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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.
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Inductores de las Enzimas del Citocromo P-450/farmacología , Sistema Enzimático del Citocromo P-450/genética , Medicamentos Herbarios Chinos/farmacología , Isoenzimas/genética , Animales , Sistema Enzimático del Citocromo P-450/sangre , Femenino , Interacciones de Hierba-Droga , Isoenzimas/sangre , Masculino , Medicina Tradicional China , ARN Mensajero/metabolismo , Ratas Wistar , Receptores Citoplasmáticos y Nucleares/sangre , Receptores Citoplasmáticos y Nucleares/genéticaRESUMEN
Very little is known about how metabolic health status, insulin resistance or metabolic challenges modulate the endocannabinoid (eCB) or polyunsaturated fatty acid (PUFA)-derived oxylipin (OxL) lipid classes. To address these questions, plasma eCB and OxL concentrations were determined at rest, 10 and 20 min during an acute exercise bout (30 min total, ~45% of preintervention VÌO2peak , ~63 W), and following 20 min recovery in overnight-fasted sedentary, obese, insulin-resistant women under controlled diet conditions. We hypothesized that increased fitness and insulin sensitivity following a ~14-week training and weight loss intervention would lead to significant changes in lipid signatures using an identical acute exercise protocol to preintervention. In the first 10 min of exercise, concentrations of a suite of OxL diols and hydroxyeicosatetraenoic acid (HETE) metabolites dropped significantly. There was no increase in 12,13-DiHOME, previously reported to increase with exercise and proposed to activate muscle fatty acid uptake and tissue metabolism. Following weight loss intervention, exercise-associated reductions were more pronounced for several linoleate and alpha-linolenate metabolites including DiHOMEs, DiHODEs, KODEs, and EpODEs, and fasting concentrations of 9,10-DiHODE, 12,13-DiHODE, and 9,10-DiHOME were reduced. These findings suggest that improved metabolic health modifies soluble epoxide hydrolase, cytochrome P450 epoxygenase (CYP), and lipoxygenase (LOX) systems. Acute exercise led to reductions for most eCB metabolites, with no evidence for concentration increases even at recovery. It is proposed that during submaximal aerobic exercise, nonoxidative fates of long-chain saturated, monounsaturated, and PUFAs are attenuated in tissues that are important contributors to the blood OxL and eCB pools.
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Terapia por Ejercicio/métodos , Obesidad/terapia , Oxilipinas/sangre , Programas de Reducción de Peso/métodos , Adulto , Citocromo P-450 CYP2J2 , Sistema Enzimático del Citocromo P-450/sangre , Epóxido Hidrolasas/sangre , Femenino , Humanos , Resistencia a la Insulina , Ácido Linoleico/sangre , Lipooxigenasa/sangre , Persona de Mediana Edad , Obesidad/sangre , Conducta SedentariaRESUMEN
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 >25â¯000 injections with minor OPI pollution and constant signals (%RSD = 8.5%, FWHM of 177 ms ± 8.5%, n = 10â¯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.
