Ultrahigh-Throughput ESI-MS: Sampling Pushed to Six Samples per Second by Acoustic Ejection Mass Spectrometry.

We present an acoustic ejection mass spectrometry (AEMS) setup for contactless electrospray ionization mass spectrometry (ESI-MS)-based sample injection at a sampling rate faster than current ESI and matrix-assisted laser desorption ionization (MALDI) techniques. For the direct transfer of samples out of 384-well plates into a modified ESI source, an open port interface (OPI) was combined with a modified acoustic droplet ejection (ADE) system. AEMS has the potential to eliminate bottlenecks known from classical MS approaches, such as speed, reproducibility, carryover, ion suppression, as well as sample preparation and consumption. This setup provided a drastically reduced transfer distance between OPI and ESI electrode for optimum throughput performance and broadens the scope of applications for this emerging technique. To simulate label-free applications of drug metabolism and pharmacokinetics (DMPK) analysis and high-throughput screening (HTS) campaigns, two stress tests were performed regarding ion suppression and system endurance in combination with minor sample preparation. The maximum sampling rate was 6 Hz for dextromethorphan and d3-dextrorphan (each 100 nM) for 1152 injections in 63 s at full width at half-maximum (FWHM) of 105 ms and a relative standard deviation (%RSD) of 7.7/7.5% without internal standard correction. Enzyme assay buffer and crude dog plasma caused signal suppression of 51/73% at %RSD of 5.7/6.7% (n = 120). An HTS endurance buffer was used for >25 000 injections with minor OPI pollution and constant signals (%RSD = 8.5%, FWHM of 177 ms ± 8.5%, n = 10 557). The optimized hardware and method setup resulted in high-throughput performance and enables further implementation in a fully automated platform for ESI-MS-based high-throughput screening.

Vitamin D-related genes and cardiometabolic markers in healthy children: a Mendelian randomisation study.

Observational studies show associations between low serum 25-hydroxyvitamin D (25(OH)D) and cardiometabolic risk markers. This Mendelian randomisation study examined associations between cardiometabolic markers in children and SNP in genes related to vitamin D metabolism (DHCR7; group-specific complement (GC); cytochrome P450 subfamily IIR1 (CYP2R1); and CYP24A1) and action (CYP27B1 and VDR). In 699 healthy 8-11-year-old children, we genotyped eleven SNP. We generated a genetic risk score based on SNP associated with low 25(OH)D and investigated associations between this and blood pressure, plasma lipids and insulin. Furthermore, we examined whether SNP related to vitamin D actions modified associations between 25(OH)D and the cardiometabolic markers. All GC and CYP2R1 SNP influenced serum 25(OH)D. A risk score based on four of the six SNP was associated with 3·4 (95 % CI 2·6, 4·2) mmol/l lower 25(OH)D per risk allele (P < 0·001), but was not associated with the cardiometabolic markers. However, interactions were indicated for the three VDR SNP (Pinteraction < 0·081) on associations between 25(OH)D and TAG, systolic blood pressure and insulin, which all decreased with increasing 25(OH)D only in major allele homozygotes (ß -0·02 (95 % CI -0·04, -0·01) mmol/l; ß -0·5 (95 % CI -0·9, -0·1) mmHg; and ß -0·5 (95 % CI -1·4, 0·3) pmol/l, respectively). In conclusion, genetic variation affected 25(OH)D substantially, but the genetic score was not associated with cardiometabolic markers in children. However, VDR polymorphisms modified associations with vitamin D, which warrants further investigation of VDR's role in the relationship between vitamin D and cardiometabolic risk.

Similarities in mRNA expression of peripheral blood drug metabolizing enzymes and cancer marker genes with biopsy samples of head and neck cancer patients.

