Antimitochondrial antibodies (AMA) directed against the lipoyl domain of the E2 subunit of pyruvate dehydrogenase (PDC-E2) are detected in 95% of patients with PBC and are present before onset of clinical disease. The recent demonstration that AMA recognize xenobiotic modified PDC-E2 with higher titers than native PDC-E2, raises the possibility that the earliest events involved in loss of tolerance are related to xenobiotic modification. We hypothesized that reactivity to such xenobiotics would be predominantly IgM and using sera from a large cohort of PBC patients and controls (n=516), we examined in detail sera reactivity against either SAc-conjugated bovine serum albumin (BSA), recombinant PDC-E2 (rPDC-E2) or BSA alone. Further, we also defined the relative specificity to the SAc moiety using inhibition ELISA; SAc conjugate and rPDC-E2 specific affinity purified antibodies were also examined for antigen specificity, isotype and cross-reactivity. Reactivity to SAc conjugates is predominantly IgM; such reactivity reflects a footprint of previous xenobiotic exposure. Indeed, this observation is supported by both direct binding, cross reactivity, and inhibition studies. In both early and late stage PBC, the predominant Ig isotype to SAc is IgM, with titers higher with advanced stage disease. We also note that there was a higher level of IgM reactivity to SAc in early stage versus late stage PBC. Interestingly, this finding is particularly significant in light of the structural similarity between SAc and the reduced form of lipoic acid, a step which is similar to the normal physiological oxidation of lipoic acid. We submit that specific modifications of the disulfide bond within the lipoic-acid-conjugated PDC-E2 moiety, i.e. by an electrophilic agent renders PDC-E2 immunogenic in a genetically susceptible host.

Our laboratory has hypothesized that xenobiotic modification of the native lipoyl moiety of the major mitochondrial autoantigen, the E2 subunit of the pyruvate dehydrogenase complex (PDC-E2), may lead to loss of self-tolerance in primary biliary cirrhosis (PBC). This thesis is based on the finding of readily detectable levels of immunoreactivity of PBC sera against extensive panels of protein microarrays containing mimics of the inner lipoyl domain of PDC-E2 and subsequent quantitative structure-activity relationships (QSARs). Importantly, we have demonstrated that murine immunization with one such mimic, 2-octynoic acid coupled to bovine serum albumin (BSA), induces antimitochondrial antibodies (AMAs) and cholangitis. Based upon these data, we have focused on covalent modifications of the lipoic acid disulfide ring and subsequent analysis of such xenobiotics coupled to a 15mer of PDC-E2 for immunoreactivity against a broad panel of sera from patients with PBC and controls. Our results demonstrate that AMA-positive PBC sera demonstrate marked reactivity against 6,8-bis(acetylthio)octanoic acid, implying that chemical modification of the lipoyl ring, i.e. disruption of the S-S disulfide, renders lipoic acid to its reduced form that will promote xenobiotic modification. This observation is particularly significant in light of the function of the lipoyl1oiety in electron transport of which the catalytic disulfide constantly opens and closes and, thus, raises the intriguing thesis that common electrophilic agents, i.e. acetaminophen or non-steroidal anti-inflammatory drugs (NSAIDs), may lead to xenobiotic modification in genetically susceptible individuals that results in the generation of AMAs and ultimately clinical PBC.