Simultaneous determination of iguratimod and its metabolite in rat plasma using a UPLC-MS/MS method: Application for drug-drug interaction.

This aim of the work was to establish an acceptable sensitive assay based on ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) for quantitatively analyzing the plasma concentrations of iguratimod (IGR) and its metabolite M2 in rats, and to further investigate the effect of fluconazole on the pharmacokinetics of IGR and M2. The mobile phase consisted of acetonitrile and water with 0.1% formic acid, was used to separate IGR, M2 and internal standard (IS) fedratinib on a UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 µm) with the flow rate of 0.4 mL/min. Positive ion mode and multiple reaction monitoring (MRM) were used to construct the quantitative analysis. The calibration standard of IGR and M2 covered 2-10000 and 1-1000 ng/mL respectively, with the lower limit of quantification (LLOQ) as 2 ng/mL and 1 ng/mL respectively. In addition, selectivity, recovery, accuracy, precision, matrix effect and stability of the method validation program were well accepted in this work. Subsequently, this approach was used to assess the effect of fluconazole on the pharmacokinetics of IGR and M2 in rats. In the presence of 20 mg/kg fluconazole (experimental group), we found the main pharmacokinetic parameters were significantly altered when compared with 2.5 mg/kg IGR alone (control group). Among them, AUC(0-∞) and Cmax of IGR in the experimental group was 1.43 and 1.08 times higher than that of the control group, respectively. Moreover, we also found that the other main pharmacokinetic parameters of M2 had no significant changes, except t1/2z and Tmax. In conclusion, fluconazole significantly altered the main pharmacokinetics of IGR and M2 in rats. It implys that we should pay more attention to the adverse reaction of IGR when the concomitant use of fluconazole and IGR occur in the future clinical practice.

An Investigation of Pharmacokinetic Interaction of Vericiguat with Apigenin based on a Newly Developed Ultra-performance Liquid Chromatography-tandem Mass Spectrometry Assay.

BACKGROUND: Vericiguat, as a new stimulator of soluble guanylate cyclase (sGC), was recently approved as a first-in-class treatment for reducing risks in patients with ejection fraction less than 45 percent and heart failure (HF) in the USA. OBJECTIVE: The main aim of the present experiment was to establish an acceptable, sensitive assay based on ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) for quantitatively analyzing the plasma concentration levels of vericiguat in rats, and to further evaluate the effect of apigenin on the metabolism of vericiguat in vivo. METHOD: In sample processes, acetonitrile was finally chosen for quickly precipitating protein. The levels of vericiguat in plasma were analyzed by a Xevo TQ-S triple quadrupole tandem mass spectrometry (Milford, MA, USA) in a positive ion mode. RESULTS: The scope of the calibration standard for vericiguat ranged from 0.5 to 1000 ng/mL, where a great linearity was acceptable. The lower limit of quantification (also called LLOQ) of vericiguat presented the sensitivity of this assay was evaluated as low as 0.5 ng/mL. Additionally, selectivity, accuracy and precision, extraction recovery, matrix effect, and stability were all verified. Subsequently, this approach also supported to assess the plasmatic concentrations of vericiguat from an interaction survey on herb-- drug, in which oral administration of apigenin (20 mg/kg) obviously increased the plasmatic levels of vericiguat and altered the pharmacokinetics of vericiguat in rats. CONCLUSION: These results would help us to further understand the pharmacokinetic properties of vericiguat when co-administration with apigenin, and to avoid unexpected clinical risks in the future.

