Identification of Serum Biomarkers to Distinguish Hazardous and Benign Aminotransferase Elevations

Abstract

The standard circulating biomarker of liver injury in both clinical settings and drug safety testing is alanine aminotransferase (ALT). However, ALT elevations sometimes lack specificity for tissue damage. To identify novel serum biomarkers with greater specificity for injury, we combined unique animal models with untargeted proteomics, followed by confirmation with immunoblotting. Using proteomics, we identified 109 proteins in serum from mice with acetaminophen (APAP)-induced liver injury that were not detectable in serum from mice with benign ALT elevations due to high-dose dexamethasone (Dex). We selected 4 (alcohol dehydrogenase 1A1 [Aldh1a1], aldehyde dehydrogenase 1 [Adh1], argininosuccinate synthetase 1 [Ass1], and adenosylhomocysteinase [Ahcy]) with high levels for further evaluation. Importantly, all 4 were specific for injury when using immunoblots to compare serum from Dex-treated mice and mice with similar lower ALT elevations due to milder models of APAP or bromobenzene-induced liver injury. Immunoblotting for ALDH1A1, ADH1, and ASS1 in serum from APAP overdose patients without liver injury and APAP overdose patients with mild liver injury revealed that these candidate biomarkers can be detected in humans with moderate liver injury as well. Interestingly, further experiments with serum from rats with bile duct ligation-induced liver disease indicated that Aldh1a1 and Adh1 are not detectable in serum in cholestasis and may therefore be specific for hepatocellular injury and possibly even drug-induced liver injury, in particular. Overall, our results strongly indicate that ALDH1A1, ADH1, and ASS1 are promising specific biomarkers for liver injury. Adoption of these biomarkers could improve preapproval drug safety assessment.

Keywords: drug safety; drug-induced liver injury; hepatotoxicity; regulatory science; transaminitis.

Figure 1.

Dexamethasone (Dex) increased serum alanine aminotransferase (ALT). Mice were treated i.p. with 100 mg/kg Dex or DMSO vehicle (DMSO). Blood and tissue were collected 24 h later. A, Serum ALT activity. B, H&E-stained liver sections. C, Serum creatine kinase (CK) activity. D, Correlation between ALT and CK in serum from Dex-treated mice. E, H&E-stained muscle sections. Data expressed as mean ± SE for n = 9–10 mice per group. *p < .05 versus DMSO.

Figure 2.

Dexamethasone (Dex) increased hepatic alanine aminotransferase (ALT). Mice were treated i.p. with 100 mg/kg Dex or DMSO vehicle (DMSO). Liver tissue was collected 24 h later. A, Gpt1 mRNA in liver. B, Gpt2 mRNA in liver. C, Immunoblot for ALT isoforms in liver tissue. D, Densitometry from immunoblot. Data expressed as mean ± SE for n = 5 mice per group for mRNA and n = 3–4 per group mice for immunoblot. *p < .05 versus DMSO.

Figure 3.

Proteomics revealed 109 candidate biomarkers that were only in serum from acetaminophen (APAP) overdose mice. Mice were treated i.p. with 100 mg/kg dexamethasone (Dex), 250 mg/kg APAP, DMSO vehicle, or phosphate-buffered saline (PBS) vehicle. Blood was collected 24 h later (Dex, DMSO) or 6 h later (APAP, PBS). Serum aliquots were used for proteomics. n = 5 mice per group.

Figure 4.

Aldh1A1 and Adh1 are specific for liver injury in mice. A, Fed mice were treated i.p. with 100 mg/kg dexamethasone (Dex), 400 mg/kg acetaminophen (APAP), or DMSO vehicle, and blood was collected 24 h later. Fasted mice were treated with 175 mg/kg APAP or phosphate-buffered saline (PBS) vehicle, and blood was collected 6 h later. Serum aliquots were used for immunoblotting for Aldh1a1, Adh1, Ass1, and Ahcy. B, Representative H&E-stained liver sections from the APAP-treated mice showing central veins with characteristic features of necrosis (swelling, karyolysis, karyorrhexis, and eosinophilia) of many surrounding hepatocytes. C, Fed mice were treated i.p. with 1.1 g/kg bromobenzene or vehicle. Blood was collected 24 h later. Serum aliquots were used for immunoblotting for Aldh1a1 and Adh1. Serum ALT values for each animal are displayed above the blots.

Figure 5.

ALDH1A1, ADH1, and ASS1 may be specific for liver injury in humans. Immunoblotting for ALDH1A1, ADH1, ASS1, and AHCY was done using aliquots of serum or plasma collected from acetaminophen (APAP) overdose patients without liver injury (peak ALT < 100 U/l) and with modest liver injury (peak ALT ≥ 100 U/l). Circulating ALT values for each subject or patient in the tested specimen are displayed above the blots.

Figure 6.

Time course of Aldh1a1 and Adh1 in serum from mice after acute injury. Fasted mice were treated i.p. with 250 mg/kg acetaminophen (APAP) or phosphate-buffered saline (PBS) vehicle. Blood was collected 6, 24, and 48 h later. A, Immunoblotting was done to detect Aldh1a1 and Adh1 in serum. Total serum protein is also displayed. B, Densitometry from immunoblotting. Data expressed as mean ± SE for n = 3 mice per group.

Figure 7.

Ald1a1 and Adh1 do not significantly increase in a rat model of cholestasis. Rats were subjected to either bile duct ligation (BDL) or sham surgery (Sham), then allowed to recover. Blood was collected 21 days after surgery. A, Serum ALT activity. B, H&E-stained liver sections. C, Immunoblots for Aldh1a1 and Adh1in serum. D, Detection of both mouse (M) and rat (R) Aldh1a1 and Adh1 using the same antibody. Total protein is also displayed. Data expressed as mean ± SE for n = 3 rats per group. *p < .05 versus Sham.