Purpose: To develop peripheral blood mRNA expression profiles of drug metabolizing enzymes (DMEs) as a surrogate to monitor tobacco induced head and neck squamous cell carcinoma (HNSCC), attempts were made to investigate (i) similarities in alterations with the cancer marker genes in biopsy samples and (ii) if alterations similar to that seen in biopsy samples are reflected in peripheral blood. Methods: Total RNA from eight soft gingival tissues and eight biopsy samples of HNSCC patients and total DNA and RNA from blood of healthy controls (n = 150) and HNSCC patients (n = 150) was processed for expression and genotyping studies. Blood from patients receiving chemo-radiotherapy was processed for follow-up study. Results: qRT-PCR revealed significant increase in mRNA expression of DMEs in biopsy and blood samples of HNSCC patients when compared to controls. Similar alterations were observed in cancer marker genes in these samples. Patients with variant genotypes of DMEs showed greater magnitude of alterations in mRNA expression when compared to wild type controls. Responders of chemo-radiotherapy showed significant decline in induction of mRNA expression of DMEs and cancer marker genes Conclusions: The data suggest that peripheral blood expression profiles could be used to monitor tobacco-induced HNSCC as well as the treatment response.

Functional expression and purification of CYP93C20, a plant membrane-associated cytochrome P450 from Medicago truncatula.

Plants possess very large numbers of biosynthetic cytochrome P450 enzymes. In spite of the importance of these enzymes for the synthesis of bioactive plant secondary metabolites, only two plant P450 structures has been obtained to date. Isoflavone synthase (IFS) is a membrane-associated cytochrome P450 enzyme catalyzing the entry-point reaction into isoflavonoid biosynthesis. IFS from the model legume Medicago truncatula (CYP93C20) was engineered by deleting the membrane-spanning domain and inserting a hydrophilic polypeptide in the N-terminus and a four histidine tag at the C-terminus. The truncated form exhibited dramatically enhanced expression and solubility. The engineered enzyme was expressed in Escherichia coli XL1-blue cells and was purified by Ni2+-NTA affinity chromatograph and size-exclusion chromatograph. The purified enzyme was characterized by enzyme assay, reduced carbon monoxide difference spectroscopy and peptide mass fingerprinting. The engineered soluble enzyme exhibited the same activity as the full length membrane-associated enzyme expressed in yeast. These studies suggest an approach for engineering plant membrane-associated P450s with enhanced expression and solubility for mechanistic and structural studies.

[Effect of B-vitamin deficiency on biochemical, immunologic markers and trace element status of rats and mice of various lines].

Biochemical, vitamin, trace element and immunological changes were searched for the combined nutritional deficiency of vitamins B1, B2, B6 on in vivo models in rats and mice. Female rats of Wistar (W) strain and hybrids of the 1st generation of Dark Aguti and Wistar (DA x W) strains, female mice of BALB/c strain and DBCB tetrahybrids were used in experiment. Animals received for 35 days a balanced diet (control) according to AIN-93 or a similar diet with the exception of vitamins B1, B2, B6 (experimental groups). The content of vitamins B1, B2 in liver, riboflavin blood plasma level and urinary excretion of thiamine, riboflavin and 4-pyridoxic acid were determined, as well as in rats: blood and liver content of α-tocopherol and retinol, blood biochemical indices of lipid and nitrogen metabolism, activity of cytochrome P isoforms-450 (CYP) in liver; in mice: the circulating levels of pro- and anti-inflammatory cytokines of blood plasma, in animals of both species - the content of essential and toxic elements in the kidneys. DAxW rats compared to W and DBCB mice compared to BALB/c were more sensitive to the development of B-vitamin deficiency judging by the B-vitamin status indicators. In the rats of the experimental groups, there were signs of a deterioration in blood and liver levels of vitamin E, multidirectional shifts in vitamin A sufficiency, increased activity of the CYP3A isoform (6ß-TG), a decrease in triglycerides, total protein and albumin fraction levels with an increase in urea level. Manifestation degree of these effects depended on the choice of the animal's line. In mice, the B-vitamin deficiency was characterized by an increase in the levels of proinflammatory cytokines TNF-α, IL-10, IL-Ιß, IL-6 and a decrease in IFN-γ and IL-17A. The content of magnesium, copper, zinc, chromium and silver was lowered, of cesium - was increased in the kidneys of the rats of the experimental groups. In mice, B-vitamin deficiency resulted in diminishment of magnesium, copper, zinc, chromium, selenium, cadmium and lead content, excess accumulation of cobalt and cesium. Some of these biomarkers are supposed to be used in pre-clinical evaluation of the effectiveness of new vitamin complexes, specialized foods and dietary supplements, as well as studies of interactions of various vitamins.