Effects of Dietary Flavonoids on the Metabolism of Vortioxetine and its Potential Mechanism

INTRODUCTION: Quercetin and apigenin are two common dietary flavonoids widely found in foods and fruits. Quercetin and apigenin can act as the inhibitors of CYP450 enzymes, which may affect the pharmacokinetics of clinical drugs. Vortioxetine (VOR), approved for marketing by the Food and Drug Administration (FDA) in 2013, is a novel clinical drug for treating major depressive disorder (MDD). OBJECTIVE: This study aimed to evaluate the effects of quercetin and apigenin on the metabolism of VOR in in vivo and in vitro experiments. METHOD: Firstly, 18 Sprague-Dawley rats were randomly divided into three groups: control group (VOR), group A (VOR + 30 mg/kg quercetin) and group B (VOR + 20 mg/kg apigenin). We collected the blood samples at different time points before and after the final oral administration of 2 mg/kg VOR. Subsequently, we further used rat liver microsomes (RLMs) to investigate the half-maximal inhibitory concentration (IC50) of the metabolism of vortioxetine. Finally, we evaluated the inhibitory mechanism of two dietary flavonoids on VOR metabolism in RLMs. RESULTS: In animal experiments, we found AUC (0-∞) (area under the curve from 0 to infinity) and CLz/F (clearance) to be obviously changed. Compared to controls, AUC (0-∞) of VOR in group A and group B was 2.22 and 3.54 times higher, respectively, while CLz/F of VOR in group A and group B was significantly decreased down to nearly two-fifth and one-third. In in vitro studies, the IC50 value of quercetin and apigenin in the metabolic rate of vortioxetine was 5.322 µM and 3.319 µM, respectively. Ki value of quercetin and apigenin was found to be 0.279 and 2.741, respectively, and the αKi value of quercetin and apigenin was 0.066 and 3.051 µM, respectively. CONCLUSION: Quercetin and apigenin exhibited inhibitory effects on the metabolism of vortioxetine in vivo and in vitro. Moreover, quercetin and apigenin non-competitively inhibited the metabolism of VOR in RLMs. Thus, we should pay more attention to the combination between these dietary flavonoids and VOR in the future clinical use.

Effects of different doses glimepiride intake on the pharmacokinetics of benzbromarone in rats.

Benzbromarone (BNR) is prescribed for the management of hyperuricemia, whereas glimepiride (GLM) for the treatment of Type 2 Diabetes Mellitus. Both drugs are certified to be mainly metabolized via cytochrome P450 (CYP) 2C9 in vivo and may have the potential drug-drug interactions. This study aims to investigate the possible influence of orally administered low- and high-dose glimepiride (GLM) on pharmacokinetic characteristics (PK) of benzbromarone (BNR) in rats. Fifteen rats were randomly assigned to group A, B and C (n=5) and administered 0.5% sodium carboxymethyl cellulose (CMC), 0.5mg/kg GLM (low-dose) and 1.0 mg/kg GLM (high-dose) once daily for 8 days, respectively, which were all followed with a single oral dose of BNR (9.0 mg/kg) on the day 8th. Blood samples were obtained from retro orbital plexus at the time points of 0.25, 0.5, 1, 2, 4, 6, 8, 10, 12 and 24h and BNR in plasma was quantitated by HPLC-MS/MS assay. Resultantly a slight influence of GLM on PK of BNR could be found in rats. When compared with Group A, the half-life time (t1/2z) of BNR in Group B and C significantly decreased 52.39% and 73.49%, respectively, although other major PK parameters were negligibly changed by co-administration of GLM. On the whole, the combinational therapy of GLM at low or high dose would notably alter the elimination of BNR and the effect was dose-dependent.

Effect of Baicalein on the Pharmacokinetics of Cilostazol and Its Two Metabolites in Rat Plasma Using UPLC-MS/MS Method.

