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A simple, low‐cost method for preparing glucuronic acid‐conjugated metabolites was developed using fentanyl, a potent synthetic opioid, as a model drug. Five glucuronic acid‐conjugated metabolites of fentanyl were measured in the culture medium of fresh human hepatocytes incubated with fentanyl. These glucuronides were also formed by incubation of their corresponding substrates (e.g., 4′‐hydroxy‐fentanyl and β‐hydroxy‐fentanyl) with uridine 5′‐diphosphoglucuronic acid and human liver microsomes (HLM). Experiments using liver microsomes of several animals revealed that significant species differences exist in the glucuronide formation patterns; fentanyl glucuronide was only formed in HLM, and 4′‐hydroxy‐fentanyl glucuronide was formed much more in rat liver microsomes (RLM) than HLM and dog liver microsomes. Furthermore, surprisingly, HLM and RLM showed opposite substrate selectivity for the enantiomers of β‐hydroxy‐fentanyl. Submilligram amounts of three of these metabolites, namely, 4′‐hydroxy‐fentanyl glucuronide and two glucuronides of β‐hydroxy‐fentanyl, were prepared by using HLM or RLM. The products were readily purified with a reversed‐phase/anion‐exchange mixed‐mode solid‐phase extraction cartridge, and then, their chemical structures were confirmed by 1D/2D nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry data. In addition, the products were quantitated by quantitative NMR, and the yields were 3.6–69%. [ABSTRACT FROM AUTHOR]

Background: Glucuronidation is an essential metabolic pathway for a variety of drugs. IMM-H004 is a novel neuroprotective agent against ischemic stroke, and its glucuronide metabolite IMM-H004G exhibits similar pharmacological activity. Despite possessing a higher molecular weight and polarity, brain exposure of IMM-H004G is much higher than that of IMM-H004. This study aimed to investigate the brain metabolism and transport mechanisms of IMM-H004 and IMM-H004G. Methods: First, the possibility of IMM-H004 glucuronidation in the brain was evaluated in several human brain cell lines and rat homogenate. Subsequently, the blood–brain barrier carrier-mediated transport mechanism of IMM-H004 and IMM-H004G was studied using overexpression cell models. In addition, intracerebroventricular injection, in situ brain perfusion model, and microdialysis/microinjection techniques were performed to study the distribution profiles of IMM-H004 and IMM-H004G. Results: IMM-H004 could be metabolized to IMM-H004G in both rat brain and HEB cells mediated by UGT1A7. However, IMM-H004G could not be hydrolyzed back into IMM-H004. Furthermore, the entry and efflux of IMM-H004 in the brain were mediated by the pyrilamine-sensitive H+/OC antiporter and P-gp, respectively, while the transport of IMM-H004G from the blood to the brain was facilitated by OATP1A2 and OATP2B1. Ultimately, stronger concentration gradients and OATP-mediated uptake played a critical role in promoting greater brain exposure of IMM-H004G. Conclusions: The active glucuronide metabolite of the brain protectant IMM-H004 with poor blood–brain barrier permeability demonstrates a high partition in the rat brain via multiple mechanisms, and our findings deepen the understanding of the mechanisms underlying the blood–brain barrier metabolism and transport of active glucuronide conjugates. [ABSTRACT FROM AUTHOR]

AbstractThis critical review examines evidence for beneficial effects of quercetin phase-2 conjugates from clinical intervention studies, volunteer feeding trials, and in vitro work. Plasma concentrations of quercetin-3-O-glucuronide (Q3G) and 3′-methylquercetin-3-O-glucuronide (3′MQ3G) after supplementation may produce beneficial effects in macrophages and endothelial cells, respectively, especially if endogenous deglucuronidation occurs, and lower blood uric acid concentration via quercetin-3′-O-sulfate (Q3′S). Unsupplemented diets produce much lower concentrations (<50 nmol/l) rarely investigated in vitro. At 10 nmol/l, Q3′S and Q3G stimulate or suppress, respectively, angiogenesis in endothelial cells. Statistically significant effects have been reported at 100 nmol/l in breast cancer cells (Q3G), primary neuron cultures (Q3G), lymphocytes (Q3G and3′MQ3G) and HUVECs (QG/QS mixture), but it is unclear whether these translate to a health benefit in vivo. More sensitive and more precise methods to measure clinically significant endpoints are required before a conclusion can be drawn regarding effects at normal dietary concentrations. Future requirements include better understanding of inter-individual and temporal variation in plasma quercetin phase-2 conjugates, their mechanisms of action including deglucuronidation and desulfation both in vitro and in vivo, tissue accumulation and washout, as well as potential for synergy or antagonism with other quercetin metabolites and metabolites of other dietary phytochemicals. [ABSTRACT FROM AUTHOR]

