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L R Pohl

Publications and source records attributed to L R Pohl.

At least 55 records · Page 3Linked to original sources
PubMed1990

Antibody assays for the detection of patients sensitized to halothane.

Sera from patients with a clinical diagnosis of halothane hepatitis have been shown to contain antibodies that react with liver microsomal proteins (100, 76, 59, 57, and 54 kDa) covalently altered by the trifluoroacetyl (TFA) halide metabolite of halothane. In the present study, rapid and sensitive enzyme-linked immunosorbent assays for the detection of these antibodies have been evaluated. A recently described method that utilizes TFA-rabbit serum albumin as test antigen was studied employing a large population of halothane hepatitis and control patients. Several problems were discovered with the assay that were not previously recognized. The assay was then compared directly with methods that utilize as test antigens either liver microsomes or purified TFA proteins from halothane-treated rats. Sixty-seven percent of patients with a clinical diagnosis of halothane hepatitis tested positive for antibodies when the test antigens were either TFA-rabbit serum albumin or liver microsomes. This value was increased to 79% when the purified TFA-57 kDa, TFA-76 kDa, and TFA-100 kDa proteins were used as test antigens. These results indicate that the specificity and sensitivity of enzyme-linked immunosorbent assay methods for the detection of patients' antibodies may be increased significantly by utilizing the purified TFA microsomal proteins as test antigens.

Animals↗
PubMed1989

Involvement of the carboxyl-terminal propeptide of beta-glucuronidase in its compartmentalization within the endoplasmic reticulum as determined by a synthetic peptide approach.

The proenzyme form of beta-glucuronidase is compartmentalized in large quantities within the endoplasmic reticulum by binding to the esterase, egasyn. Also, the propeptide of the proenzyme form of beta-glucuronidase is likely located at the carboxyl terminus. We have, therefore, tested if this carboxyl-terminal peptide is important in binding to egasyn. A polyclonal antibody to a 30-mer synthetic peptide, corresponding to the carboxyl-terminal 30 amino acids of pro-beta-glucuronidase, provided evidence that egasyn binds to the carboxyl terminus of beta-glucuronidase. This antibody interacted with proenzyme beta-glucuronidase-egasyn complexes in which one, two, or three egasyn molecules were bound to the beta-glucuronidase tetramer, but not with those complexes (M4) which contained four egasyn molecules. We interpret these results as indicating that all available carboxyl termini of the beta-glucuronidase proenzyme tetramer are shielded by egasyn in the M4 complexes. The same antibody did not recognize the mature lysosomal form of beta-glucuronidase, indicating that only the proenzyme form of microsomal beta-glucuronidase contains the original carboxyl terminus. Also, the synthetic 30-mer was found to be a specific and potent inhibitor (50% inhibition at 1.3 microM) of the esterase activity of purified egasyn but exhibited little inhibitory activity toward other purified esterases including a rat trifluoroacetylated esterase or egasyn esterase from another species. Together, these data describe a potent interaction of the exposed carboxyl terminus of precursor glucuronidase with the esterase catalytic site of egasyn, which in turn results in the specific localization of glucuronidase within the lumen of the endoplasmic reticulum.

Amino Acid Sequence↗
PubMed1989

Human anti-endoplasmic reticulum antibodies in sera of patients with halothane-induced hepatitis are directed against a trifluoroacetylated carboxylesterase.

Previous studies have demonstrated that patients with halothane-induced hepatitis have serum antibodies that are directed against novel liver microsomal neoantigens and have suggested that these neoantigens may play an immunopathological role in development of the patients' liver damage. These investigations have further revealed that the antibodies 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 metabolite 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 immunoaffinity column of anti-TFA IgG. Antibodies were raised against the 59-kDa-TFA protein and were used to purify the native protein from liver microsomes of untreated 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 3.1.1.1). A similar strategy may be used to purify and characterized neoantigens associated with other drug toxicities that are believed to have an immunopathological basis.