This study aimed to explore the effect of baicalein on the pharmacokinetics of cilostazol (CLZ) and its two metabolites 3,4-dehydro cilostazol (3,4-CLZ) and 4'-trans-hydroxy cilostazol (4'-CLZ) in rats using a newly established ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method. Ticagrelor was used as an internal standard (IS), then cilostazol and its two metabolites were separated by means of a UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 µm) using gradient elution method with 0.4 ml/min of flow rate. Acetonitrile as organic phase and water with 0.1% formic acid as aqueous phase constructed the mobile phase. Selective reaction monitoring (SRM) mode and positive ion mode were preferentially chosen to detect the analytes. Twelve SD rats were divided into two groups (n = 6) when CLZ was administered orally (10 mg/kg) with or without oral baicalein (80 mg/kg). The selectivity, linearity, recovery, accuracy, precision, matrix effect and stability of UPLC-MS/MS assay were satisfied with the standards of United States Food and Drug Administration guidelines. In control group, AUC0-∞ and Cmax of CLZ were 2,169.5 ± 363.1 ng/ml*h and 258.9 ± 82.6 ng/ml, respectively. The corresponding results were 3,767.6 ± 1,049.8 ng/ml*h and 308.6 ± 87.9 ng/ml for 3, 4-CLZ, 728.8 ± 189.9 ng/ml*h and 100.3 ± 51.3 ng/ml for 4'-CLZ, respectively. After combination with baicalein, AUC0-∞ and Cmax of CLZ were 1.48, 1.38 times higher than the controls. Additionally, AUC0-∞ and Cmax were separately decreased by 36.12 and 19.54% for 3,4-CLZ, 13.11 and 44.37% for 4'-CLZ. Baicalein obviously alters the pharmacokinetic parameters of CLZ, 3,4-CLZ and 4'-CLZ in rats. These results suggested that there was a potential drug-drug interaction between baicalein and CLZ. Therefore, it must raise the awareness when concomitant use of CLZ with baicalein, the dosage regimen of CLZ should be taken into consideration, if this result is confirmed in clinical studies.

A study on UHPLC-MS/MS analyses of DNA and RNA oxidative damage metabolites in patients with cervical carcinoma: 8-oxoG in urine as a potential biomarker of cervical carcinoma.

Objective: The purpose of this study was to compare the levels of 8-oxoG and 8-oxodG in urine of patients with cervical carcinoma and healthy women to evaluate their influences on cervical carcinoma. Methods: In this study, urine samples were collected from 70 patients with cervical carcinoma, 24 patients with one-year follow-up, and 100 healthy women. The contents of 8-oxodG and 8-oxoG in urine were assayed by ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS). Results: The levels of 8-oxoG and 8-oxodG were higher in patients with cervical carcinoma (P < 0.000), while AUC of 8-oxoG and 8-oxodG was higher than 0.7. Specifically, the high-risk HPV (HR-HPV) positive group had higher 8-oxoG levels (P < 0.000), but there was no difference in 8-oxodG levels. Yet, 8-oxoG level was associated with lymphatic metastasis, lymph-vascular space infiltration (LVSI) and stromal infiltration, while 8-oxodG level was affected by the differentiation degree and stromal infiltration. According to statistics, the distinct cut-off index of lymphatic metastasis was 7.282 nmol/mmol creatinine. After operation, the concentrations of 8-oxoG and 8-oxodG dropped significantly (8-oxoG P < 0.000, 8-oxodG P = 0.004). Except for chemotherapy group, the urinary 8-oxoG dose of all treatment groups and 8-oxodG dose of chemo-radiotherapy group declined obviously. Conclusions: 8-oxoG may be a potential biomarker for cervical carcinoma.

Effect of Sophocarpine on the Pharmacokinetics of Umbralisib in Rat Plasma Using a Novel UPLC-MS/MS Method.