Baicalin is a biologically active flavone glucuronide with poor water solubility that can be enhanced via glucosylation. In this study, the transglucosylation of baicalin was successfully achieved with CGTases from Thermoanaerobacter sp. and Bacillus macerans using α-cyclodextrin as a glucosyl donor. The synthesis of baicalin glucosides was optimized with CGTase from Thermoanaerobacter sp. Enzymatically modified baicalin derivatives were α-glucosylated with 1 to 17 glucose moieties. The two main glucosides were identified as Baicalein-7-O-α-D-Glucuronidyl-(1→4′)-O-α-D-Glucopyranoside (BG1) and Baicalein-7-O-α-D-Glucuronidyl-(1→4′)-O-α-D-Maltoside (BG2), thereby confirming recent findings reporting that glucuronyl groups are acceptors of this CGTase. Optimized conditions allowed for the attainment of yields above 85% (with a total glucoside content higher than 30 mM). BG1 and BG2 were purified via centrifugal partition chromatography after an enrichment through deglucosylation with amyloglucosidase. Transglucosylation increased the water solubility of BG1 by a factor of 188 in comparison to that of baicalin (molar concentrations), while the same value for BG2 was increased by a factor of 320. Finally, BG1 and BG2 were evaluated using antioxidant and anti-glycation assays. Both glucosides presented antioxidant and anti-glycation properties in the same order of magnitude as that of baicalin, thereby indicating their potential biological activity. [ABSTRACT FROM AUTHOR]

Background: Glucuronidation is an essential metabolic pathway for a variety of drugs. IMM-H004 is a novel neuroprotective agent against ischemic stroke, and its glucuronide metabolite IMM-H004G exhibits similar pharmacological activity. Despite possessing a higher molecular weight and polarity, brain exposure of IMM-H004G is much higher than that of IMM-H004. This study aimed to investigate the brain metabolism and transport mechanisms of IMM-H004 and IMM-H004G. Methods: First, the possibility of IMM-H004 glucuronidation in the brain was evaluated in several human brain cell lines and rat homogenate. Subsequently, the blood–brain barrier carrier-mediated transport mechanism of IMM-H004 and IMM-H004G was studied using overexpression cell models. In addition, intracerebroventricular injection, in situ brain perfusion model, and microdialysis/microinjection techniques were performed to study the distribution profiles of IMM-H004 and IMM-H004G. Results: IMM-H004 could be metabolized to IMM-H004G in both rat brain and HEB cells mediated by UGT1A7. However, IMM-H004G could not be hydrolyzed back into IMM-H004. Furthermore, the entry and efflux of IMM-H004 in the brain were mediated by the pyrilamine-sensitive H+/OC antiporter and P-gp, respectively, while the transport of IMM-H004G from the blood to the brain was facilitated by OATP1A2 and OATP2B1. Ultimately, stronger concentration gradients and OATP-mediated uptake played a critical role in promoting greater brain exposure of IMM-H004G. Conclusions: The active glucuronide metabolite of the brain protectant IMM-H004 with poor blood–brain barrier permeability demonstrates a high partition in the rat brain via multiple mechanisms, and our findings deepen the understanding of the mechanisms underlying the blood–brain barrier metabolism and transport of active glucuronide conjugates.

Quantitative analysis of postmortem urine, instead of blood, for buprenorphine and metabolites may provide additional evidence for the diagnosis of fatal buprenorphine poisoning. In this study, 247 autopsy urine samples, previously testing positive for buprenorphine or norbuprenorphine, were quantitatively reanalysed with a recently developed liquid chromatography–tandem mass spectrometry (LC‐MS/MS) method for unconjugated buprenorphine (BUP), norbuprenorphine (NBUP), naloxone (NAL), and their respective conjugated metabolites, buprenorphine glucuronide (BUPG), norbuprenorphine glucuronide (NBUPG), and naloxone glucuronide (NALG). The cases were divided, according to medical examiners' decision, to buprenorphine poisonings and other causes of death. The groups were compared for urinary concentrations and metabolite concentration ratios of the six analytes. All median concentrations were higher in the buprenorphine poisoning group. The median concentration of BUPG was significantly higher and the median metabolite ratios NBUP/BUP, NBUPG/BUPG, and NBUPtotal/BUPtotal were significantly lower in poisonings than in other causes of death. Naloxone‐related concentrations and ratios were not significantly different between the groups. [ABSTRACT FROM AUTHOR]

