Protocol for the study of hepatic bilirubin uptake in the isolated perfused rat liver

We present a protocol for the study of bilirubin uptake in the isolated and perfused rat liver. The liver is perfused with an albumin-free saline buffered solution supplemented with glucose, pyruvate and lactate, in the absence of oxygen, at a physiologically low flow rate. Fractions of the venous effluent are collected and analyzed for bilirubin, bilirubin glucuronide and biomarkers of liver integrity. The liver preparation is viable and intact for 1 h after isolation from the general circulation, with constant levels of both bilirubin and bilirubin glucuronide (< 2 nM) in the effluent. Up to 12 boli of 10 nmol bilirubin can be sequentially injected into the portal vein without and with molecules that target sinusoidal membrane transporters of organic anions. Selective inhibition of bilirubin or bilirubin glucuronide uptake is detected as transient peaks in the effluent (Cmax up 6 to 60 nM). This protocol allows collecting repeated observations in the same liver, thus reducing the animal number by a factor of 10.

Investigation of MDMA Inhibitory Effect on CytochromeP450 3A4 in Isolated Perfused Rat Liver Model Using Tramadol

Purpose: MDMA (methylenedioxymethamphetamine) is a synthetic compound, which is a structurally derivative of amphetamine. Also, it acts like an amphetamine, structurally, and functionally. MDMA uses mechanism-based inhibition, to inhibit isoenzyme CYP2D6. It can also inhibit other isoenzymes contributing to its metabolism, including CYP3A4 which is the most important member of the cytochrome P450 superfamily. Since more than 50% of drugs are metabolized by CYP3A4, its inhibition may cause harmful and even lethal drug interactions. Tramadol, as an opioid-like analgesic, is mainly metabolized into O-desmethyl tramadol (M1), by CYP2D6 and undergoes N-demethylation to M2, by CYP2B6 and CYP3A4. Due to the significant potential of abusing tramadol, either alone or in combination with MDMA, the rate of its toxicity and side effects may increase following possible MDMA relevant enzyme inhibition. Methods: Different doses of MDMA (1-10 mg/Kg) were intraperitoneally administered to Wistar male rats of both control and treatment groups. Then, after one hour, their isolated livers were perfused by perfusion buffer containing tramadol (1 µg/mL). Afterward, perfusate samples were collected. They were analyzed by HPLC to determine the concentrations of tramadol and its metabolites. Results: MDMA administration in treatment groups reduced M1 production. On the other hand, by following the treatment with different MDMA doses, the M2 metabolic ratio increased by 46 to 101%. Conclusion: it seems that the regular doses of MDMA cannot inhibit the CYP3A4 activity.

Heavy Water (D2O) Containing Preservation Solution Reduces Hepatic Cold Preservation and Reperfusion Injury in an Isolated Perfused Rat Liver (IPRL) Model

Background: Heavy water (D2O) has many biological effects due to the isotope effect of deuterium. We previously reported the efficacy of D2O containing solution (Dsol) in the cold preservation of rat hearts. Here, we evaluated whether Dsol reduced hepatic cold preservation and reperfusion injury. Methods: Rat livers were subjected to 48-hour cold storage in University of Wisconsin (UW) solution or Dsol, and subsequently reperfused on an isolated perfused rat liver. Graft function, injury, perfusion kinetics, oxidative stress, and cytoskeletal integrity were assessed. Results: In the UW group, severe ischemia and reperfusion injury (IRI) was shown by histopathology, higher liver enzymes leakage, portal resistance, and apoptotic index, oxygen consumption, less bile production, energy charge, and reduced glutathione (GSH)/oxidized glutathione (GSSG) ratio (versus control). The Dsol group showed that these injuries were significantly ameliorated (versus the UW group). Furthermore, cytoskeletal derangement was progressed in the UW group, as shown by less degradation of α-Fodrin and by the inactivation of the actin depolymerization pathway, whereas these changes were significantly suppressed in the Dsol group. Conclusion: Dsol reduced hepatic IRI after extended cold preservation and subsequent reperfusion. The protection was primarily due to the maintenance of mitochondrial function, cytoskeletal integrity, leading to limiting oxidative stress, apoptosis, and necrosis pathways.