Umbralisib is a dual inhibitor of phosphatidylinositol 3-kinase delta (PI3Kδ) and casein kinase 1 epsilon (CK1ε) for treating marginal zone lymphoma (MZL) and follicular lymphoma (FL). This study aimed to develop a fast and stable ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for quantitative analysis of umbralisib in rat plasma and its application for evaluating the effect of sophocarpine on the pharmacokinetics of umbralisib. A direct protein preparation with acetonitrile was used to deal with rat plasma. Umbralisib and duvelisib (internal standard, IS) were isolated on a Waters Acquity UPLC BEH C18 column with mobile phase consisted of acetonitrile and 0.1% formic acid in water. The linear range was from 0.5 to 1,000 ng/ml. Both of the precision (RSD%) and accuracy (RE%) were less than 15% in a permissible range. The mean recovery and matrix effect of umbralisib were 86.3-96.2% and 97.8-112.0%, respectively. When umbralisib was combined with sophocarpine, AUC0→∞ of umbralisib was significantly reduced to 2462.799 ± 535.736 ng/ml•h from 5416.665 ± 1,451.846 ng/ml•h, and Cmax also was markedly diminished. Moreover, CLz/F was increased more than two times. This developed, optimized and technical UPLC-MS/MS method was extremely suitable for detecting the concentrations of umbralisib in rat plasma after an oral administration, and sophocarpine significantly changed the pharmacokinetics of umbralisib in rats. This obvious pharmacokinetic changes indicates that there seems to exist herb-drug interaction between sophocarpine and umbralisib.

A novel bioanalytical method for the quantification of pemigatinib in rat plasma by UPLC-MS/MS.

Pemigatinib is an oral, selective, potent, competitive inhibitor acting on fibroblast growth factor receptor (FGFR)1, FGFR2, and FGFR3, which has obtained accelerated approval in the USA through a test approved by the USA FDA. It is not only significant in the therapy of adult recurrent, unresectable, metastatic or locally advanced cholangiocarcinoma, but also plays an important role in treating adult patients with FGFR2 fusion or other rearrangements. The aim of our research was to establish and verify a reliable and quick ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) assay to determine the level of pemigatinib in rat plasma. The analyte was prepared using a simple and convenient approach with acetonitrile for protein crash, and then separated from the matrix on a Waters Acquity UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 µm) in a gradient elution program, where the mobile phase was consisted of acetonitrile and 0.1 % formic acid in water and was set at 0.40 mL/min flow rate. Selective reaction monitoring (SRM) was used to conducted for UPLC-MS/MS dectection with ion transitions at m/z 488.01 → 400.98 for pemigatinib and m/z 447.00 → 361.94 for erdafitinib (Internal standard, IS), respectively. This method had good linearity in a 0.5-1000 ng/mL calibration range for pemigatinib, where the lower limit of quantification (LLOQ) was validated at 0.5 ng/mL. The precision of pemigatinib for intra- and inter-day was less than 13.3 %, and the accuracy was determined to be from -4.8%-11.2%. During the assay in plasma samples, the analyte was found to be stable. Besides, matrix effect and recovery of the analyte and IS were acceptable. The novel optimized UPLC-MS/MS assay was also suitable for determining the concentration level of pemigatinib in a pharmacokinetic study after a single dose of 1.35 mg/kg pemigatinib orally to the rats.

Controllable Synthesis and Biological Application of Schiff Bases from d-Glucosamine and Terephthalaldehyde.

Theoretically, the two aldehydes of terephthalaldehyde (TPA) are equivalent, so the single or double Schiff base from TPA and d-glucosamine (Glc) may be formed at the same time. However, it is preferred to produce separately a single Schiff base (L1 ) or double Schiff base (L2 ) for different synthesis systems of anhydrous methanol or water-methanol. We calculated the Δr G of the formation of compounds L1 and L2 by density functional theory (DFT). In an anhydrous methanol system, the Δr G values of L1 and L2 are both below zero and L2 is lower, suggesting the spontaneous formation of the two Schiff bases. Though adjusting the molar ratio of Glc to TPA, L1 and L 2 both were separately formed in anhydrous methanol. However, in the water-methanol system, L2 was absent, which is most likely due to higher Δr G (4.95 eV) and better water solubility. The results also exhibits that the positive charge of C in -CHO for TPA is smaller in a mixed solvent than that in methanol, which confirms that the nucleophilic reaction of the Schiff base is more difficult in a mixed solvent. Therefore, we could realize to control the synthesis of a pure single or double Schiff base from Glc and TPA by adjusting the molar ratio and solvent. The as-prepared two kinds of Schiff bases have strong optical properties, high bacteriostatic activity, and can be used as fluorescent probes for tumor cell imaging.