Baicalin is a biologically active flavone glucuronide with poor water solubility that can be enhanced via glucosylation. In this study, the transglucosylation of baicalin was successfully achieved with CGTases from Thermoanaerobacter sp. and Bacillus macerans using α-cyclodextrin as a glucosyl donor. The synthesis of baicalin glucosides was optimized with CGTase from Thermoanaerobacter sp. Enzymatically modified baicalin derivatives were α-glucosylated with 1 to 17 glucose moieties. The two main glucosides were identified as Baicalein-7-O-α-D-Glucuronidyl-(1→4′)-O-α-D-Glucopyranoside (BG1) and Baicalein-7-O-α-D-Glucuronidyl-(1→4′)-O-α-D-Maltoside (BG2), thereby confirming recent findings reporting that glucuronyl groups are acceptors of this CGTase. Optimized conditions allowed for the attainment of yields above 85% (with a total glucoside content higher than 30 mM). BG1 and BG2 were purified via centrifugal partition chromatography after an enrichment through deglucosylation with amyloglucosidase. Transglucosylation increased the water solubility of BG1 by a factor of 188 in comparison to that of baicalin (molar concentrations), while the same value for BG2 was increased by a factor of 320. Finally, BG1 and BG2 were evaluated using antioxidant and anti-glycation assays. Both glucosides presented antioxidant and anti-glycation properties in the same order of magnitude as that of baicalin, thereby indicating their potential biological activity.

A reactive metabolite of nonsteroidal anti-inflammatory drugs (NSAIDs), acyl-β-D-glucuronide (AG), covalently binds to endogenous proteins. The covalent adduct formation of NSAIDs-AG may lead to the dysfunction of target proteins. Therefore, it is important to clarify the detailed characterization of the formation of covalent protein adducts of NSAID-AG. UDP-glucuronosyltransferase (UGT) catalyzes the conversion of NSAIDs to NSAIDs-AG. The aim of this study was to perform a quantitative analysis of the covalent adduct formation of NSAIDs-AG with UGT. Diclofenac-AG and ketoprofen-AG formed covalent adducts with organelle proteins. Next, the number of covalent adducts formed between NSAIDs-AG and UGT isoforms (UGT1A1, UGT1A9, UGT2B4, and UGT2B9) was determined. The capacity of diclofenac-AG to form covalent adducts with UGT1A9 or UGT2B7 was approximately 10 times higher than that of mefenamic acid-AG. The amounts of covalent adducts of AG of propionic acid derivative NSAIDs with UGT2B were higher than those with UGT1A. Stereoselectivity was observed upon covalent binding to UGT. A significant negative correlation between the half-lives of NSAIDs-AG in phosphate buffers and the amount of covalent adduct with UGT2B7 was observed, suggesting the more labile NSAID-AG forms higher irreversible bindings to UGT. This report provides comprehensive information on the covalent adduct formation of NSAIDs-AGs with UGT. [ABSTRACT FROM AUTHOR]

Mass spectral library annotation of liquid chromatography-high resolution tandem mass spectrometry (LC-HRMS/MS) data is a reliable approach for fast identification of organic contaminants and toxicants in complex environmental and biological matrices. While determining the exposure of humans or mammals, it is indispensable to include phase I and phase II metabolites (conjugates) along with the parent compounds, but often, tandem mass spectra for these are unavailable. In this study, we present and evaluate a strategy for annotating glucuronide conjugates in LC-HRMS/MS scans by applying a neutral loss search for detection, then truncating the spectra which we refer to as in silico deconjugation, and finally searching these against mass spectral libraries of the aglycones. The workflow was tested on a dataset of in vitro–generated glucuronides of reference standard mixtures and a dataset of 51 authentic urine samples collected from patients with known medication status, acquired on different instrumentations. A total number of 75 different glucuronidated molecular structures were identified by in silico deconjugation and spectral library annotation. We also identified specific molecular structures (sulfonamides, ether bonds, di-glucuronides), which resulted in slightly different fragmentation patterns between the glucuronide and the unconjugated compound. This led to a decreased spectral matching score and in some cases to a false-negative identification. Still, by applying this method, we revealed a reliable annotation of most common glucuronides, leading to a new strategy reducing the need for deconjugation steps or for recording many reference glucuronide spectra for screening approaches. [ABSTRACT FROM AUTHOR]