Animals↗
PubMed1988

Rat liver carboxylesterase: cDNA cloning, sequencing, and evidence for a multigene family.

A cDNA clone was isolated from a rat liver lambda gt11 expression library by screening with polyclonal antibodies raised against a rat liver microsomal carboxylesterase. This clone of 1.8 kb contained an open reading frame encoding a mature protein of 531 amino acids with a predicted molecular weight of 58,084. The 5' portion of the clone coded for 9 amino acids of a putative signal peptide. The 3' end of the clone included an untranslated region and a poly (A) tail. Carboxylesterase active site regions, five potential N-linked glycosylation sites, and 2 postulated cystine disulfide bridges were found in the cDNA-deduced amino acid sequence. Sequences obtained from tryptic peptides and the NH2-terminus of the purified native carboxylesterase were aligned with the deduced amino acid sequence, and the overall identity was 84%. Southern blot analysis suggested the presence of multiple genes. Thus it is concluded that we have cloned a rat liver carboxylesterase, and that this enzyme is a member of a multigene family.

Amino Acid Sequence↗
PubMed1988

Enflurane metabolism produces covalently bound liver adducts recognized by antibodies from patients with halothane hepatitis.

The existence of a rare syndrome of "enflurane hepatitis" similar to that described for halothane and of a cross-sensitization between halothane and enflurane has been controversial, largely due to equivocal clinical case reports and a lack of a plausible molecular mechanism for the hepatotoxicity. The present study suggests a possible hypersensitivity basis for enflurane hepatitis and the apparent cross-sensitization between halothane and enflurane involving covalently bound liver microsomal adducts. Immunoblotting studies have revealed that antibodies in the sera of six patients with halothane hepatitis recognize liver microsomal antigens of Mr = 100,000, or both 100,000 and 76,000, formed in rats treated with enflurane or halothane. These antigens were not detected in microsomes from isoflurane- or sesame oil-treated rats. The recognition of these antigens could be abolished by preincubation of the sera with microsomes from halothane-treated rats. These data suggest that the difluoromethoxydifluoroacetyl halide metabolite of enflurane, as well as the trifluoroacetyl halide metabolite of halothane, covalently bind to similar hepatic proteins, and may become immunogens in susceptible patients. This mechanism may also account for the apparent cross-sensitization between halothane and enflurane anesthesia, and the development of hepatic necrosis.

Animals↗
PubMed1988

Metabolic basis for a drug hypersensitivity: antibodies in sera from patients with halothane hepatitis recognize liver neoantigens that contain the trifluoroacetyl group derived from halothane.

Previous studies have demonstrated that antibodies in sera from patients with halothane hepatitis recognize halothane-induced liver microsomal polypeptide neoantigens, and have suggested that these antibodies may play a role in the pathogenesis of the hepatitis. In the present study, the mechanism of neoantigen generation was investigated. Liver microsomes from rats treated in vivo with halothane or deuterated halothane were tested by immunoblotting for reactivity with patients' sera and with an antiserum specific for the covalently bound trifluoroacetyl (TFA) halide metabolite of halothane. Rat liver microsomes incubated aerobically or anaerobically with halothane or deuterated halothane in vitro, +/- NADPH and/or NADH, were also analyzed. The results obtained demonstrate that neoantigen expression involves oxidative halothane metabolism by cytochromes P-450 to TFA halide and covalent binding of the TFA group to the proteins. Incubation of microsomes from halothane-treated rats with 1 M piperidine cleaved the TFA groups from the proteins and abolished antigenicity, confirming this conclusion. Recognition of the neoantigens by the patients' antibodies was inhibited only partially using the hapten derivative N-E-TFA-L-lysine. It appears that the patients' antibodies recognize epitopes consisting of the TFA group plus associated structural features of the protein carriers (100 kDa, 76 kDa, 59 kDa, 57 kDa and 54 kDa), not the TFA hapten alone. To our knowledge, this constitutes the first characterization of drug metabolite-tissue protein neoantigens implicated in a drug hypersensitivity. The approach described may be of general utility for characterization of drug-induced neoantigens associated with other drug hypersensitivities.