Specific packaging and circulation of cytochromes P450, especially 2E1 isozyme, in human plasma exosomes and their implications in cellular communications.

Cytochrome P450 (CYP) enzymes metabolize the majority of xenobiotics and are mainly found in hepatic and some extra-hepatic cells. However, their presence and functional role in exosomes, small extracellular vesicles that are secreted from various cells into extracellular fluids including plasma, is unknown. In this study, we analyzed the expression and biological activity of CYP enzymes in human plasma exosomes. First, we optimized isolation of plasma exosomes and characterized them for their physical properties and quality. The results showed that the purity of exosomes (<200 nm) improved upon prior filtration of plasma using a 0.22 micron filter. We then analyzed the relative level of exosomal CYP mRNAs, proteins, and enzyme activity. The results showed that the relative level of CYP enzymes in exosomes is higher than in plasma, suggesting their specific packaging in exosomes. Of the seven CYP enzymes tested, the mRNA of CYP1B1, CYP2A6, CYP2E1, and CYP3A4 were detectable in exosomes. Interestingly, the relative level of CYP2E1 mRNA was >500-fold higher than the other CYPs. The results from the Western blot showed detectable levels of CYP1A1, CYP1B1, CYP2A6, CYP2E1, and CYP3A4. Our results also demonstrated that exosomal CYP2E1 and CYP3A4 show appreciable activity relative to their respective positive controls (CYP-induced baculosomes). Our results also showed that CYP2E1 is expressed relatively higher in plasma exosomes than hepatic and monocytic cells and exosomes derived from these cells. In conclusion, this is the first evidence of the specific packaging and circulation of CYP enzymes, especially CYP2E1, in human plasma exosomes. The findings have biological and clinical significance in terms of their implications in cellular communications and potential use of plasma exosomal CYPs as biomarkers.

Integrating multi-omics biomarkers and postprandial metabolism to develop personalized treatment for anorexia nervosa.

BACKGROUND: Anorexia Nervosa (AN) is a serious mental illness characterized by emaciation, an intense fear of gaining weight despite being underweight, and distorted body image. Few treatments reverse the core symptoms in AN such as profound aversion to food and food avoidance. Consequently, AN has a chronic and relapsing course and the highest mortality rate of any psychiatric illness. A more complete understanding of the disease pathogenesis is needed in order to develop better treatments and improve AN outcome. The pathogenesis and psychopathophysiology of AN can be better elucidated by combining longitudinal phenotyping with multiple "omics" techniques, including genomics, proteomics, lipidomics, and metabolomics. DESIGN: This paper summarizes the key findings of a series of interrelated studies including new experimental data and previously published data, and describes our current initiatives and future directions. RESULTS: Exon sequencing data was analyzed in 1205 AN and 1948 controls. Targeted metabolomics, lipidomics, and proteomics data were collected in two independent convenience samples consisting of 75 subjects with eating disorders and 61 sex- and age-matched healthy controls. Study participants were female and the mean age was 22.9 (4.9 [SD]) years. Epoxide hydrolase 2 (EPHX2) genetic variations were significantly associated with AN risk, and epoxide hydrolase (sEH) activity was elevated in AN compared to controls. The polyunsaturated fatty acids (PUFAs) and eicosanoids data revealed that cytochrome P450 pathway was implicated in AN, and AN displayed a dysregulated postprandial metabolism of PUFAs and sEH-dependent eicosanoids. IMPLICATION AND CURRENT INITIATIVES: Collectively, our data suggest that dietary factors may contribute to the burden of EPHX2-associated AN susceptibility and affect disease outcome. We are implementing new investigations using a longitudinal study design in order to validate and develop an EPHX2 multi-omics biomarker system. We will test whether sEH-associated postprandial metabolism increases AN risk and affects treatment outcome through an ω-6 rich breakfast challenge. Participants will include 100 ill AN patients, 100 recovered AN patients, and 100 age- and race-matched healthy women. These data will allow us to investigate 1) how genetic and dietary factors independently and synergistically contribute to AN risk and progression, and 2) if clinical severity and treatment response in AN are affected by sEH activity and eicosanoid dysregulation. Results of our study will 1) identify clinically relevant biomarkers, 2) unravel mechanistic functions of sEH, and 3) delineate contributory roles of dietary PUFAs and cytochrome P450 pathway eicosanoids for the purpose of developing novel AN treatments and improving disease prognosis.