[Display omitted] • Unconjugated EHS is significantly less stable in urine compared to glucuronidated EHS. • Lower stability of unconjugated EHS occurred at both 8 °C and −20 °C. • The hydroxylated metabolites of EHS showed no relevant stability issues. • Only small ratio of unconjugated EHS found in urine samples after dermal exposure. • Low stability of unconjugated EHS shall be considered for calibration and quality control samples. 2-Ethylhexyl salicylate (EHS) is used as a UV filter in personal care products, especially sunscreens. The elimination of EHS and its metabolite 2-ethyl-5-hydroxyhexyl salicylate (5OH–EHS) after dermal exposure has been previously investigated and showed that both are excreted in high amounts. However, the low stability of the reference substance for EHS highly affected the analysis of this parameter. Since EHS was assumed to be excreted primarily as a conjugate to glucuronic acid in vivo , we compared the stability of unconjugated EHS to that of glucuronidated EHS (EHS-GlcA) in urine at 8 °C and —20 °C. The relative recovery of unconjugated EHS decreases significantly when stored at both 8 °C and –20 °C. Stored as EHS-GlcA, the relative recovery remains steady over the course of the experiment. In addition to the stability experiment, the fraction of unconjugated EHS in human urine samples directly after dermal exposure to EHS was assessed, and only a low effective portion of unconjugated EHS was found. The same samples were reassessed after several months of storage at —20 °C and showed relative recoveries within the acceptance criteria, which also endorses the low initial level of unconjugated EHS. The hydroxylated EHS metabolites, 5OH-EHS, 4OH-EHS and 2OH-EHS, were also included in the experiment and showed slightly decreasing relative recoveries at 8 °C, but stable relative recoveries at –20 °C. Despite unconjugated EHS being excreted only in a low portion in vivo , its low stability in urine may affect the effectiveness of EHS biomonitoring, because only the use of EHS glucuronide guarantees a stable and reliable calibration in urine. However, the hydroxylated EHS metabolites have proven to be suitable standards for calibration as well as parameters for biomonitoring when stored at —20 °C. [ABSTRACT FROM AUTHOR]

Phase II metabolites play an important role in diazepam-related cases. The study aimed to assess the stability of diazepam's phase II metabolites in dried blood spots on filter paper. A piece of filter paper was spotted with 100 µL of whole blood (added 1% sodium fluoride as needed) obtained from participant who received 5 mg diazepam orally, air dried for 2 h at room temperature, and then stored at different conditions. Whole spots were cut at 0.1 cm from the outer edge of blood spots at post-consumption time-points of prior (zero), 5, 16, 35, 61, 120 days and 1, 1.5 years. Analytes were extracted with methanol/water mixture (8:2, v/v) and determined using HPLC-MS/MS. Decomposition rules were analyzed by a statistical software "SPSS". Temazepam glucuronide remained stable (0.5–18.6% loss) at 20 ℃ and at 20 ℃ with 1% sodium fluoride for 16 days, while it was unstable after 5 days at 4 ℃ (21.1–26.2% loss) and − 20 ℃ (28.9 - 34.4% loss). After 35 days, temazepam glucuronide concentrations began to fluctuate significantly under all conditions, and an obvious increase (290.4–355.1%) was observed in 1.5 years. Oxazepam glucuronide was always unstable after 5 days, the percentage loss was even 100% when it was stored for 61 days and 1.5 years. Dried blood spots on ordinary filter paper are recommended to be stored at 20 ℃ or 20 ℃ with 1% sodium fluoride within 16 days. Samples should be analyzed immediately or stored in sterile and dry media. • TG was stable on filter paper at 20 ℃ with or without 1 % NaF within 16 days, and it is a more reliable marker in real cases. • OG was always unstable after 5 days, the percentage loss was even 100% when it was stored for 61 days and 1.5 years. • DBS on filter paper in diazepam-related cases are recommended to be stored at 20 ℃ with or without 1 % NaF within 16 days. [ABSTRACT FROM AUTHOR]

