Study On Inhibition Of UGT1A1-Mediated Bilirubin Metabolism By Traditional Chinese Medicine Injection

Traditional Chinese medicine (TCM) injection is widely used in clinical practice. Recent years, clinical case reports on TCM injection leading to bilirubin related adverse reactions (e.g. jaundice) have been emerging successively. Unfortunately, these literatures or reports only remained at the level of case reports without conducting the experimental research and providing clear clarification of the cause and mechanism of bilirubin toxicity.Bilirubin, an essential endogenous substance in human body, was metabolized mainly via the liver. Unconjugated bilirubin (UCB) is the main form of bilirubin in plasma. After hepatic uptake, UCB is mainly metabolized selectively to hydrosoluble bilirubin glucuronides (BG) by UGT1A1 which located at the smooth endoplasmic reticulum, subsequently BG is secreted into the bile. When UGT1A1 was inhibited by xenobiotics (e.g.drugs), it is difficult for UCB to be metabolized to BG. It results in the concertation increasing of UCB in plasma and metabolic disorder of bilirubin, further leads to bilirubin related adverse reactions (e.g. jaundice, hyperbilirubinemia and kernicterus), even causes the death of the patients.Many TCM injections contain flavonoids or phenolic acids. These components have glucuronidation groups (e.g. hydroxy or carboxy), therefore they are the potential substrates and/or inhibitors of UGT1A1 and can cause metabolic disorder of bilirubin. Consequently, we investigated the inhibition effect of TCM injection and its components on bilirubin metabolism and reveal the mechanism of bilirubin poisoning from the perspective of UGT1A1 regulation.Objective(1) To establish a roust HPLC method for the determination of bilirubin (UCB) and its metabolites bilirubin monoglucuronides (BMGs) and bilirubin diglucuronide (BDG) in rat liver microsomes (RLM), human liver microsomes (HLM) and recombinant human UGT1A1 enzyme (UGT1A1) incubation mixture.(2) To establish the in vitro incubation system of bilirubin glucuronidation, and reveal its enzyme kinetic mechanism.(3) To investigate the inhibition effect of TCM injection and its components on bilirubin glucuronidation, and disclose the cause and mechanism of TCM injection leading to bilirubin-related adverse reactions (e.g. jaundice).Methods(1) An HPLC method for the determination of UCB and its three glucuronides in the three in vitro incubation system. Chromatographic condition:a Diamonsil C18 (2) column (5 μm,200 mm×4.6 mm) was used. The mobile phase consisted of 0.1% formic acid in water (B) and 100% acetonitrile (A) at a flow rate of 1 ml/min. A linear gradient elution program:mobile phase A,0-9 min,60-25%; 9-18 min,25-5%; 18-26 min,5%; 26-28 min,5-60%; 28-30 min,60%.The UV detection was at 450nm and the column temperature was 45 ℃. The method validation included the specificity, sensitivity, precision, accuracy, extraction recovery and stability, and so on.(2) Method of bilirubin glucuronidation.UGT1A1 (HLM or RLM, final protein concentration 12.5 μg/ml), potassium phosphate buffer (50 mM), UCB (0.2-2uM), MgCl2 (0.88 mM), alamethicin (0.022 mg/ml), Uridine 5’-diphosphoglucuronic acid (UDPGA,3.5 mM, finally added) were mixed in order. The mixture (200 μL) was incubated in a 37℃ shaking water bath for 15 min. Then the reaction was terminated by the addition of 600 μL ice-cold methanol containing 200 mM ascorbic acid.(3) Method of inhibition of bilirubin glucuronidation. Based on the method of bilirubin glucuronidation, UCB and TCM injection (or its components) were co-incubated at the same time. The inhibition kinetics parameters were calculated, respectively.Results(1) The calibration curve of bilirubin showed good linearity in the range of 0.01-2 (J.M. The limit of quantification and the limit of detection were 0.01 μM and 0.005 μM, respectively. The RSD (%) of the intra-and inter-precision was less than 3.7% (n=5) and accuracy was in the range of 92.8%～104.3% (n=5). And the extraction recoveries> 92.3%(n=5).UCB and its glucuronides were stable under 37℃, room temperature or-70℃.(2) Bilirubin glucuronidation obeyed Michaelis-Menten equation in the range of 0.2～2μM UCB. The kinetic parameters of TBG were as followed:Km of 0.80±0.10, 0.37±0.02,0.44±0.02 μM, Vmax of 3.67±0.19,1.82±0.03,2.95±0.04 nmol/mg/min, and CLint of 4.58±0.26、4.98±0.01、6.72±0.27 ml/mg/min in the RLM, HLM, UGT1A1 incubation system,respectively.(3) Ginkgo Biloba extract injection, danshen injection, xiangdan injection, shuanghuanglian for injection, shengmai injection, compound angelica injection, shenmai injection, breviscapine for injection, xuesaitong injection, yinzhihuang injection and qingkailing injection all displayed strong inhibitory effect on UGT1A1-mediated bilirubin glucuronidation, IC50 value (calculated by the component of high content and strong inhibitory effect in the injection) of these TCM injection was 0.31,0.45,0.59,22.14,5.61,6.05,11.35,16.10,20.23,24.66,42.90μM, respectively. The above TCM injections contain one or more components (e.g. quercetin, kaempferol, isorhamnetin, salvianolic acid A, salvianolic acid B, baicalin, baicalein, ginsenoside Rb1 and so on)exhibiting strong inhibitory effect on bilirubin glucuronidation.ConclusionThe HPLC method is simple, accurate, specific, sensitive, and the in vitro incubation system is stable and reliable, they can be used to study bilirubin metabolism and its regulation. The kinetics of bilirubin glucuronidation obeyed the Michaelis-Menten equation and showed species differences.TCM injections containing flavonoids, phenolic acids or saponins are the potential substrates and/or inhibitors of UGT1A1 and showed quite strong inhibitory effect on bilirubin glucuronidation, which will cause metabolic disorder of bilirubin and lead to bilirubin-related adverse reactions.