A tyrosinase-induced fluorescence immunoassay for detection of tau protein using dopamine-functionalized CuInS2/ZnS quantum dots.

Rapid, highly sensitive detection of tau protein and other neurodegenerative biomarkers remains a significant hurdle for diagnostic tests for Alzheimer's disease. In this work, we developed a novel tyrosinase (TYR)-induced tau aptamer-tau-tau antibody (anti-tau) sandwich fluorescence immunoassay to detect tau protein that used dopamine (DA)-functionalized CuInS2/ZnS quantum dots as the fluorophore. CuInS2/ZnS core/shell quantum dots with high luminescence, low toxicity, and excellent biocompatibility were successfully fabricated and decorated with DA through amide conjugation. Meanwhile, TYR was conjugated with anti-tau by a click reaction. When DA-functionalized CuInS2/ZnS quantum dots were added to the sandwich system, TYR catalyzed the transformation of DA to dopamine quinone, which acted as an effective electron acceptor and triggered fluorescence quenching. The fluorescence intensity of the immunoassay based on DA-functionalized CuInS2/ZnS quantum dots shows good performance in terms of linearity with the logarithm of tau protein concentration, with a linear concentration range from 10 pM to 200 nM. This work is the first to use a TYR-induced fluorescence immunoassay for the rapid detection of tau protein, paving a new way for the detection of disease biomarkers. Graphical abstract.

A New UPLC-MS/MS Method Validated for Quantification of Jervine in Rat Plasma and the Study of Its Pharmacokinetics in Rats.

The aim of this study was to develop an ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method to assess the concentration of jervine in rat plasma and its pharmacokinetics. Diazepam was used as internal standard (IS). The chromatographic separation of jervine and IS was carried out on an UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 µm) with a flow rate of 0.4 mL/min. A mixture of acetonitrile and water (0.1% formic acid) was used as a mobile phase. The UPLC-MS/MS was equipped with an electrospray ionization (ESI), adopting multiple reactive monitoring mode to determine jervine in rat plasma. The retention times of jervine and the internal standard were 1.71 and 2.13 min, respectively. The calibration curve of jervine ranged between 1 and 1000 ng/mL. The lower limit of quantitation (LLOQ) was 1 ng/mL, and the lower limit of determination (LLOD) was 0.2 ng/mL. The accuracy was ±6%; the interday precision and intraday precision were no more than 9%. The recovery was higher than 90.3%, and the matrix effect was lower than 10%. The UPLC-MS/MS method was successfully developed and used for the application of the pharmacokinetic study. The primary pharmacokinetic parameters of jervine in this study were as follows: the AUC(0-∞) was 969.3 ± 277.7 ng/mL·h, the C max was 506.6 ± 192.8 ng/mL, the CL/F was 1.7 ± 0.5 L/h/kg, and the t 1/2 was 3.4 ± 1.2 h.

Oxidative DNA and RNA damage and their prognostic values during Salmonella enteritidis-induced intestinal infection in rats.

