Abstract
Glutamate dehydrogenase (GLDH) is a liver-specific biomarker of hepatocellular damage currently undergoing qualification as a drug development tool. Since GLDH is located within the mitochondrial matrix, it has been hypothesized that it might also be useful in assessing mitotoxicity as an initiating event during drug-induced liver injury. According to this hypothesis, hepatocyte death that does not involve primary mitochondrial injury would result in release of intact mitochondria into circulation that could be removed by high speed centrifugation and result in lower GLDH activity measured in spun serum vs un-spun serum. A single prior study in mice has provided some support for this hypothesis. We sought to repeat and extend the findings of this study. Accordingly, mice were treated with the known mitochondrial toxicant, acetaminophen (APAP), or with furosemide (FS), a toxicant believed to cause hepatocyte death through mechanisms not involving mitotoxicity as initiating event. We measured GLDH levels in fresh plasma before and after high speed centrifugation to remove intact mitochondria. We found that both APAP and FS treatments caused substantial hepatocellular necrosis that correlated with plasma alanine aminotransferase (ALT) and GLDH elevations. The plasma GLDH activity in both the APAP- and FS- treated mice was not affected by high-speed centrifugation. Interestingly, the ratio of GLDH:ALT was 5-fold lower during FS compared to APAP hepatotoxicity. Electron microscopy confirmed that both APAP- and FS-treatments had resulted in mitochondrial injury. Mitochondria within vesicles were only observed in the FS-treated mice raising the possibility that mitophagy might account for reduced release of GLDH in the FS-treated mice. Although our results show that plasma GLDH is not clinically useful for evaluating mitotoxicity, the GLDH:ALT ratio as a measure of mitophagy needs to be further studied.
Highlights
Drug-induced liver injury (DILI) is a serious concern for pharmaceutical companies and clinicians alike, representing the number one cause of acute liver failure in the United States [1]
As done by McGill et al, we compared Glutamate dehydrogenase (GLDH) release following hepatotoxicity induced by APAP, a model hepatotoxicant that causes primary mitochondrial dysfunction with hepatotoxicity induced by FS, which is believed to occur without inducing primary mitochondrial injury [10]
To exclude the possibility that GLDH contained within intact mitochondria is not measured by the assay, we repeated our assays after three freeze/thaw cycles that should disintegrate mitochondria (Fig 1)
Summary
Drug-induced liver injury (DILI) is a serious concern for pharmaceutical companies and clinicians alike, representing the number one cause of acute liver failure in the United States [1]. Because GLDH has a shorter half-life in circulation compared to ALT in humans, this biomarker may give a more accurate reflection of the concurrent liver injury [7,8,9]. GLDH has shown value as a prognostic biomarker of outcome in the clinic following acetaminophen (APAP) overdose [13]. Based on these and other observations, a Qualification Plan for GLDH was accepted by the FDA in 2020, encouraging submission of a Full Qualification Package for GLDH use as a biomarker of liver injury in patients with underlying muscle injury [15]
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