Time course of acetaminophen-protein adducts and acetaminophen metabolites in circulation of overdose patients and in HepaRG cells

Abstract

1.?It has been suggested that acetaminophen (APAP)-protein adducts can be measured in circulation to diagnose APAP-induced liver injury. However, the full-time course of plasma adducts has not been studied specifically in early-presenting overdose patients. In fact, surprisingly little work has been done on the metabolism of APAP after overdose in general.

2.?We measured APAP, five APAP metabolites and APAP-protein adducts in plasma samples from early- and late-presenting overdose patients, and APAP-protein adducts in culture medium from HepaRG cells.

3.?In contrast to earlier rodents studies, we found that APAP-protein adducts were lower at early time points and peaked around the time of peak liver injury, suggesting that these adduct levels may take longer to become elevated or remain elevated than previously thought.

4.?APAP and its major metabolites were elevated in plasma at early time points and rapidly decreased.

5.?Although clinical measurement of APAP-protein adducts holds promise as a diagnostic tool, we suggest caution in its interpretation in very early-presenting patients. Our data also support the idea that sulfation is saturated even at low doses but glucuronidation has a much higher capacity, highlighting the importance of glucuronidation in APAP metabolism.     

一种岩藻糖基化硫酸软骨素三糖的合成

目的 通过化学合成的方法制备结构确证的FCS寡糖以研究其作用机制及构效关系。方法 基于天然海参中FCS结构,从单糖砌块开始,通过线性合成策略进行FCS三糖化合物的合成。结果 从功能化的单糖砌块开始,以12步反应23%的总收率制备了岩藻糖3,4位硫酸化的FCS三糖重复单元β-D-GalNAc（4,6-diS）（1-4）［α-L-Fuc（3,4-diS）（1-3）］-β-D-GlcA-OMP。结论 在合成中,建立了选择性O-硫酸化以及立体选择性糖基化的保护基策略,同时以高产率实现了各砌块间的偶联,为FCS类寡糖的合成研究提供了参考。化合物结构经1H NMR,13C NMR和HR-MS（ESI）表征。     

Sulfation of a high endothelial venule-expressed ligand for L-selectin. Effects on tethering and rolling of lymphocytes.

During lymphocyte homing, L-selectin mediates the tethering and rolling of lymphocytes on high endothelial venules (HEVs) in secondary lymphoid organs. The L-selectin ligands on HEV are a set of mucin-like glycoproteins, for which glycosylation-dependent cell adhesion molecule 1 (GlyCAM-1) is a candidate. Optimal binding in equilibrium measurements requires sulfation, sialylation, and fucosylation of ligands. Analysis of GlyCAM-1 has revealed two sulfation modifications (galactose [Gal]-6-sulfate and N-acetylglucosamine [GlcNAc]-6-sulfate) of sialyl Lewis x. Recently, three related sulfotransferases (keratan sulfate galactose-6-sulfotransferase [KSGal6ST], high endothelial cell N-acetylglucosamine-6-sulfotransferase [GlcNAc6ST], and human GlcNAc6ST) were cloned, which can generate Gal-6-sulfate and GlcNAc-6-sulfate in GlyCAM-1. Imparting these modifications to GlyCAM-1, together with appropriate fucosylation, yields enhanced rolling ligands for both peripheral blood lymphocytes and Jurkat cells in flow chamber assays as compared with those generated with exogenous fucosyltransferase. Either sulfation modification results in an increased number of tethered and rolling lymphocytes, a reduction in overall rolling velocity associated with more frequent pausing of the cells, and an enhanced resistance of rolling cells to detachment by shear. All of these effects are predicted to promote the overall efficiency of lymphocyte homing. In contrast, the rolling interactions of E-selectin transfectants with the same ligands are not affected by sulfation.     

百合多糖的化学结构表征和生物活性研究进展

百合是传统中医药中一味重要的滋阴药,具有悠久的用药历史,同时还是一味营养丰富的食材。多糖作为百合中一类重要的活性成分,具有免疫调节、抗肿瘤、抗氧化、降血糖等多重作用。通过对国内外百合多糖的研究报道进行系统整理,综述不同来源百合多糖的结构特征、结构修饰与生物学活性,为百合多糖的研究与开发提供参考与借鉴。     

Sulfated modification can enhance the adjuvanticity of lentinan and improve the immune effect of ND vaccine

The purpose of this research is to study the effect of sulfated lentinan (sLNT) on immune effect of Newcastle disease (ND) vaccine. In immune response test, 14-day-old chickens were vaccinated with ND vaccine then grouped respectively to inject three sLNTs at two doses, once a day for three successive days, taking non-sulfated lentinan (LNT) as the control. The changes of serum antibody titer and peripheral lymphocyte proliferation were determined before and after vaccination. In immune protection test, 35-day-old chickens were challenged with ND virus (NDV) after treated similar to above mentioned. The morbidity and mortality of chickens were observed, and the changes of serum antibody titer and peripheral lymphocyte proliferation were measured before and after challenge. The results showed that three modified sLNTs could significantly enhance serum antibody titer and promote lymphocyte proliferation in two experimental chickens, and reduce morbidity and mortality of chickens challenged with NDV, which were better than that of non-modified LNT. Their high doses in enhancing antibody titer and low doses in promoting lymphocyte proliferation were more preferable. These results indicated that sulfated modification could enhance the adjuvanticity of LNT and improve the immune effect of ND vaccine. sLNT₂ possessed the best efficacy and would be expected as the candidate of a new-type immune adjuvant.     