A reactive metabolite of nonsteroidal anti-inflammatory drugs (NSAIDs), acyl-β-D-glucuronide (AG), covalently binds to endogenous proteins. The covalent adduct formation of NSAIDs-AG may lead to the dysfunction of target proteins. Therefore, it is important to clarify the detailed characterization of the formation of covalent protein adducts of NSAID-AG. UDP-glucuronosyltransferase (UGT) catalyzes the conversion of NSAIDs to NSAIDs-AG. The aim of this study was to perform a quantitative analysis of the covalent adduct formation of NSAIDs-AG with UGT. Diclofenac-AG and ketoprofen-AG formed covalent adducts with organelle proteins. Next, the number of covalent adducts formed between NSAIDs-AG and UGT isoforms (UGT1A1, UGT1A9, UGT2B4, and UGT2B9) was determined. The capacity of diclofenac-AG to form covalent adducts with UGT1A9 or UGT2B7 was approximately 10 times higher than that of mefenamic acid-AG. The amounts of covalent adducts of AG of propionic acid derivative NSAIDs with UGT2B were higher than those with UGT1A. Stereoselectivity was observed upon covalent binding to UGT. A significant negative correlation between the half-lives of NSAIDs-AG in phosphate buffers and the amount of covalent adduct with UGT2B7 was observed, suggesting the more labile NSAID-AG forms higher irreversible bindings to UGT. This report provides comprehensive information on the covalent adduct formation of NSAIDs-AGs with UGT.

Nitric oxide (NO) is an important signaling molecule involved in many pathophysiological processes. NO mediates vasodilation and blood flow in the arteries, and its action contributes to maintaining vascular homeostasis by inhibiting vascular smooth muscle contraction and growth, platelet aggregation, and leukocyte adhesion to the endothelium. Dietary antioxidants and their metabolites have been found to be directly and/or indirectly involved in the modulation of the intracellular signals that lead to the production of NO. The purpose of this study was to investigate the contribution of conjugated metabolites of hydroxytyrosol (HT) and tyrosol (TYR) to the release of NO at the vascular level, and the related mechanism of action, in comparison to their parental forms. Experiments were performed in human aortic endothelial cells (HAEC) to evaluate the superoxide production, the release of NO and production of cyclic guanosine monophosphate (cGMP), the activation of serine/threonine-protein kinase 1 (Akt1), and the activation state of endothelial nitric oxide synthase (eNOS). It was observed that the tested phenolic compounds enhanced NO and cGMP concentration, inhibiting its depletion caused by superoxide overproduction. Moreover, some of them enhanced the activation of Akt (TYR, HT metabolites) and eNOS (HT, HVA, TYR-S, HT-3S). Overall, the obtained data showed that these compounds promote NO production and availability, suggesting that HT and TYR conjugated metabolites may contribute to the effects of parental extra virgin olive oil (EVOO) phenolics in the prevention of cardiovascular diseases.

This critical review examines evidence for beneficial effects of quercetin phase-2 conjugates from clinical intervention studies, volunteer feeding trials, and in vitro work. Plasma concentrations of quercetin-3- O -glucuronide (Q3G) and 3'-methylquercetin-3- O- glucuronide (3'MQ3G) after supplementation may produce beneficial effects in macrophages and endothelial cells, respectively, especially if endogenous deglucuronidation occurs, and lower blood uric acid concentration via quercetin-3'- O -sulfate (Q3'S). Unsupplemented diets produce much lower concentrations (<50 nmol/l) rarely investigated in vitro. At 10 nmol/l, Q3'S and Q3G stimulate or suppress, respectively, angiogenesis in endothelial cells. Statistically significant effects have been reported at 100 nmol/l in breast cancer cells (Q3G), primary neuron cultures (Q3G), lymphocytes (Q3G and3'MQ3G) and HUVECs (QG/QS mixture), but it is unclear whether these translate to a health benefit in vivo. More sensitive and more precise methods to measure clinically significant endpoints are required before a conclusion can be drawn regarding effects at normal dietary concentrations. Future requirements include better understanding of inter-individual and temporal variation in plasma quercetin phase-2 conjugates, their mechanisms of action including deglucuronidation and desulfation both in vitro and in vivo, tissue accumulation and washout, as well as potential for synergy or antagonism with other quercetin metabolites and metabolites of other dietary phytochemicals.