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Acústica , Sistema Enzimático del Citocromo P-450/sangre , Dextrometorfano/análisis , Dextrorfano/análisis , Ensayos Analíticos de Alto Rendimiento , Animales , Sistema Enzimático del Citocromo P-450/metabolismo , Perros , Electrodos , Femenino , Ensayos Analíticos de Alto Rendimiento/instrumentación , Masculino , Tamaño de la Partícula , Espectrometría de Masa por Ionización de Electrospray/instrumentación , Factores de TiempoRESUMEN
Measuring in vivo changes in the drug metabolizing activity of cytochrome P450 (CYP) enzymes is critical to understanding and assessing drug-drug, drug-diet and drug-disease interactions. The sensitivity and specificity of ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) makes it an ideal tool for analyzing drugs and their metabolites in biological matrices, and has demonstrated utility in CYP phenotyping across varied applications. Published CYP phenotyping cocktail assays often require large plasma sample volumes (0.5-1 mL), have relatively low sensitivity and multi-step complex sample preparation and extraction procedures. Further, variability exists in the way that recovery and matrix effects are investigated and reported, and some studies fail to report these data altogether. Therefore, the aim of this study was to develop, validate and optimize a simplified assay for the probe drugs caffeine (metabolized by CYP1A2), omeprazole (CYP2C19), losartan (CYP2C9), dextromethorphan (CYP2D6), midazolam (CYP3A4) and their respective enzyme-specific metabolites in small volumes (100 µL) of human plasma, that addresses the issues noted. Analyte extraction involved protein precipitation with acetonitrile and solid-phase extraction (SPE). Samples were analyzed using an Agilent 1290 infinity LC system in tandem with 6460A triple quadrupole mass spectrometers. The assay met FDA guideline-recommended requirements for specificity, sensitivity (analyte LLOQs 0.78-23.4 ng/mL), accuracy (intra-day RE% nominal concentration 90.7-110.2%; inter-day RE% 87.0-110.5%) and precision (intra-day analyte RSD% 0.46-11.4%; inter-day RSD% 1.36-11.2%). Recovery and matrix effects were thoroughly investigated and excluded as potential interferers with assay performance. This assay has been used successfully to phenotype CYP activity in a human clinical trial participant. Importantly, the authors provide a contemporary commentary on commonly found issues in the CYP phenotyping cocktail assay literature, and make recommendations concerning best-practice approaches and the standardization of data reporting in this area.
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Sistema Enzimático del Citocromo P-450/sangre , Sistema Enzimático del Citocromo P-450/clasificación , Técnicas Biosensibles/métodos , Cafeína/metabolismo , Cromatografía Líquida de Alta Presión , Dextrometorfano/metabolismo , Interacciones Farmacológicas , Humanos , Límite de Detección , Losartán/metabolismo , Midazolam/metabolismo , Omeprazol/metabolismo , Fenotipo , Sensibilidad y Especificidad , Espectrometría de Masas en TándemRESUMEN
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.
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Sistema Enzimático del Citocromo P-450/sangre , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/sangre , Receptores de Calcitriol/sangre , Deficiencia de Vitamina D/genética , Vitamina D/análogos & derivados , 25-Hidroxivitamina D3 1-alfa-Hidroxilasa/sangre , Alelos , Biomarcadores/sangre , Presión Sanguínea/genética , Factores de Riesgo Cardiometabólico , Niño , Colestanotriol 26-Monooxigenasa/sangre , Familia 2 del Citocromo P450/sangre , Femenino , Genotipo , Voluntarios Sanos , Homocigoto , Humanos , Insulina/sangre , Lípidos/sangre , Masculino , Análisis de la Aleatorización Mendeliana , Polimorfismo de Nucleótido Simple , Medición de Riesgo , Vitamina D/sangre , Vitamina D3 24-Hidroxilasa/sangreRESUMEN
BACKGROUND: The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor, which is critically involved in the pathogenesis of a variety of skin diseases. The aim of this study was to detect AhR and its downstream regulators including cytochrome P450 (CYP1A1), AhR nuclear translocation (ARNT), and aryl hydrocarbon receptor repressor (AhRR) in serum, peripheral blood mononuclear cells (PBMCs), and skin lesions in patients with atopic dermatitis (AD). METHODS: Twenty-nine AD patients defined according to the criteria of Hanifin and Rajka and Chinese criteria of AD were included. Subjects without allergic and chronic diseases were recruited as controls. Patients and controls were selected from the dermatology outpatient clinic of Peking University People's Hospital from August 1 to December 31 in 2018. Enzyme-linked immunosorbent assay was performed to detect serum AhR level. The mRNA of AhR, AhRR, ARNT, and CYP1A1 in PBMCs were measured by real-time quantitative polymerase chain reaction. AhR expression in skin lesions was measured by immunohistochemistry. RESULTS: AhR was significantly higher expressed in serum (41.26â±â4.52 vs. 33.73â±â2.49 pmol/L, tâ=â6.507, Pâ<â0.001) and skin lesions (0.191â±â0.041 vs. 0.087â±â0.017, tâ=â10.036, Pâ<â0.001) of AD patients compared with those of controls. The mRNA levels of AhR (1.572â±â0.392 vs. 1.000â±â0.173, tâ=â6.819, Pâ<â0.001), AhRR (2.402â±â1.716 vs. 1.000â±â0.788, tâ=â3.722, Pâ<â0.001), CYP1A1 (2.258â±â1.598 vs. 1.000â±â0.796, tâ=â3.400, Pâ=â0.002) in PBMCs of AD patients were higher compared with those of controls. The difference in mRNA levels of ARNT was not statistically significant between the patients and controls (1.383â±â0.842 vs. 1.000â±â0.586, tâ=â1.653, Pâ=â0.105). AhR mRNA levels in PBMCs positively correlated with eczema area and severity index score and serum interleukin-6 levels. CONCLUSION: AhR and its downstream regulators were highly expressed in serum, PBMCs, and skin of AD patients, which might contribute to the pathogenesis of AD.