In‐vitro sulfation of piceatannol by human liver cytosol and recombinant sulfotransferases

Objectives The aim of this study was to investigate the concentration‐dependent sulfation of piceatannol, a dietary polyphenol present in grapes and wine and known for its promising anticancer and anti‐inflammatory activity. Methods Sulfation of piceatannol was investigated in human liver cytosol as well as using a panel of recombinant sulfotransferase isoforms. Furthermore, the chemical structures of novel sulfates were identified by liquid chromatography/mass spectrometry (LC/MS). Key findings In the presence of 3′‐phosphoadenosine‐5′‐phosphosulfate, three metabolites could be detected whose structures were identified by LC/MS/MS as piceatannol disulfate (M1) and two monosulfates (M2, M3). The kinetics of M1 formation exhibited a pattern of substrate inhibition with a K_i of 21.8 ± 11.3 μM and a V_max/K_m of 7.63 ± 1.80 μl/mg protein per min. Formation of M2 and M3 showed sigmoidal kinetics with apparent K_m and V_max values of 27.1 ± 2.90 μM and 118.4 ± 4.38 pmol/mg protein per min, respectively, for M2; and 35.7 ± 2.70 μM and 81.8 ± 2.77 pmol/mg protein per min, respectively, for M3. Incubation in the presence of human recombinant sulfotransferases (SULTs) demonstrated that M1 was formed equally by SULT1A1*1 and SULT1B1 and to a lesser extent by SULT1A1*2. M2 was preferentially catalysed by SULT1A1*2, 1A3 and 1E1. The formation of M3, however, was mainly catalysed by SULT1A2*1 and SULT1A3. Conclusions Our results elucidate the importance of piceatannol sulfation in human liver, which must be taken into account in humans after dietary intake of piceatannol.     

Tyrosine-sulfated glycopeptide involved in cellular proliferation and expansion in Arabidopsis

Posttranslational modification can confer special functions to peptides. Based on exhaustive liquid chromatography mass spectrometry analysis targeting tyrosine-sulfated peptides, we identified an 18-aa tyrosine-sulfated glycopeptide in Arabidopsis cell suspension culture medium. This peptide, which we named PSY1, significantly promotes cellular proliferation and expansion at nanomolar concentrations. PSY1 is widely expressed in various Arabidopsis tissues, including shoot apical meristem, and is highly up-regulated by wounding. Perception of PSY1 depends on At1g72300, which is a leucine-rich repeat receptor kinase (LRR-RK) whose two paralogs are involved in the perception of phytosulfokine (PSK), which is a 5-aa tyrosine-sulfated peptide that primarily promotes cellular proliferation. Multiple loss-of-function mutations in these three paralogous LRR-RKs significantly enhanced phenotypes, compared with single disruptants, suggesting that these LRR-RKs have overlapping functions. Triple mutations in these LRR-RKs resulted in dwarfism because of decreases in cell number and cell size and caused insufficiency in tissue repair after wounding. The present results suggest that this paralogous LRR-RK family integrates growth-promoting signals mediated by two structurally distinct sulfated peptides: PSY1 and PSK.     

Para-nitrophenol glucuronidation and sulfation in rat and human liver slices

Para-nitrophenol (PNP) is a well-known substrate for both phase I (hydroxylation at cytochrome P450) and phase II reactions (glucuronidation and sulfation). HPLC separation of PNP conjugates has already been described, but not for respective studies with liver slices, which nowadays have proven to be a suitable model for metabolic studies. Therefore we adapted an HPLC method for the simultaneous measurement of PNP glucuronidation (PNP-G) and sulfation (PNP-S) in this in vitro system. Both activities are substantially maintained over an incubation period of 24 h. PNP-G activity, however, seems to be better preserved, as indicated by stable values for PNP-G but reduced PNP-S values after 48 h liver slice preincubation. 24 h exposure of the slices to beta-naphthoflavone or phenobarbital does not change PNP-G or PNP-S activities.     