A rapid preparation method for the analysis of the urine from a cannabis user was established. Generally, 11-nor-9-carboxy-∆9-tetrahydrocannabinol (THC-COOH), which is one of the main metabolites of ∆9-tetrahydorocannabinol (THC), must be detected from a user's urine to verify cannabis use. However, existing preparation methods are usually multistep and time-consuming processes. Before the analysis by liquid-chromatography tandem mass spectrometry (LC-MS/MS), deconjugation by treatment with β-glucuronidase or alkaline solution, liquid-liquid extraction or solid-phase extraction (SPE), and evaporation are generally performed. In addition, subsequent derivatization (silylation or methylation) are certainly necessary for gas-chromatography mass spectrometry (GC/MS) analysis. Here, we focused on the phenylboronic-acid (PBA) SPE, which selectively binds compounds with a cis -diol moiety. THC-COOH is metabolized as a glucuronide conjugate (THC-COOGlu) which has cis -diol moieties, therefore, we investigated the conditions of its retention and elution to reduce the operating time. We developed four elution conditions, which afford the following derivatives: acidic elution for THC-COOGlu, alkaline elution for THC-COOH, methanolysis elution for the THC-COOH methyl ester (THC-COOMe), and methanolysis elution and following methyl etherification for O -methyl-THC-COOMe (O -Me-THC-COOMe). All repeatability and recovery rates were evaluated by LC-MS/MS in this study. As a result, these four pathways required short times (within 10–25 min) and exhibited good repeatability and recovery rates. Detection limits of pathway I-IV were 10.8, 1.7, 18.9, and 13.8 ng mL-1, respectively. Lower limits of quantification were 62.5, 31.25, 57.3, and 62.5 ng mL-1, respectively. When proof of cannabis use is required, any elution condition can be selected to match the possessing reference standards and analytical instruments. To our knowledge, this is the first report of using PBA SPE for the preparation of the urine samples containing cannabis and achieving partial derivatization when eluting from a PBA carrier. Our method can provide a new and practical solution for the preparation of the urine samples from cannabis users. Although the PBA SPE method cannot recover THC-COOH in urine because of its lack of a 1,2-diol moiety, this method has technological advantages for simplifying the process and reducing the operating time, thereby avoiding human errors. • We developed a quick preparation method for the analysis of urinary cannabis by phenylboronic acid solid-phase extraction. • Four pathways can provide four different kinds of 11-nor-9-carboxy-∆9-tetrahydrocannabinol derivatives, respectively. • All pathways showed good recovery rates and repeatability. • This method can simplify the process and shorten the operating time, resulting in contributing to reduce human errors. [ABSTRACT FROM AUTHOR]

Drug-drug interactions (DDIs) arising from the perturbation of drug metabolising enzyme activities represent both a clinical problem and a potential economic loss for the pharmaceutical industry. DDIs involving glucuronidated drugs have historically attracted little attention and there is a perception that interactions are of minor clinical relevance. This review critically examines the scope and aetiology of DDIs that result in altered exposure of glucuronidated drugs. Interaction mechanisms, namely inhibition and induction of UDP-glucuronosyltransferase (UGT) enzymes and the potential interplay with drug transporters, are reviewed in detail, as is the clinical significance of known DDIs. Altered victim drug exposure arising from modulation of UGT enzyme activities is relatively common and, notably, the incidence and importance of UGT induction as a DDI mechanism is greater than generally believed. Numerous DDIs are clinically relevant, resulting in either loss of efficacy or an increased risk of adverse effects, necessitating dose individualisation. Several generalisations relating to the likelihood of DDIs can be drawn from the known substrate and inhibitor selectivities of UGT enzymes, highlighting the importance of comprehensive reaction phenotyping studies at an early stage of drug development. Further, rigorous assessment of the DDI liability of new chemical entities that undergo glucuronidation to a significant extent has been recommended recently by regulatory guidance. Although evidence-based approaches exist for the in vitro characterisation of UGT enzyme inhibition and induction, the availability of drugs considered appropriate for use as 'probe' substrates in clinical DDI studies is limited and this should be a research priority. [ABSTRACT FROM AUTHOR]

