived from ERp58 and P450 2E1, may cause volatile anesthetic hepatitis. In this regard, antigen-specific cytotoxic T cells, but not autoantibodies, appear to have a role in the pathogenesis of liver injury caused by hepatitis B infection (20).

In contrast, humoral reactions, cellular immune reactions, or both against native ERp58 and P450 2E1 may not have a role in volatile anesthetic-induced hepatitis. Perhaps only TFA-altered forms of these autoantigens can be targets of pathogenic antibodies or cytotoxic T cells. In this regard, previous studies have demonstrated that only halothane hepatitis patients, but not patients exposed to halothane who did not develop hepatitis, or patients with other forms of liver disease, have serum antibodies that react with TFA liver microsomal antigens (7). Furthermore, other cellular targets of the reactive acyl halide metabolites of volatile anesthetics that also become TFA modified, such as a carboxylesterase, protein disulfide isomerase, ERp72, and glucose-related proteins 78 and 94, could potentially become the immunogens that lead to volatile anesthetic-induced hepatitis (7).

In conclusion, we found significantly higher levels of ERp58 and P450 2E1 serum autoantibodies in pediatric anesthesiologists when compared with general anesthesiologists. The mathematical regression models verify that female anesthesiologists, both pediatric and general, have higher levels of autoantibodies to ERp58 than male anesthesiologists, and female pediatric anesthesiologists tend to have higher levels of P450 2E1 autoantibodies than male pediatric anesthesiologists. Still, only one of the female pediatric anesthesiologists developed symptoms of anesthetic hepatitis, even though these autoantibodies have been associated with volatile anesthetic-induced hepatitis. These findings suggest that ERp58 and P450 2E1 serum autoantibodies may not have a role in volatile anesthetic-induced hepatitis and that other immune mechanisms may determine whether halogenated volatile anesthetic-induced hepatitis occurs.

	Acknowledgments

 
DBN is supported, in part, by the Foundation for Anesthesiology Education and Research.

	Footnotes

 
Presented, in part, at the Fourth Annual Society of Pediatric Anesthesiology Winter Meeting, February 14, 1998, Phoenix, AZ and at the International Anesthesiology Research Society 73rd Clinical and Scientific Congress, March 13, 1999, Los Angeles, CA.

	References

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Anesthesia & Analgesia® is published for the International Anesthesia Research Society® by Lippincott Williams & Wilkins with the assistance of Stanford University Libraries' HighWire Press®. Copyright 2006 by the International Anesthesia Research Society. Online ISSN: 1526-7598   Print ISSN: 0003-2999