Emerging evidence suggests that microbial pathogens may induce oxidative stress in infected hosts. The aim of the present study was to investigate the relationship between changes in oxidative stress and intestinal infection with and without antibiotic treatment in animal models. Sprague-Dawley (SD) rats were divided into three groups: rats infected with Salmonella enterica serovar Enteritidis (S. enteritidis), rats infected with S. enteritidis followed by norfloxacin treatment, and the control group. To evaluate oxidative stress changes, levels of 8-oxo-7,8-dihydroguanosine (8-oxo-Gsn) and 8-oxo-7,8-dihydro-2-deoxyguanosine (8-oxo-dGsn), which represented oxidative damage to RNA and DNA, respectively, were analysed in urine and tissue samples. In urine, the level of 8-oxo-Gsn increased significantly after oral exposure to S. enteritidis (p ≤ 0.001) and returned to baseline after recovery. Notably, norfloxacin treatment decreased the level of 8-oxo-Gsn in urine significantly (p = 0.001). Changes of 8-oxo-Gsn measured in tissues from the small intestine, colon, liver and spleen were consistent with 8-oxo-Gsn measured in urine. Our study suggested that 8-oxo-Gsn in urine may serve as a highly sensitive biomarker for evaluating the severity of S. enteritidis infection and the effectiveness of antibiotic treatment against infection.

Development of CdSe⁻ZnO Flower-Rod Core-Shell Structure Based Photoelectrochemical Biosensor for Detection of Norovirous RNA.

In this work, the CdSe⁻ZnO flower-rod core-shell structure (CSZFRs) was prepared by ion-exchange method. The surface of CSZFRs was modified by 3-mercaptopropionic acid (MPA), and then the DNA probe was immobilized on the surface via chemical bond between -NH2 of DNA probe and -COOH of MPA. Finally, the target norovirous (NV) RNA was combined with the probe according to the principle of complementary base pairing, resulting in a decrease of the photocurrent. The results show that the absorbance spectrum of visible light is enhanced for CSZFRs compared with pure ZnO. Under visible light irradiation, the photocurrent of CSZFRs is up to 0.1 mA, which can improve the sensitivity of the photoelectrochemical (PEC) biosensor. In the measurement range of 0⁻5.10 nM, the measured concentrations (c) have a good linear relationship with the output photocurrent of the biosensor. The linear regression equation is expressed as I = 0.03256 - 0.0033c (R² = 0.99, S/N = 3) with a detection limit of 0.50 nM. Therefore, this work realizes a rapid and sensitive method for the detection of NV RNA.

Pharmacokinetics and Bioavailability Study of Tubeimoside I in ICR Mice by UPLC-MS/MS.

The aim of this study is to establish and validate a rapid, selective, and sensitive ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method to determine tubeimoside I (TBMS-I) in ICR (Institute of Cancer Research) mouse whole blood and its application in the pharmacokinetics and bioavailability study. The blood samples were precipitated by acetonitrile to extract the analytes. Chromatographic separation was performed on a UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 µm). The mobile phase consisted of water with 0.1% formic acid and methanol (1 : 1, v/v) at a flow rate of 0.4 mL/min. The total eluting time was 4 min. The TBMS-I and ardisiacrispin A (internal standard (IS)) were quantitatively detected by a tandem mass spectrometry equipped with an electrospray ionization (ESI) in a positive mode by multiple reaction monitoring (MRM). A validation of this method was in accordance with the US Food and Drug Administration (FDA) guidelines. The lower limit of quantification (LLOQ) of TBMS-I was 2 ng/mL, and the calibration curve was linearly ranged from 2 to 2000 ng/mL (r2 ≥ 0.995). The relative standard deviation (RSD) of interday precision and intraday precision was both lower than 15%, and the accuracy was between 91.7% and 108.0%. The average recovery was >66.9%, and the matrix effects were from 104.8% to 111.0%. In this assay, a fast, highly sensitive, and reproducible quantitative method was developed and validated in mouse blood for the first time. The absolute availability of TBMS-I in the mouse was only 1%, exhibiting a poor oral absorption.

ZnO flower-rod/g-C3N4-gold nanoparticle-based photoelectrochemical aptasensor for detection of carcinoembryonic antigen.