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Dermatitis Atópica/sangre , Dermatitis Atópica/metabolismo , Leucocitos Mononucleares/metabolismo , Receptores de Hidrocarburo de Aril/sangre , Receptores de Hidrocarburo de Aril/metabolismo , Enfermedades de la Piel/sangre , Enfermedades de la Piel/metabolismo , Adulto , Anciano , Translocador Nuclear del Receptor de Aril Hidrocarburo/sangre , Translocador Nuclear del Receptor de Aril Hidrocarburo/metabolismo , Sistema Enzimático del Citocromo P-450/sangre , Sistema Enzimático del Citocromo P-450/metabolismo , Femenino , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , ARN Mensajero/metabolismo , Proteínas Represoras/sangre , Proteínas Represoras/metabolismoRESUMEN
Epoxides derived from arachidonic acid (AA) are released during exercise and may contribute to vasodilation. However, exercise may also affect circulating levels of other epoxides derived from cytochromes P450 (CYP) monooxygenase and lipoxygenase (LOX) pathways, many of whose exhibit cardiovascular activity in vitro. The effects of exercise on their levels have not been documented. We tested the hypothesis that acute, maximal exercise would influence the plasma concentrations of these vasoactive substances. We measured plasma CYP and LOX mediators derived from both the n - 3 and n - 6 fatty acid (FA) classes in healthy volunteers before, during and after short-term exhaustive exercise. Lipid mediators were profiled by means of LC-MS/MS tandem mass spectrometry. A maximal Bruce treadmill test was performed to voluntary exhaustion. Exhaustive exercise increased the circulating levels of epoxyoctadecenoic (12,13-EpOME), dihydroxyeicosatrienoic (5,6-DHET), dihydroxyeicosatetraenoic acids (5,6-DiHETE, 17,18-DiHETE), but had no effect on the majority of CYP and LOX metabolites. Although our calculations of diol/epoxide ratios revealed preferred hydrolysis of epoxyeicosatrienoic acids (EEQs) into their diols (DiHETEs), this hydrolysis was resistant to maximal exercise. Our study is the first documentation that bioactive endogenous n - 3 and n - 6 CYP lipid mediators are released by short-term exhaustive exercise in humans. In particular, the CYP epoxy-metabolite status, 12,13-EpOME/DiHOME, 5,6-EET/DHET, 5,6-EEQ/DiHETE and 17,18-EEQ/DiHETE may contribute to the cardiovascular response during maximal exercise.