Use of RNA-seq to determine variation in canine cytochrome P450 mRNA expression between blood, liver, lung, kidney and duodenum in healthy beagles.

RNA sequencing (RNA-seq) is a powerful tool for the evaluation and quantification of transcriptomes and expression patterns in animals, tissues, or pathological conditions. The purpose of this study was to determine the physiologic expression of cytochrome P450 (CYP) mRNA transcripts in whole blood, kidney, duodenum, liver, and lung in healthy, adult male (n = 4) and female (n = 4) beagles via RNA-seq. mRNA expression was above background (transcripts per million) for 45 canine CYPs, with liver, duodenum, and lung expressing a high number of xenobiotic metabolizing CYPs, while prominent endogenous metabolizing CYP expression was present in blood and kidney. The relative expression pattern of CYP2A13, 2B11, 2C21, 2D15, 2E1, 3A12, and 27A1 in liver, lung, and duodenum was verified through qPCR. This is the first global profiling of physiologic CYP mRNA expression in multiple canine tissues, providing a platform for further studies characterizing canine CYPs and changes in gene expression in disease states.

Cytochrome P450-derived epoxyeicosatrienoic acids and coronary artery disease in humans: a targeted metabolomics study.

Cytochrome P450 (CYP)-derived epoxyeicosatrienoic acids (EETs) exhibit potent cardiovascular protective effects in preclinical models, and promoting the effects of EETs has emerged as a potential therapeutic strategy for coronary artery disease (CAD). The relationship between circulating EET levels and CAD extent in humans, however, remains unknown. A panel of free (unesterified) plasma eicosanoid metabolites was quantified in 162 patients referred for coronary angiography, and associations with extent of CAD [no apparent CAD (N = 39), nonobstructive CAD (N = 51), and obstructive CAD (N = 72)] were evaluated. A significant relationship between free EET levels and CAD extent was observed (P = 0.003) such that the presence of obstructive CAD was associated with lower circulating EET levels. This relationship was confirmed in multiple regression analysis where CAD extent was inversely and significantly associated with EET levels (P = 0.013), and with a biomarker of EET biosynthesis (P < 0.001), independent of clinical and demographic factors. Furthermore, quantitative enrichment analysis revealed that these associations were the most pronounced compared with other eicosanoid metabolism pathways. Collectively, these findings suggest that the presence of obstructive CAD is associated with lower EET metabolite levels secondary to suppressed EET biosynthesis. Novel strategies that promote the effects of EETs may have therapeutic promise for patients with obstructive CAD.

Usefulness of oxidative stress biomarkers evaluated in the snout scraping, serum and Peripheral Blood Cells of Crocodylus moreletii from Southeast Campeche for assessment of the toxic impact of PAHs, metals and total phenols.

In this study, we assessed the effects of inorganic and organic pollutants [As, Cu, Fe, Mn, Pb, Zn, PAHs (11 compounds) and total phenols] from a panel of biomarkers [O2, H2O2, thiobarbituric acid reactive substances (TBARS), carbonyl proteins (RCO), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and total cytochrome P450 activities] evaluated in the Snout Scraping (SS), Serum (S) and Peripheral Blood Cells (PBC) of the Morelet's crocodile (Crocodylus moreletii) inhabiting the reference locality (Lake Mocu) and polluted locality (Champoton River) using Principal Component Analysis (PCA). In male crocodiles from the reference site, only H2O2 in PBC was related to levels of fluoranthene on the Keel of Caudal Scales (KCS), but, in females, no association was detected. In contrast, a sex-linked response was detected in specimens from the polluted locality. Levels of benzo[a]pyrene, benzo[a]anthracene, chrysene, pyrene, phenanthrene, acenaphthene, Zn, Cu, and Pb in KCS of the female crocodil were related to the oxidative stress biomarkers on PBC, incluing the total CYP450 activity and levels of O2, H2O2 in serum. However, in male crocodiles, the oxidative stress in SS and in the serum (TBARS, RCO, CAT, GPx), and SOD in PBC was related to As, Pb, Cu, Fe, and benzo[a]pyrene water concentrations and to the burdens of As, Fe, Mn, indeno[1,2,3cd]pyrene in KCS. These results confirm the usefulness of minimal or non-invasive methods of evaluating the oxidative stress response for the environmental monitoring program on the wild Morelet's crocodile that is subject to special protection in Mexican guidelines.