Animals↗
PubMed1986

Carbon tetrachloride and 2-isopropyl-4-pentenamide-induced inactivation of cytochrome P-450 leads to heme-derived protein adducts.

When CCl4 was incubated with rat liver microsomes from phenobarbital-treated rats in an aerobic or anaerobic atmosphere, over 69% of the heme moiety of cytochrome P-450 was destroyed. At least 45% of the degraded heme under both reaction conditions was accounted for as heme-derived products irreversibly bound to microsomal proteins. Furthermore, 33% of the irreversibly bound products were bound specifically to a 54-kDa form of cytochrome P-450. A structurally different compound, 2-isopropyl-4-pentenamide, also destroyed the heme moiety of cytochrome P-450 and produced heme-derived adducts of microsomal proteins that accounted for 28% of the destroyed heme. These results represent a novel mechanism for the destruction of cytochromes P-450 by xenobiotics.

Acetamides↗
PubMed1986

Inactivation of cytochrome P-450 by 2-isopropyl-4-pentenamide and other xenobiotics leads to heme-derived protein adducts.

When cytochrome P-450 in phenobarbital-induced rat liver microsomes was destroyed by 2-isopropyl-4-pentenamide (AIA) in vitro, 50% of the degraded heme was recovered as heme-derived products irreversibly bound to microsomal proteins. In contrast, less than 50% of the degraded heme was accounted for as N-alkylated porphyrins. Furthermore, 64% of the irreversibly bound products was bound specifically to a 54-kD form of cytochrome P-450. Several other compounds which have been reported to destroy cytochrome P-450 by forming N-alkylated porphyrins also produced heme-derived protein adducts. These findings indicate that the formation of heme-derived protein adducts may represent an important pathway for the irreversible degradation of cytochrome P-450 by many xenobiotics.

Acetamides↗
PubMed1985

Two-dimensional J-resolved nuclear magnetic resonance spectral study of two bromobenzene glutathione conjugates.

The application of two-dimensional J-resolved nuclear magnetic resonance spectroscopy to determine the structure of two bile metabolites isolated from rats injected interperitoneally with bromobenzene is described. The structures of the two molecules are obtained unambiguously from the proton-proton spin coupling constants. This paper discusses the fundamentals of the technique and demonstrates the resolution of small long-range coupling constants.

Animals↗
PubMed1985

Immunological studies on the mechanism of halothane-induced hepatotoxicity: immunohistochemical evidence of trifluoroacetylated hepatocytes.

The fulminant hepatotoxicity caused by halothane has been thought to have an immunological basis because this toxicity occurs most often after repeated administration of halothane and because sera from patients recovering from severe halothane hepatotoxicity contain antibodies that bind to the surface membranes of hepatocytes of rabbits treated with halothane. In order to determine whether the major reactive metabolite of halothane, trifluoroacetyl halide, covalently binds to hepatocytes, we have developed specific and sensitive peroxidase enzyme-linked immunosorbent assays and an indirect immunofluorescence staining method for identifying trifluoroacetylated (TFA)-hepatocytes. Liver sections prepared from rats at 4 hr after halothane administration were stained preferentially in the centrilobular region with anti-TFA serum whereas livers of control rats showed no staining. The specificity of the assay for the TFA group was confirmed by the complete inhibition of the staining with 200 microM N-epsilon-TFA-L-lysine in the diluted antiserum. On the other hand, 2 mM halothane or L-lysine did not inhibit this staining. Moreover, treatment of rats with deuterated halothane resulted in significantly less staining than did halothane. At 24 hr after halothane administration, hepatocytes isolated and stained by indirect immunofluorescence showed a linear and granular pattern on their surface membranes. These results indicate that trifluoroacetyl halide either reacts directly with constituents of the plasma membranes or with other cellular components which become incorporated into the plasma membranes.

Animals↗