Acute high-dose diazepam reduces total bilirubin in bile: an experimental mouse study

Acute drug intoxication may impair hepatic conjugation before overt liver failure. This study examined potential conjugation disturbances by assessing changes in serum and biliary bilirubin together with vital signs after an acute high dose of diazepam in mice. Male ICR mice (n = 9 per group) received an oral dose of diazepam (720 mg/kg) or vehicle (0.5% carboxymethylcellulose sodium) and were monitored for 120 min. Pulse rate, peripheral capillary oxygen saturation (SpO₂), and respiratory rate were recorded every 5 min using a pulse oximeter. At 120 min, the mice were euthanized by cervical dislocation, and blood and bile were collected. Total bilirubin concentrations in serum and bile were measured using a colorimetric assay with a lower limit of quantification of 0.16 mg/dL. Diazepam-treated mice showed a greater decline in pulse rate than vehicle-treated mice, whereas SpO₂ and respiratory rate showed no significant between-group differences. Serum total bilirubin remained below the lower limit of quantification in all animals. By contrast, biliary total bilirubin concentrations were significantly lower in diazepam-treated mice than in vehicle controls (9.16 ± 0.76 vs. 5.31 ± 0.80 mg/dL, p = 0.01), despite comparable bile weight between groups. Acute high-dose diazepam selectively reduced biliary total bilirubin without marked hypoxemia or respiratory depression. These findings suggest that biliary bilirubin content may serve as a candidate marker of altered hepatic conjugation during acute diazepam intoxication and warrant further evaluation in analyses of human autopsy bile in forensic toxicology.