Gene-environment interaction in preterm delivery with special reference to organochlorine pesticides.

We investigated the association between glutathione S-transferases mu1 (GSTM1), theta 1 (GSTT1), Cytochrome P450IA1-T6235C (rs4646903, CYP1A1m1) and CYP1A1-1462V (rs1048943, CYP1A1m2) gene polymorphisms, and organochlorine pesticides (OCPs) level with risk of preterm delivery (PTD). Maternal and cord blood samples of PTD (n = 156) cases and subjects of full-term delivery (FTD, n = 151) were collected at the time of delivery/after delivery. Women occupationally exposed to OCPs and other high-risk factors such as anemia, hypertension and dietary habit were excluded. The OCP levels were estimated by gas chromatography, and polymorphic analysis of GSTM1/GSTT1 and CYP450 genes was carried out using multiplex PCR and PCR-restriction fragment length polymorphism, respectively. The frequency of GSTM1/GSTT1 (null) genotype was significantly higher in PTD cases than in the controls. Significantly high levels of α-hexachlorocyclohexane (HCH), γ-HCH and Dichlorodiphenyldichloroethylene (p'p'-DDE) were observed in maternal blood, while significantly high levels of p,p'-dichlorodiphenyltrichloroethane and p'p'-DDE were found in the cord blood of PTD cases compared with the controls. A significant association was seen between ß-HCH and GSTM1 genotype when interaction between GSTM1 gene polymorphism, maternal blood OCP levels and period of gestation (POG) was ascertained. A significant reduction in POG was observed. Similarly, cord blood dieldrin levels were significantly associated with CYP1A1m2 (Aa/aa) with reduction in POG. Our observations indicate that higher levels of OCPs in pregnant women may be associated with increased risk of 'idiopathic' PTD. Furthermore, this study shows that the interaction between high OCPs levels and polymorphism in CYP1A1m2 and GSTM1 null genotypes may magnify the risk of PTD, thus providing evidence for a gene-environment interaction in pregnant women.

The role of 14,15-dihydroxyeicosatrienoic acid levels in inflammation and its relationship to lipoproteins.

BACKGROUND: 14,15-Epoxyeicosatrienoic acids (14,15-EETs) generated from arachidonic acid by cytochrome P450 epoxygenases have beneficial effects in certain cardiovascular diseases, and increased 14,15-EET levels protect the cardiovascular system. 14,15-EETs are rapidly hydrolyzed by soluble epoxide hydrolase (sEH) to the corresponding 14,15-dihydroxyeicosatrienoic acids (14,15-DHETs), which are generally less biologically active but more stable metabolite. A functionally relevant polymorphism of the CYP2J2 gene is independently associated with an increased risk of coronary heart disease (CHD), and the major CYP2J2 product is 14,15-EETs. 14,15-DHETs can be considered a relevant marker of CYP2J2 activity. Therefore, the aim of the present study was to evaluate the plasma 14,15-DHET levels to reflect the 14,15-EET levels in an indirectly way in patients with CHD, and to highlight the growing body of evidence that 14,15-EETs also play a role in anti-inflammatory and lipid-regulating effects in patients with CHD. This was achieved by investigating the relationship between 14,15-DHETs and high-sensitivity C-reactive protein (hs-CRP) and blood lipoproteins. METHODS: Samples of peripheral venous blood were drawn from 60 patients with CHD and 60 healthy controls. A 14,15-DHET enzyme-linked immunosorbent assay kit (14,15-DHET ELISA kit) was used to measure the plasma 14,15-DHET levels. Hs-CRP, total cholesterol, triglyceride, high-density lipoprotein cholesterol, and low-density lipoprotein-cholesterol levels were measured. RESULTS: 14,15-DHET levels (2.53 ± 1.60 ng/mL) were significantly higher in patients with CHD as compared with those of the healthy controls (1.65 ± 1.54 ng/mL, P < 0.05). There was a significant positive correlation between 14,15-DHETs and hs-CRP levels (R = 0.286, P = 0.027). However, there was no significant correlation between 14,15-DHETs and blood lipoproteins (all, P > 0.05). CONCLUSIONS: Increased plasma 14,15-DHET levels reflect the decreased of 14,15-EET levels in an indirectly way. Indicated that decreased plasma 14,15-EET levels might be involved in the inflammatory reaction process in atherosclerosis.

