RETRACTED: Molecular forms of HMGB1 and keratin-18 as mechanistic biomarkers for mode of cell death and prognosis during clinical acetaminophen hepatotoxicity

Abstract

Background & Aims Full length keratin-18 (FL-K18) and High Mobility Group Box-1 (HMGB1) represent circulating indicators of necrosis during acetaminophen (APAP) hepatotoxicity in vivo. In addition, the caspase-cleaved fragment of K18 (cK18) and hyper-acetylated HMGB1 represent serum indicators of apoptosis and immune cell activation respectively. The study aim was to assess their mechanistic utility to establish the balance between apoptosis, necrosis and immune cell activation throughout the time course of clinical APAP hepatotoxicity. Methods HMGB1 (total, acetylated) and K18 (apoptotic, necrotic) were identified and quantified by novel LC-MS/MS assays in APAP overdose patients (n=78). Results HMGB1 (total; 15.4±1.9ng/ml, p<0.01, acetylated; 5.4±2.6ng/ml, p<0.001), cK18 (5649.8±721.0U/l, p<0.01) and FL-K18 (54770.2±6717.0U/l, p<0.005) were elevated in the sera of APAP overdose patients with liver injury compared to overdose patients without liver injury and healthy volunteers. HMGB1 and FL-K18 correlated with alanine aminotransferase (ALT) activity (R2=0.60 and 0.58 respectively, p<0.0001) and prothrombin time (R2=0.62 and 0.71 respectively, p<0.0001). Increased total and acetylated HMGB1 and FL-K18 were associated with worse prognosis (King’s College Criteria) or patients that died/required liver transplant compared to spontaneous survivors (all p<0.05-0.001), a finding not reflected by ALT and supported by ROC analysis. Acetylated HMGB1 was a better predictor of outcome than the other markers of cell death. Conclusion K18 and HMGB1 represent blood-based tools to investigate the cell death balance clinical APAP hepatotoxicity. Activation of the immune response was seen later in the time course as shown by the distinct profile of acetylated HMGB1 and was associated with worse outcome.

Refining Liver Safety Risk Assessment: Application of Mechanistic Modeling and Serum Biomarkers to Cimaglermin Alfa (GGF2) Clinical Trials

“…The time course of the elevations was similar to that of miR‐122 and appeared to be returning to baseline levels at the final measurement. The absolute values for the increase in both M30 (cc‐K18) and M65 (cc‐K18 and FL‐K18) were higher than previously reported in healthy adult volunteers receiving recurrent therapeutic doses of acetaminophen12 but were significantly lower than those observed in patients with serious DILI due to acetaminophen overdose 15. The mathematical ratio of M30 to M65 is termed the “apoptotic index” and has been recently proposed to estimate the relative proportions of cell death by apoptosis and necrosis 4.…”

“…The Plasma compartment represents systemic circulation. Creation of the Intermediate compartment was required to optimize serum ALT dynamics observed in clinical response to several hepatotoxic compounds15, 23, 24, 25 and may represent the ALT in the interstitial space, the Space of Disse, or the liver sinusoids. The clearance of ALT is driven by a specified half‐life 26.…”

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

“…Other biomarkers such as miR-122 [244–247], full-length cytokeratin-18 [247–250], high mobility group box 1 (HMGB1) protein [247–249, 251], and argininosuccinate synthetase (ASS) [252] are generally considered indicators of mainly necrotic cell death [253]. On the other hand, increased plasma levels of the caspase-cleaved form of cytokeratin-18 [247, 248, 250, 254, 255] and activated caspases detected as enzyme activity and/or the protein by western blotting [164, 247, 249, 254], are markers of apoptotic cell death. However, due to the sensitivity of the assay, cleaved cytokeratin-18 levels should always be compared to the full-length version in order to assess the relative importance of apoptosis [247, 248, 254].…”

“…On the other hand, increased plasma levels of the caspase-cleaved form of cytokeratin-18 [247, 248, 250, 254, 255] and activated caspases detected as enzyme activity and/or the protein by western blotting [164, 247, 249, 254], are markers of apoptotic cell death. However, due to the sensitivity of the assay, cleaved cytokeratin-18 levels should always be compared to the full-length version in order to assess the relative importance of apoptosis [247, 248, 254]. The clinical utility of some of these biomarkers may be that they are highly specific for hepatocyte cell death, e.g.…”

“…miR-122, and that they are even more sensitive indicators of cell injury than the routine parameters of liver cell death plasma ALT and AST activities [252, 256]. The enhanced sensitivity is not only reflected in an earlier increase in the plasma levels of these biomarkers but also in a more rapid decline [248, 252], which makes most of these compounds more accurate indicators of the time course of acute cell death compared to ALT and AST [253]. Another use of biomarkers such as plasma miRNA profiles can be used to distinguish between clinical conditions that are very similar such as acute liver injury after an APAP overdose and hypoxic hepatitis [257].…”