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Ácido 8,11,14-Eicosatrienoico/sangre , Sistema Enzimático del Citocromo P-450/sangre , Ejercicio Físico , Lipooxigenasa/sangre , Ácido 8,11,14-Eicosatrienoico/metabolismo , Adulto , Prueba de Esfuerzo , Femenino , Humanos , Lipidómica , Masculino , Persona de Mediana EdadRESUMEN
BACKGROUND: Per- and polyfluoroalkyl substances (PFASs) are widely used in China, but little is known about the association between prenatal PFASs exposure and fetal reproductive development as well as its potential mechanism. OBJECTIVE: We investigated the effects of cord blood PFASs on fetal reproductive hormones and its potential mechanism in relation to steroidogenic enzymes. METHODS: Ten selected PFASs (nâ¯=â¯351) including PFOS, PFOA, PFBS, PFDA, PFDoA, PFHpA, PFHxS, PFNA, PFOSA, and PFUA, and two reproductive hormones estradiol (E2) (nâ¯=â¯351) and testosterone (T) (nâ¯=â¯349) were measured in 351 cord blood serum samples from a Chinese birth cohort between 2010 and 2013. Three steroidogenic enzymes including P450arom (nâ¯=â¯125), 3ß-HSD1 (nâ¯=â¯123), and 17ß-HSD1 (nâ¯=â¯116) were measured in 125 placental tissue samples. Linear regression tested the associations between cord blood PFASs and reproductive hormones in cord blood. Mediation analysis assessed the role of placental steroidogenic enzymes between cord blood PFASs and reproductive hormones. RESULTS: The positive associations between PFOA, PFHxS and E2 levels, PFOS, PFUA, PFNA and T levels, and PFOS, PFUA and T/E2 ratio were significant. PFUA, PFNA, PFDA, PFHxS, and ∑PFASs were associated with higher P450arom levels. PFHxS was also associated with increased 3ß-HSD1 and 17ß-HSD1 levels. These associations were more pronounced in females than males when stratified by gender. Furthermore, 17ß-HSD1 demonstrated mediating effects in the positive association between cord blood PFHxS and E2 levels in females. CONCLUSION: Our findings suggested the potential impacts of cord blood PFASs on fetal reproductive hormones, in which steroidogenic enzymes may play important roles. These associations were more pronounced in females than males.
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Sistema Enzimático del Citocromo P-450/sangre , Contaminantes Ambientales/sangre , Estradiol/sangre , Sangre Fetal/química , Fluorocarburos/sangre , Testosterona/sangre , China , Femenino , Humanos , Masculino , Placenta , EmbarazoRESUMEN
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.
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Biomarcadores de Tumor/genética , Sistema Enzimático del Citocromo P-450/genética , Neoplasias de Cabeza y Cuello/genética , Proteínas de Neoplasias/genética , ARN Mensajero/genética , Carcinoma de Células Escamosas de Cabeza y Cuello/genética , Adulto , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Biomarcadores de Tumor/sangre , Biopsia , Estudios de Casos y Controles , Sistema Enzimático del Citocromo P-450/sangre , ADN de Neoplasias/genética , ADN de Neoplasias/metabolismo , Rayos gamma/uso terapéutico , Expresión Génica , Perfilación de la Expresión Génica , Encía/metabolismo , Encía/patología , Neoplasias de Cabeza y Cuello/etiología , Neoplasias de Cabeza y Cuello/metabolismo , Neoplasias de Cabeza y Cuello/terapia , Humanos , India , Masculino , Persona de Mediana Edad , Proteínas de Neoplasias/sangre , ARN Mensajero/metabolismo , ARN Neoplásico/genética , ARN Neoplásico/metabolismo , Carcinoma de Células Escamosas de Cabeza y Cuello/etiología , Carcinoma de Células Escamosas de Cabeza y Cuello/metabolismo , Carcinoma de Células Escamosas de Cabeza y Cuello/terapia , Uso de Tabaco/efectos adversosRESUMEN