The complete understanding of the excretion of surplus 25-hydroxyvitamin D 3 [25(OH)D 3 ] in humans remains to be accomplished. In our previous study, 24,25-dihydroxyvitamin D 3 [24,25(OH) 2 D 3 ] 24-glucuronide was identified as a major urinary vitamin D 3 metabolite, while the glucuronide of 23,25-dihydroxyvitamin D 3 [23,25(OH) 2 D 3 ] is another metabolite of interest but has not been sufficiently evaluated. Although the quantitative analysis of 24,25(OH) 2 D 3 liberated in urine by the treatment with β-glucuronidase (GUS) has been conducted, no information was provided about the amount of the glucuronidated 23,25(OH) 2 D 3 in the urine. In this study, we first developed and validated a liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS)-based method for the simultaneous quantification of 23,25(OH) 2 D 3 and 24,25(OH) 2 D 3 liberated in urine by GUS. The analysis of the urine samples revealed that the amount of 23,25(OH) 2 D 3 was almost as much as that of 24,25(OH) 2 D 3 , in contrast to the fact that the plasma concentration of 23,25(OH) 2 D 3 was much lower than that of 24,25(OH) 2 D 3. These results strongly suggested that 23,25(OH) 2 D 3 is more susceptible to glucuronidation and more promptly excreted into urine than 24,25(OH) 2 D 3. Furthermore, the amount ratios of 23,25(OH) 2 D 3 to 24,25(OH) 2 D 3 in the urine samples did not markedly vary during the day (morning/evening) and even by a week-long vitamin D 3 supplementation (1000 IU/body/day). We concluded that the C-23 hydroxylation plays a crucial role in the urinary excretion of surplus 25(OH)D 3. [Display omitted] • LC/ESI-MS/MS method for the quantification of 23,25(OH) 2 D 3 and 24,25(OH) 2 D 3. • Almost the same amounts of the two metabolites were liberated in urine by GUS treatment. • 23,25(OH) 2 D 3 was virtually absent in the plasma as opposed to 24,25(OH) 2 D 3. • C-23 hydroxylation is inferred to have a crucial role in the 25(OH)D 3 excretion. • Vitamin D intake had less impact on the ratio of the two metabolites in the urine. [ABSTRACT FROM AUTHOR]

Stilbene metabolites are attracting great interest because many of them exhibit similar or even stronger biological effects than their parent compounds. Furthermore, the metabolized forms are predominant in biological fluids; therefore, their study is highly relevant. After hemisynthesis production, isolation, and structural elucidation, three glucuronide metabolites for oxyresveratrol (ORV) were formed: trans -ORV-4'- O -glucuronide, trans -ORV-3- O -glucuronide, and trans -ORV-2'- O -glucuronide. In addition, two glucuronide metabolites were obtained for gnetol (GN): trans -GN-2'- O -glucuronide and trans -GN-3- O -glucuronide. When the metabolism of ORV and GN is studied in vitro by human and rat hepatic enzymes, four of the five hemisynthesized compounds were identified and quantified. Human enzymes glucuronidated preferably at the C-2' position, whereas rat enzymes do so at the C-3 position. In view of these kinetic findings, rat enzymes have a stronger metabolic capacity than human enzymes. Finally, ORV, GN, and their glucuronide metabolites (mainly at the C-3 position) decreased nitric oxide, reactive oxygen species, interleukin 1β, and tumor necrosis factor α production in lipopolysaccharide-stimulated macrophages.

[Display omitted] • Analysis of long-term metabolites of oral-turinabol via LC-HRMSMS. • Identification of three new phase-II metabolites of dehydrochloromethyltestosterone. • Structure determination by fractionation and derivatization experiments. Dehydrochloromethyltestosterone (DHCMT) is one of the most detected illicit used anabolic–androgenic steroids in professional sports. Therefore, a fast and accurate analysis of this substance is of great importance for a constructive fight against doping abuse. The conventional method for the analysis of this drug, GC-MSMS, is very sensitive and selective but also very time- and resource-consuming. With the presented work, a new approach for simple detection with LC-HRMSMS without any sample preparation is introduced. The method is based on the direct analysis of two newly described phase-II metabolites of the DHCMT long-term metabolite 4-chloro-18-nor-17β-hydroxymethyl-17α-methyl-5β-androst-13-en-3α-ol (M3). LC-HRMSMS, GC-MSMS, fractionation and derivatization experiments are combined to identify and characterize for the first time two different glucuronide-acid conjugates of this metabolite in positive human urine samples. In addition, a third glucuronide metabolite was identified, however without isomeric structure determination. The detection of these metabolites is particularly interesting for confirmation analyses, as the method is rapid and requires little sample material. [ABSTRACT FROM AUTHOR]