In vitro metabolism of TAK-438, vonoprazan fumarate,a novel potassium-competitive acid blocker

1.?TAK-438, vonoprazan fumarate, is a novel orally active potassium-competitive acid blocker, developed as an antisecretory drug. In this study, we investigated the in vitro metabolism of ¹⁴C-labeled TAK-438. In human hepatocytes, M-I, M-II, M-III and M-IV-Sul were mainly formed, and these were also detected in clinical studies. N-demethylated TAK-438 was also formed as an in vitro specific metabolite. Furthermore, CYP3A4 mainly contributed to the metabolism of TAK-438 to M-I, M-III, and N-demethylated TAK-438, and CYP2B6, CYP2C19 and CYP2D6 partly catalyzed the metabolism of TAK-438. The sulfate conjugation by SULT2A1 also contributed to the metabolism of TAK-438 to form TAK-438 N-sulfate, and CYP2C9 mediated the formation of M-IV-Sul from TAK-438 N-sulfate. The metabolite M-IV, which could be another possible intermediate in the formation of M-IV-Sul, was not observed as a primary metabolite of TAK-438 in any of the in vitro studies.

2.?In conclusion, TAK-438 was primarily metabolized by multiple metabolizing enzymes including CYP3A4, CYP2B6, CYP2C19, CYP2D6, and a non-CYP enzyme SULT2A1, and the influence of the CYP2C19 genotype status on gastric acid suppression post TAK-438 dosing could be small. The multiple metabolic pathways could also minimize the effects of co-administrated CYP inhibitors or inducers on the pharmacokinetics of TAK-438.     

Hepatic modeling of metabolite kinetics in sequential and parallel pathways: Salicylamide and gentisamide metabolism in perfused rat liver

Previous data on salicylamide (SAM) metabolism in the perfused rat liver had indicated that SAM was metabolized by three parallel (competing) pathways: sulfation, glucuronidation, and hydroxylation, whereas sequential metabolism of the hydroxylated metabolite, gentisamide (GAM), was solely via 5-glucuronidation to form GAM-5G. However, under comparable conditions, preformed GAM formed mainly two monosulfate conjugates at the 2- and 5-positions (GAM-2S and GAM-5S); 5-glucuronidation was a minor pathway. In the present study, the techniques of normal (N) and retrograde (R) rat liver perfusion with SAM and mathematic modeling on SAM and GAM metabolism were used to explore the role of enzymic distributions in determining the dissimilar fates of GAM, as a generated metabolite of SAM or as preformed GAM. Changes in the steady-state extraction ratio of SAM (E) and metabolite formation ratios between N and R perfusions were used as indices of the uneven distribution of enzyme activities. Two SAM concentrations (134 and 295 M) were used for single-pass perfusion: the lower SAM concentration exceeded the apparent K_m for SAM sulfation but was less than those for SAM glucuronidation and hydroxylation; the higher concentration exceeded the apparent K_m 's for SAM sulfation and glucuronidation but was less than the K_m for hydroxylation. Simulation of SAM metabolism data was carried out with various enzyme distribution patterns and extended to include GAM metabolism. At both input concentrations, E washigh (0.94 at 134 Mand 0.7 at 295 M) and unchanged during N and R, with SAM-sulfate (SAM-S) as the major metabolite and GAM-5G as the only detectable metabolite of GAM. Saturation of SAM sulfation occurred at the higher input SAM concentration as shown by a decrease in Eand a proportionally less increase in sulfation rates and proportionally more than expected increases in SAM hydroxylation and glucuronidation rates. At both SAM concentrations, the steady-state ratio of metabolite formation rates for SAM-S/SAM-G decreased when flow direction changed from N to R. An insignificant decrease in SAM-S/SAM-OH was observed at the low input SAM concentration, due to the small amount of SAM-OH formed and hence large variation in the ratio among the preparations, whereas at the high input SAM concentration, the decrease in SAM-S/SAM-OH with a change in flow direction from N to R was evident. The metabolite formation ratio, SAM-G/SAM-OH, however, was unchanged at both input concentrations and flow directions. The observed data suggest an anterior SAM sulfation system in relation to the glucuronidation and hydroxylation systems, which are distributed similarly. When the observations were compared to predictions from the enzyme-distributed models, the best prediction on SAM metabolism was given by a model which described sulfation activities anteriorly, glucuronidation activities evenly, and hydroxylation activities posteriorly (perivenous). When the model was used to predict data for SAM and GAM metabolism in once-through perfused rat livers at different input SAM concentrations, in the absence or presence of the sulfation inhibitor, 2,6-dichloro-4-nitrophenol (DCNP), the predictions were in close agreement with previously observed SAM data but failed to predict the exclusive formation of GAM-SG; rather, GAM-2S and GAM-5S were predicted as major sequential metabolites of SAM. The poor correlation for GAM metabolic data may be explained on the basis of subcellular enzyme localizations: the cytochromes P-450 and UDP-glucuronyltransferases, being membrane-bound enzymes, are more coupled for GAM formation and glucuronidation, when GAM was generated intracellularly. The present study suggests that subcompartmentalization of enzymes may need to be considered in hepatic modeling for better prediction of metabolic events.This work was presented in part at the ASPET meeting, Montreal, 1988, and was conducted in partial fulfilment of Xin Xu's Ph.D. thesis. This work was supported by the Medical Research Council of Canada and a grant from the Canadian Liver Foundation.