Background Hibiscus sabdariffa beverage (HSB) is widely consumed as a medicinal herb and sometimes used concomitantly with drugs. This study evaluated the in vitro inhibitory potential of the aqueous extract of H. sabdariffa calyces (AEHS) on selected cytochrome P450 (CYP) isozymes and the effect of HSB on the pharmacokinetics of caffeine in vivo. Methods In vitro inhibitions of eight major CYP isozymes by AEHS were estimated by monitoring CYP-specific model reactions of 10 CYP probe substrates using N-in-one assay method. Subsequently, an open, randomized, two-period crossover design was used to evaluate the effect of HSB on the pharmacokinetics of single-dose 200 mg caffeine in six healthy human volunteers. Blood samples were obtained at specific times over a 24 h period. Probe drugs and metabolites were analyzed in their respective matrices with ultra-performance liquid chromatography/mass spectrometer/mass spectrometer and reversed-phase high-performance liquid chromatography/ultraviolet detection. Results The H. sabdariffa aqueous extract weakly inhibited the selected CYP isozymes in vitro, with IC50 of >100 µgmL-1 in the order of CYP1A2 > CYP2C8 > CYP2B6 >> CYP2D6 > CYP2C19 > CYP3A4 > CYP2A6 > CYP2C9. HSB decreased terminal t1/2 and Tmax of caffeine by 13.6% and 13.0%, respectively, and increased Cmax by 10.3%. Point estimates of primary pharmacokinetic endpoints, Cmax = 1.142 (90% confidence interval (CI) = 0.882, 1.480) and AUC0-∞ = 0.992 (90% CI = 0.745, 1.320), were outside the 90% CI of 0.8-1.25 bioequivalence limits. Conclusion The aqueous extract of H. sabdariffa weakly inhibited eight CYP isozymes in vitro, but HSB modified the exposure to caffeine in human. Caution should be exercised in administering HSB with caffeine or similar substrates of CYP1A2 until more clinical data are available.
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Cafeína/farmacocinética , Sistema Enzimático del Citocromo P-450/sangre , Interacciones de Hierba-Droga , Hibiscus/química , Extractos Vegetales/farmacología , Cafeína/sangre , Estudios Cruzados , Voluntarios Sanos , Humanos , Isoenzimas/sangre , Especificidad por SustratoRESUMEN
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.
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Avitaminosis/inmunología , Oligoelementos/inmunología , Complejo Vitamínico B , Animales , Avitaminosis/sangre , Biomarcadores/sangre , Sistema Enzimático del Citocromo P-450/sangre , Sistema Enzimático del Citocromo P-450/inmunología , Citocinas/inmunología , Femenino , Masculino , Ratones , Ratones Endogámicos BALB C , Ratas Wistar , Especificidad de la Especie , Oligoelementos/sangreRESUMEN
AIM: To develop an LC-MS/MS assay to quantitate well-tolerated substrates; midazolam (CYP3A), omeprazole (CYP2C19), dextromethorphan (CYP2D6), losartan (CYP2C9) and their respective metabolites' concentrations in plasma samples. PATIENTS & METHODS: A solid-phase extraction method was optimized to extract analytes of interest simultaneously from human plasma samples. The assay analyzed plasma samples collected from patients who received equal or lower than therapeutic doses of CYP substrates. RESULTS: This assay was validated based on the European Medicines Agency guideline for bioanalytical method validation and was sensitive, linear, accurate and precise with acceptable recovery and matrix effects. CONCLUSION: Small sample volume and dose of cytochrome P450 substrates, short-run time, using stable isotope internal standards and being cost effective are the major advantages of the assay.