Effects of Panax notoginseng saponins on the activities of CYP1A2, CYP2C9, CYP2D6 and CYP3A4 in rats in vivo.

The aim of this study was to assess the influence of the Panax notoginseng saponins (PNS) on the activities of the drug-metabolizing enzymes cytochrome P450 (CYP450) 1A2, 2 C9, 2D6 and 3A4 in rats. The activities of CYP1A2, 2 C9, 2D6 and 3A4 were measured using specific probe drugs. After pretreatment for 1 week with PNS or physiological saline (control group), probe drugs caffeine (10 mg/kg; CYP1A2 activity), tolbutamide (15 mg/kg; CYP2C9 activity), metoprolol (20 mg/kg; CYP2D6 activity) and dapsone (10 mg/kg; CYP3A4 activity) were administered to rats by intraperitoneal injection. The blood was then collected at different times for ultra performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) analysis. The data showed that PNS exhibited an induction effect on CYP1A2 by decreasing caffeine C(max) (36.3%, p < 0.01) and AUC(0-∞) (22.77%, p < 0.05) and increasing CL/F (27.03%, p < 0.05) compared with those of the control group. Western blot analysis was used to detect the effect of PNS on the protein level of CYP1A2, and the results showed that PNS could upregulate the protein expression of CYP1A2. However, no significant changes in CYP2C9, 2D6 or 3A4 activities were observed. In conclusion, the results indicate that PNS could induce CYP1A2, which may affect the disposition of medicines primarily dependent on the CYP1A2 pathway. Our work may be the basis of related herb-drug interactions in the clinic.

[Effects of polyvitamin deficiency on the activity of xenobiotic-metabolizing enzymes in rat liver].

The activity of xenobiotic-metabolizing enzymes was studied in the liver of male Wistar rats, which were fed for 4 weeks diets, containing 100 (control), 50 and 20% of vitamin adequate level. Moderate (50%) polyvitamin deficiency increased activity of EROD (by 13%), MROD (by 34%; p<0,05), 4-nitrophenol hydroxylase (by 16%), 6beta-testosterone hydroxylase (by 17%), UDP-glucuronosyle transferase (by 26%, p<0,05) and quinone reductase (by 55%, p<0,05). Deep (20%) polyvitamin deficiency decreased in liver activity of MROD (to 78% of control level, p<0,05), 4-nitrophenol hydroxylase (to 74%, p<0,05), heme oxygenase-1 (to 83%, p<0,05) and quinone reductase (to 60%, p<0,05). At the same time a 22% increase in the UDP-glucuronosyle transferase activity compared to the control group was found; activities of EROD, PROD, 6beta-testosterone hydroxylase and the total activity of glutathione S-transferase were unchanged. Deep polyvitamin deficiency had no significant effect on CYP1A1 mRNA and AhR mRNA level, whereas the expression of CYP1A2 mRNA and CYP3A1 mRNA were decreased to 62 and 79%, respectively, as compared with control.

Cytochrome P450 metabolites of arachidonic acid are elevated in stroke patients compared with healthy controls.