Nitric oxide (NO) is an important signaling molecule involved in many pathophysiological processes. NO mediates vasodilation and blood flow in the arteries, and its action contributes to maintaining vascular homeostasis by inhibiting vascular smooth muscle contraction and growth, platelet aggregation, and leukocyte adhesion to the endothelium. Dietary antioxidants and their metabolites have been found to be directly and/or indirectly involved in the modulation of the intracellular signals that lead to the production of NO. The purpose of this study was to investigate the contribution of conjugated metabolites of hydroxytyrosol (HT) and tyrosol (TYR) to the release of NO at the vascular level, and the related mechanism of action, in comparison to their parental forms. Experiments were performed in human aortic endothelial cells (HAEC) to evaluate the superoxide production, the release of NO and production of cyclic guanosine monophosphate (cGMP), the activation of serine/threonine-protein kinase 1 (Akt1), and the activation state of endothelial nitric oxide synthase (eNOS). It was observed that the tested phenolic compounds enhanced NO and cGMP concentration, inhibiting its depletion caused by superoxide overproduction. Moreover, some of them enhanced the activation of Akt (TYR, HT metabolites) and eNOS (HT, HVA, TYR-S, HT-3S). Overall, the obtained data showed that these compounds promote NO production and availability, suggesting that HT and TYR conjugated metabolites may contribute to the effects of parental extra virgin olive oil (EVOO) phenolics in the prevention of cardiovascular diseases. [ABSTRACT FROM AUTHOR]

Objective: To evaluate the impact of genetic polymorphisms of UGT enzymes (UGT1A4, UGT2B7, UGT2B15 and UGT 2B17) and the transporter protein ABCB1 on Lamotrigine (LTG) metabolism., Methods: Single nucleotide polymorphisms UGT1A4*2 (P24T, c.70C>A), UGT1A4*3 (L48V c.142T>G), UGT2B7*2 (H802Y, c.802C>T), UGT2B15*2 (Y85D, c.253G>T), UGT2B17 deletion and transporters ABC 1236C> T and 3435C> T were determined in 337 Caucasian patients with epilepsy treated with LTG in Denmark. The prospectively collected data included LTG dosage, LTG plasma concentration, 2-N-GLU concentration, sex, smoking habits, concomitant medicine, oral contraceptives (OC)., Results: The non-smokers with LTG monotherapy and LTG polytherapy with other non-interacting drugs NIAEDs (n = 199) were analyzed separately in univariant analyses. LTG ratios (LTG plasma concentration/ (LTG dose/weight)) in patients carrying wild type UGT1A4*2 C-allele were 22% lower than in heterozygous C-carriers (p = 0.013). Patients with UGT2B7*2 polymorphism TT genotype had 1.2-fold higher LTG ratios (p = 0.0078) and 0.78-fold lower GLU/LTG ratio (p = 0.0275) than patients homozygous for the C allele. The similar significant findings were also seen comparing homozygotes (TT) with heterozygotes patients (CT). Individuals homozygous for the UGT2B15*2 T allele displayed 18% lower LTG ratio concentrations than individuals homozygous for the G allele (p = 0.014),while significant difference in GLU/LTG ratio was only seen comparing wild type with homozygous patients (GG versus TT, p = 0.031). A copy number variation gene deletion polymorphism of UGT2B17 showed that individuals devoid of the gene (del/del) exhibited 1.3-fold higher LTG ratio (p = 0.015). For ABCB1c.1236 C>T and ABC1B1c.3435 C>T no associations with LTG and GLU ratios were found. Sex specific differences in enzyme activity (most prominent effect in women) on LTG metabolism were found for UGT2B15, UGT2B17, UGT1A4 and UGT2B7 polymorphisms. Multiple regression analysis confirmed the significant effect of OC, VPA and UGT1A4 * 2 and UGT2B7 * 2 on LTG metabolism., Conclusion: Our study confirms the previous findings that genetic variations in UGT2B7 and UGT1A4 genes are associated with serum LTG concentrations. Furthermore, our results indicate that it is possible that different UGT genotypes may exert larger impact on LTG metabolism in women than in men., (Copyright © 2022 Elsevier B.V. All rights reserved.)