Acylcarnitine Profiles in Acetaminophen Toxicity in the Mouse: Comparison to Toxicity, Metabolism and Hepatocyte Regeneration

Abstract

High doses of acetaminophen (APAP) result in hepatotoxicity that involves metabolic activation of the parent compound, covalent binding of the reactive intermediate N-acetyl-p-benzoquinone imine (NAPQI) to liver proteins, and depletion of hepatic glutathione. Impaired fatty acid β-oxidation has been implicated in previous studies of APAP-induced hepatotoxicity. To better understand relationships between toxicity and fatty acid β-oxidation in the liver in APAP toxicity, metabolomic assays for long chain acylcarnitines were examined in relationship to established markers of liver toxicity, oxidative metabolism, and liver regeneration in a time course study in mice. Male B6C3F1 mice were treated with APAP (200 mg/kg IP) or saline and sacrificed at 1, 2, 4, 8, 24 or 48 h after APAP. At 1 h, hepatic glutathione was depleted and APAP protein adducts were markedly increased. Alanine aminotransferase (ALT) levels were elevated at 4 and 8 h, while proliferating cell nuclear antigen (PCNA) expression, indicative of hepatocyte regeneration, was apparent at 24 h and 48 h. Elevations of palmitoyl, oleoyl and myristoyl carnitine were apparent by 2–4 h, concurrent with the onset of Oil Red O staining in liver sections. By 8 h, acylcarnitine levels were below baseline levels and remained low at 24 and 48 h. A partial least squares (PLS) model suggested a direct association of acylcarnitine accumulation in serum to APAP protein adduct and hepatic glutathione levels in mice. Overall, the kinetics of serum acylcarnitines in APAP toxicity in mice followed a biphasic pattern involving early elevation after the metabolism phases of toxicity and later depletion of acylcarnitines.

Targeted Liquid Chromatography–Mass Spectrometry Analysis of Serum Acylcarnitines in Acetaminophen Toxicity in Children

“…Targeted quantitative metabolite analysis was also performed using a previously published method [18]. Samples were removed from storage at −80°C and placed at −4°C for 2 h. A total of 50 µl of serum was spiked with isotope-labeled l-carnitine, acetyl-carnitine, myristoyl-carnitine, palmitoyl-carnitine and oleoyl-carnitine.…”

“…The α-oxidation of fatty acids is facilitated by transport of activated fatty acids into the mitochondria via carnitine. With disruption of mitochondrial function, long-chain fatty acids accumulate and can be detected in blood [17,18]. …”

“…We recently reported the elevation of long-chain acylcarnitines (palmitoyl-, oleoyl- and myristoyl-carnitines) in mice treated a high, nonfatal dose of APAP [18]. In order to examine the clinical relevance of data generated in animal models, the following study quantified acylcarnitines and other known indicators of APAP metabolism and toxicity in children with APAP poisoning.…”

Mitochondrial dysfunction as a mechanism of drug-induced hepatotoxicity: current understanding and future perspectives

“…In contrast, long-chain acylcarnitines, which have been suggested as markers of liver injury and mitochondrial dysfunctions [265], increased before ALT or AST activities after APAP overdose in mice [265–267]. However, acylcarnitine levels did not increase during furosemide toxicity despite the substantial cell necrosis [266].…”

Metabolomics: A Tool Ahead for Understanding Molecular Mechanisms of Drugs and Diseases

“…Two studies done by Bhattacharyya et al [11,12] found that in both animals as well as humans, elevations of long chain acylcarnitines can predict hepatotoxicity due to paracetamol before the transaminases get elevated. Since these metabolites are involved in the b-oxidation of fatty acids and since b-oxidation takes place in mitochondria, their elevations can mean mitochondrial toxicity.…”