CYP450AAM [arachidonic acid metabolites of the CYP450 (cytochrome P450) enzyme system] have a range of biological functions. CYP450AAM are involved in the pathogenesis of hypertension, renal function and vascular function, yet their role in stroke has not been clarified. We aimed at determining the levels of circulating CYP450 metabolites in patients with acute ischaemic stroke (<96 h) compared with healthy age- and gender-matched controls. This was a retrospective case-controlled study of 44 acute ischaemic stroke patients and 44 matched controls. A subset of acute ischaemic stroke patients was available for follow-up. Acute ischaemic stroke patients had elevated plasma CYP450AAM, including 20-HETE (20-hydroxyeicosatetraenoic acid) (1921±170 compared with 1108±170 pmol/l, P<0.001), EETs (epoxyeicosatrienoic acids) (77.88±3.34 compared with 35.35±3.34 nmol/l, P<0.0001) and DiHETEs (dihydroxyeicosatetraenoic acids) (92.87±4.61 compared with 68.17±4.61 nmol/l, P<0.0001), as well as increased plasma F2-isoprostane levels (3754±538 compared with 1947±538 pmol/l, P<0.02), the latter a marker of oxidative stress, compared with controls. In a subset analysis of the stroke patients, plasma 20-HETE, EETs and F2-isoprostanes were attenuated 30 days after the stroke. Baseline 20-HETE levels were also associated with lesion size and functional indices within the stroke patients. The present study highlights the elevation in CYP450AAM and oxidative stress in acute ischaemic stroke patients. Further investigation of the effect this has on long-term clinical outcome or whether this can be modified by treatment is warranted.

Cytochrome P450 epoxygenase CYP2J2 attenuates nephropathy in streptozotocin-induced diabetic mice.

Cytochrome P450 (CYP) epoxygenases metabolize arachidonic acid into epoxyeicosatrienoic acids (EETs), which play important and diverse roles in the cardiovascular system. The anti-inflammatory, anti-apoptotic, pro-angiogenic, and anti-hypertensive properties of EETs in the cardiovascular system suggest a beneficial role for EETs in diabetic nephropathy. This study investigated the effects of endothelial specific overexpression of CYP2J2 epoxygenase on diabetic nephropathy in streptozotocin-induced diabetic mice. Endothelial CYP2J2 overexpression attenuated renal damage as measured by urinary microalbumin and glomerulosclerosis. These effects were associated with inhibition of TGF-ß/Smad signaling in the kidney. Indeed, overexpression of CYP2J2 prevented TGF-ß1-induced renal tubular epithelial-mesenchymal transition in vitro. These findings highlight the beneficial roles of the CYP epoxygenase-EET system in the pathogenesis of diabetic nephropathy.

Development and validation of a LC-MS/MS method based on a new 96-well Hybrid-SPE™-precipitation technique for quantification of CYP450 substrates/metabolites in rat plasma.

A rapid and selective high-throughput HESI-LC-MS/MS method for determining eight cytochrome P450 probe drugs in one-step extraction and single run was developed and validated. The four specific probe substrates midazolam, dextromethorphan, tolbutamide, theophylline and their metabolites 1-hydroxymidazolam, dextrorphan, hydroxyl(methyl)tolbutamide, 1,3-dimethyluric acid, together with the deuterated internal standards, were extracted from rat plasma using a novel 96-well Hybrid-SPE™-precipitation technique. The bioanalytical assay was based on reversed phase liquid chromatography coupled with tandem mass spectrometry in the positive ion mode using selected reaction monitoring for drug (-metabolite) quantification. All analytes were separated simultaneously in a single run that lasted less than 11 min. The intra- and inter-day precisions for all eight substrates/metabolites were 1.62-12.81% and 2.09-13.02%, respectively, and the relative errors (accuracy) for the eight compounds ranged from -9.62% to 7.48% and -13.84% to 8.82%. Hence, the present method provides a robust, fast and reproducible analytical tool for the evaluation of four major drug metabolising cytochrome P450 (3A4, 2C9, 1A2 and 2D6) activities with a cocktail approach in rats to clarify herb-drug interactions. The method can be used as a basic common validated high-throughput analytical assay for in vivo interaction studies.

Application of dried blood spot sampling combined with LC-MS/MS for genotyping and phenotyping of CYP450 enzymes in healthy volunteers.