A highly sensitive and selective photoelectrochemical (PEC) aptasensor was constructed for carcinoembryonic antigen (CEA) detection based on ZnO flower-rods (ZnO FRs) modified with g-C3N4-Au nanoparticle (AuNP) nanohybrids. The nanohybrids of g-C3N4-AuNPs can improve the visible light absorbance of ZnO FRs and enhance the PEC property. We designed a sandwichlike structure formed with DNA hybridization of NH2-probe1, CEA aptamer, and CuS-NH2-probe2 to detect CEA. The p-type semiconductor CuS nanocrystals (NCs) at the terminational part of sandwichlike structure work as electron traps to capture photogenerated electrons and consequently lead to a magnified photocurrent change. The results indicate that the photocurrent is increased when CEA antigen (Ag) is introduced. Since the sandwichlike structure is destroyed, CuS NCs are restricted to capture photogenerated electron. The PEC aptasensor for CEA determination is ranged from 0.01 ng·mL-1 to 2.5 ng·mL-1 with a detection of 1.9 pg·mL-1. The proposed aptasensor exhibits satisfactory PEC performances with rapid detection, great sensitivity and specificity. Specially, this PEC aptasensor shows a reliable result for the determination of CEA in invalid human serum compared with the ELISA method. The designed aptasensor may provide a new idea for a versatile PEC platform to determine various molecules. Graphical abstract ᅟ.

The Ratio of Plasma and Urinary 8-oxo-Gsn Could Be a Novel Evaluation Index for Patients with Chronic Kidney Disease.

Nucleic acid oxidation plays an important role in the pathophysiology progress of a variety of diseases. 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dGsn) and 8-oxo-7,8-dihydroguanosine (8-oxo-Gsn), which originate from DNA and RNA oxidation, were the most widely used indicators for oxidative stress. The study investigated the relation between 8-oxo-dGsn, 8-oxo-Gsn, and CKD. 146 patients with CKD were divided into five disease stages, and their fasting blood and morning urine were collected. The levels of 8-oxo-dGsn and 8-oxo-Gsn in plasma and urine were quantified by LC-MS/MS. The ratio of urinary 8-oxo-Gsn to creatinine increased from stages 1 to 4 corresponding to the increased severity of CKD, but it decreased in stage 5. And plasma 8-oxo-Gsn gradually increased with the decline of renal function. In particular, the increased ratio of plasma and urine 8-oxo-Gsn in stage 5 exceeded the concentration of creatinine. This trend was similar to the estimated glomerular filtration rate (eGFR), which indicates that 8-oxo-Gsn could be an appropriate indicator for renal function. Our finding indicates that as the disease progresses, RNA oxidation is increased. The significant increase in the ratio of plasma and urinary 8-oxo-Gsn is a novel evaluation index of end-stage renal disease.

Levels of 8-oxo-dGsn and 8-oxo-Gsn in random urine are consistent with 24 h urine in healthy subjects and patients with renal disease.

Oxidatively generated damage to nucleic acids may play an important role in the pathophysiological processes of a variety of diseases. 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dGsn) and 8-oxo-7,8-dihydroguanosine (8-oxo-Gsn) are oxidatively generated products of DNA and RNA, respectively. Our previous studies have suggested that the amounts of 8-oxo-dGsn and 8-oxo-Gsn in urine were considerably higher than other body fluid or tissue. The aim of this study was to investigate whether 8-oxo-dGsn and 8-oxo-Gsn levels in random urine samples are consistent with those in 24 h urine samples in healthy subjects and patients with renal disease. A total of 16 healthy subjects and 104 renal disease patients were enrolled in this study, and their random and 24 h urine samples were collected. The levels of urinary 8-oxo-dGsn and 8-oxo-Gsn were quantified by LC-MS/MS and corrected by creatinine. Regardless of healthy subjects or renal disease patients, the levels of oxidised nucleosides in random urine samples were consistent with 24 h urine samples. Regardless of the age bracket, there is no significant difference between random samples and 24 h urine samples. In conclusion, 8-oxo-dGsn and 8-oxo-Gsn levels in random urine samples could replace those in 24 h urine samples, and were considered as the representative of the level of systemic oxidative stress for the whole day.

