Circulating acylcarnitines as biomarkers of mitochondrial dysfunction after acetaminophen overdose in mice and humans

Abstract

Acetaminophen (APAP) is a widely used analgesic. However, APAP overdose is hepatotoxic and is the primary cause of acute liver failure in the developed world. The mechanism of APAP-induced liver injury begins with protein binding and involves mitochondrial dysfunction and oxidative stress. Recent efforts to discover blood biomarkers of mitochondrial damage have identified increased plasma glutamate dehydrogenase activity and mitochondrial DNA concentration in APAP overdose patients. However, a problem with these markers is that they are too large to be released from cells without cell death or loss of membrane integrity. Metabolomic studies are more likely to reveal biomarkers that are useful at early time points, before injury begins. Similar to earlier work, our metabolomic studies revealed that acylcarnitines are elevated in serum from mice after treatment with toxic doses of APAP. Importantly, a comparison with furosemide demonstrated that increased serum acylcarnitines are specific for mitochondrial dysfunction. However, when we measured these compounds in plasma from humans with liver injury after APAP overdose, we could not detect any significant differences from control groups. Further experiments with mice showed that N-acetylcysteine, the antidote for APAP overdose in humans, can reduce acylcarnitine levels in serum. Altogether, our data do not support the clinical measurement of acylcarnitines in blood after APAP overdose due to the standard N-acetylcysteine treatment in patients, but strongly suggest that acylcarnitines would be useful mechanistic biomarkers in other forms of liver injury involving mitochondrial dysfunction.

Figure 1. Acetaminophen-induced liver injury in mice

Fasted or fed mice were treated with 300 or 600 mg/kg APAP, respectively, for various times. (A–B) ALT activity was measured in serum. Data are expressed as mean ± SEM of n = 3. *P<0.05 (compared to t=0 h) (C) Hematoxylin and eosin (H&E) staining and TUNEL labeling were performed with liver sections from these animals. Representative sections (×100 or ×200) are shown.

Figure 2. Metabolomic analysis of serum from mice with acetaminophen-induced liver injury

Score plots (A–B) and S-plots (C–D) for fasted mice treated with 300 mg/kg APAP (A,C) or fed mice treated with 600 mg/kg APAP (B,D). Ions from acylcarnitines are shown by the arrows, with their respective molecular weights. In the Score plots (A,B), the x-axis shows differences in total ion profiles between groups while the y-axis shows differences between individual mice. In the S-plots (C,D), the x-axis indicates changes in ion average concentration after APAP treatment, while the y-axis indicates variance confidence (changes in ion concentration farther from 0 have less variation within groups).

Figure 3. Serum acylcarnitines during acetaminophen-induced liver injury in mice

Fasted or fed mice were treated with 300 or 600 mg/kg APAP, respectively, for various times. (A) Acylcarnitine levels in serum from fasted mice after APAP treatment. The control concentrations were 200 ± 20, 139 ± 19 and 467 ± 35 nM for palmitoylcarnitine (Palm), linoleoylcarnitine (Linoleo) and oleoylcarnitine (Oleo), respectively. (B) Acylcarnitine levels in serum from fed mice after APAP treatment. The control concentrations were 72 ± 2, 29 ± 1 and 166 ± 12 nM for Palm, Linoleo, and Oleo, respectively. Data are expressed as mean ± SEM of n = 3. *P < 0.05 (compared to t=0 h).

Figure 4. Furosemide-induced liver injury and serum acylcarnitines

Fed mice were treated with 500 mg/kg furosemide for various times. (A) ALT was measured in serum from these animals. (B) Acylcarnitine levels were also measured in serum. Control concentrations were 761 ± 82, 409 ± 13 and 2,805 ± 194 nM for palmitoylcarnitine, linoleoylcarnitine and oleoylcarnitine, respectively. (C) Hematoxylin and eosin-stained liver sections from these mice (100×, except where indicated). Data are expressed as mean ± SEM of n = 3. *P < 0.05 (compared to t=0 h).

Figure 5. Plasma ALT and acylcarnitines in samples from APAP overdose patients

(A) Average peak ALT values for the patients. (B–C) Acylcarnitines were measured in plasma from APAP overdose patients at the first blood draw after admission and study enrollment and at the time of peak liver injury, as indicated by ALT. (D) Average ALT and lineoylcarnitine levels in plasma from 6 overdose patients in the abnormal LT group. In panels A–C, data are expressed as mean ± SEM for 6 healthy volunteers, 14 normal liver test result (LT) overdose patients, and 16 abnormal LT overdose patients. *P<0.05 (compared to Vol.).

Figure 6. Effect of N-acetylcysteine on serum acylcarnitines during APAP-induced liver injury

Mice were treated with 140 mg/kg NAC 1.5 h after 300 mg/kg APAP. Acylcarnitines were measured 3 h post-APAP. Control concentrations were 387 ± 69, 244 ± 40 and 1,855 ± 258 nM for palmitoylcarnitine (Palm), linoleoylcarnitine (Linoleo) and oleoylcarnitine (Oleo), respectively. Data are expressed as mean ± SEM of n = 3. *P < 0.05 (compared to CTRL); ^#P < 0.05 (compared to 3 h APAP).