An early clinical development study (phase I) was conducted to determine the usefulness of dried blood spot (DBS) sampling as an alternative to venous sampling for phenotyping and genotyping of CYP450 enzymes in healthy volunteers. Midazolam (MDZ) was used as a substrate for phenotyping CYP3A4 activity; the concentrations of MDZ and its main metabolite 1'-hydroxymidazolam (1-OH MDZ) were compared between the DBS method from finger punctures, plasma and whole blood (WB), drawn by venipuncture, whereby several methodological parameters were studied (i.e. punch width, amount of dots analyzed and storage time stability). Genotyping between DBS and venous WB samples was compared for CYP2D6 (*3, *4, *6), CYP2C19 (*2, *3), CYP3A4 (*1B) and CYP3A5 (*3C). In addition, the subject's and phlebotomist's satisfaction with venous blood sampling compared with the DBS method was evaluated using a standardized questionnaire. An LC-MS/MS method for the quantification of the MDZ and 1-OH MDZ concentrations in DBS samples was developed and validated in the range of 0.100-100 ng/mL. No compromises were made for the limits of quantification of the DBS-LC-MS/MS method vs the authentic plasma and WB methods.

A critical evaluation of the cardiac toxicity of citalopram: part 2.

In August 2011, the U.S. Food and Drug Administration issued a drug safety communication that the antidepressant drug citalopram (Celexa®) should not be used at dosages greater than 40 mg per day (or greater than 20 mg per day for patients 60 and older) because higher doses have been associated with abnormal heart rhythms. Clinical studies using citalopram in patients with cardiac disease and in older patients do not confirm such a risk. The major metabolite of citalopram is demethylcitalopram, which is subsequently metabolized to the minor metabolite didemethylcitalopram (DDCT). High DDCT concentrations have been associated with QT interval prolongation in beagle dogs. Therapeutic drug monitoring study data suggest that routine or even high oral doses of citalopram are unlikely to result in cardiotoxic concentrations of the DDCT metabolite. Based on evidence taken from a wide variety of studies, the citalopram dose limitations described in the safety announcement do not have strong clinical justification.

Study on the effects of Zhuanggu Guanjie Pill, a modern Chinese medicine formula, on the activities and mRNA expression of seven CYP isozymes in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Zhuanggu Guanjie Pill (ZGGJP), a modern Chinese medicine formula, is composed of 12 herbs and has been used to treat osteoporosis in China for almost 30 years. However, no in vivo study of the influences of ZGGJP on the cytochrome P450 (CYP) activities have been reported. AIM OF THE STUDY: The aim of this study was to evaluate the effects of ZGGJP on the activities and the mRNA expression of CYP enzymes (CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A) and their corresponding nuclear receptor levels in rats. MATERIALS AND METHODS: After 7 days oral treatment of ZGGJP at low- and high-dose, cocktail solution was given to rats. Blood samples were collected at series of time points. The plasma concentrations of probe drugs and their corresponding metabolites were determined by UPLC-MS/MS. The influence of ZGGJP on the activities of seven CYPs were evaluated the metabolic ratios (Cmax and AUC0-t) for metabolites/probe drugs. In addition, the effects of ZGGJP on the mRNA expression of CYPs and their corresponding nuclear receptors in rat liver were evaluated by real-time PCR. RESULTS: ZGGJP showed significant inductive effects on CYP1A2 and CYP2B6 of both male and female rats. The influence of ZGGJP on CYP2C9 and CYP3A showed gender difference. ZGGJP could induce the activities of CYP2C9 and CYP3A in female rats, but have no influence on the activities in male rats. ZGGJP had no effects on CYP2D6, CYP2C19 and CYP2E1. The mRNA expression results of CYPs were in accordance with the pharmacokinetic results. The mRNA expression levels of constitutive androstane receptor (CAR) and vitamin D receptor (VDR) were increased significantly in female rats at high dosage, but no significant changes were observed in male rats. CONCLUSION: ZGGJP had inductive effects on CYP1A2 and CYP2B6 in both male and female rats. The results showed that ZGGJP could induce the activities of CYP2C9 and CYP3A in female rats, but had no effect in male rats. This may suggest that the influence of ZGGJP on CYP2C9 and CYP3A exhibit gender difference. The inductive effects of ZGGJP on the activities of CYPs, exhibiting gender difference, may be regulated by CAR and VDR. Therefore, co-administration of ZGGJP with other drugs, especially using CYP2C9 and CYP3A substrates in females, may need dose adjustment to avoid herb-drug interaction.