Effect of 24 cytochrome P450 2D6 variants found in the Chinese population on the N-demethylation of amitriptyline in vitro.

CONTEXT: Amitriptyline (AT), one of the tricyclic antidepressants, is still widely used for the treatment of the depression and control of anxiety states and panic disorders in the developing countries. OBJECTIVE: This study evaluates the catalytic activities of CYP2D6*1, CYP2D6*2, CYP2D6*10 and 22 novel alleles in Han Chinese population and their effects on the N-demethylation of AT in vitro. MATERIALS AND METHODS: CYP2D6*1 and 24 CYP2D6 allelic variants were highly expressed in insect cells, and all variants were characterized using AT as a substrate. Reactions were performed at 37 °C with 10-1000 µM substrate for 30 min. We established a HPLC method to quantify the levels of nortriptyline (NT). The kinetic parameters Km, Vmax and intrinsic clearance (Vmax/Km) of NT were calculated. RESULTS: Among the 24 CYP2D6 variants, all variants exhibited decreased intrinsic clearance values compared with wild-type CYP2D6.1. Kinetic parameters of two CYP2D6 variants (CYP2D6*92, *96) could not be determined because of absent enzyme activities. CONCLUSIONS: The comprehensive in vitro assessment of CYP2D6 variants provides significant insight into allele-specific activity towards AT in vivo.

The role of CYP2C9 genetic polymorphism in carvedilol O-desmethylation in vitro.

We aimed at investigating the role of CYP2C9 in carvedilol O-desmethylation and identifying the effect of 35 CYP2C9 allelic variants we found in Chinese Han population on the in vitro metabolism of carvedilol. Recombinant CYP2C9 and CYP2D6 microsomes of the wild type were used to test and verify the enzymes involved in carvedilol O-desmethylation. Recombinant CYP2C9 microsomes of distinguished genotypes were used to characterize the corresponding enzyme activity toward carvedilol. 2-100 µM carvedilol was incubated for 30 min at 37 °C. The products were detected using high-performance liquid chromatography. CYP2C9 plays a certain role in carvedilol metabolism. Compared with wild-type CYP2C9*1, the intrinsic clearance (V max/K m) values of all variants toward carvedilol O-desmethylation were significantly altered. The variants exhibited significantly decreased values (from 30 to 99.8 %) due to increased K m and/or decreased V max values. We conclude that recombinant system could be used to investigate the enzymes involved in drug metabolism and these findings complement the database where CYP2C9 polymorphism interacts with biotransformation of exogenous substances like drugs and toxins.

Effect of CYP2D6 variants on venlafaxine metabolism in vitro.

1. CYP2D6 is an important member of the cytochrome P450 (CYP450) enzyme superfamily, we recently identified 22 CYP2D6 alleles in the Han Chinese population. The aim of this study was to assess the catalytic activities of these allelic isoforms and their effects on the metabolism of venlafaxine in vitro. 2. The wild-type and 24 CYP2D6 variants were expressed in insect cells, and each variant was characterized using venlafaxine as the substrate. Reactions were performed at 37 °C with 5-500 µM substrate (three variants was adjusted to 1000 µM) for 50 min. By using high-performance liquid chromatography to detect the products, the kinetic parameters Km, Vmax, and intrinsic clearance (Vmax/Km) of O-desmethylvenlafaxine were determined. 3. Among the 22 CYP2D6 variants, the intrinsic clearance (Vmax/Km) values of all variants were significantly decreased (from 0.2% to 84.5%) compared with wild-type CYP2D6*1. In addition, the kinetic parameters of two CYP2D6 variants could not be detected because they have no detectable enzyme activity. 4. The comprehensive in vitro assessment of CYP2D6 variants provides significant insights into allele-specific activity towards venlafaxine in vivo.