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Cromatografía Liquida/métodos , Sistema Enzimático del Citocromo P-450/metabolismo , Pruebas de Enzimas/métodos , Espectrometría de Masas en Tándem/métodos , Citocromo P-450 CYP2C19/sangre , Citocromo P-450 CYP2C19/metabolismo , Citocromo P-450 CYP2C9/sangre , Citocromo P-450 CYP2C9/metabolismo , Citocromo P-450 CYP2D6/sangre , Citocromo P-450 CYP2D6/metabolismo , Citocromo P-450 CYP3A/sangre , Citocromo P-450 CYP3A/metabolismo , Sistema Enzimático del Citocromo P-450/sangre , HumanosRESUMEN
A method for the detection and quantification of hydroxyl and epoxy arachidonic acid (AA) metabolites in human plasma was developed using liquid-liquid extraction, phospholipid saponification followed by derivatization of the acid moiety and liquid chromatographic tandem mass spectrometric detection. Derivatization with a pyridinium analog allowed for detection in the positive ion mode, greatly improving sensitivity and the stability of the more labile AA metabolites. The entire method utilizes a 96-well plate format, increasing sample throughput, and was optimized to measure 5-, 8-, 9-, 11-, 12-, 15-, 19-, and 20- hydroxyeicosatetraenoic acid (HETE), 5,6-, 8,9-, 11,12-, and 14,15- dihydroxyeicosatrienoic acid (DHET), and the regio- and cis-/ trans- isomers of 5,6-, 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acid (EET). The method was validated for its applicability over the FA concentration range found in human plasma. Using 100 µL aliquots of pooled human plasma, EET levels, particularly 5,6-EET, were observed to be higher than previously reported, with measured concentrations of 23.6 ng/ml for 5,6-EET, 5.6 ng/mL for 5,6-trans-EET, 8.0 ng/mL for 8,9-EET, 1.9 ng/mL for 8,9-trans-EET, 8.8 ng/mL for 11,12-EET, 3.4 ng/mL for 11,12-trans-EET, 10.7 ng/mL for 14,15-EET, and 1.7 ng/mL 14,15-trans- EET. This method is suitable for large population studies to elucidate the complex interactions between the eicosanoids and various disease states and may be used for quantitation of a wide variety of fattyacids beyond eicosanoids from small volumes of human plasma.
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Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Ácido Araquidónico/sangre , Ácido Araquidónico/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Ácido 8,11,14-Eicosatrienoico/sangre , Ácido 8,11,14-Eicosatrienoico/aislamiento & purificación , Ácido 8,11,14-Eicosatrienoico/metabolismo , Cromatografía Líquida de Alta Presión , Sistema Enzimático del Citocromo P-450/sangre , Humanos , Estructura Molecular , Extracción en Fase Sólida , Estereoisomerismo , Espectrometría de Masas en TándemRESUMEN
In a one-way cross-over study, we investigated the effect of Khat, a natural amphetamine-like psychostimulant plant, on catalytic activities of five major drug-metabolizing cytochrome P450 (CYP) enzymes. After a one-week Khat abstinence, 63 Ethiopian male volunteers were phenotyped using cocktail probe drugs (caffeine, losartan, dextromethorphan, omeprazole). Phenotyping was repeated after a one-week daily use of 400 g fresh Khat leaves. Genotyping for CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A5 were done. Urinary cathinone and phenylpropanolamine, and plasma probe drugs and metabolites concentrations were quantified using LC-MS/MS. Effect of Khat on enzyme activities was evaluated by comparing caffeine/paraxanthine (CYP1A2), losartan/losartan carboxylic acid (CYP2C9), omeprazole/5-hydroxyomeprazole (CYP2C19), dextromethorphan/dextrorphan (CYP2D6) and dextromethorphan/3-methoxymorphinan (CYP3A4) metabolic ratios (MR) before and after Khat use. Wilcoxon-matched-pair-test indicated a significant increase in median CYP2D6 MR (41%, p < 0.0001), and a marginal increase in CYP3A4 and CYP2C19 MR by Khat. Repeated measure ANOVA indicated the impact of CYP1A2 and CYP2C19 genotype on Khat-CYP enzyme interactions. The median MR increased by 35% in CYP1A2*1/*1 (p = 0.07) and by 40% in carriers of defective CYP2C19 alleles (p = 0.03). Urinary log cathinone/phenylpropanolamine ratios significantly correlated with CYP2D6 genotype (p = 0.004) and CYP2D6 MR (P = 0.025). Khat significantly inhibits CYP2D6, marginally inhibits CYP3A4, and genotype-dependently inhibit CYP2C19 and CYP1A2 enzyme activities.
Asunto(s)
Biocatálisis , Catha/química , Sistema Enzimático del Citocromo P-450/metabolismo , Preparaciones Farmacéuticas/metabolismo , Farmacogenética , Adulto , Alcaloides/orina , Sistema Enzimático del Citocromo P-450/sangre , Sistema Enzimático del Citocromo P-450/genética , Frecuencia de los Genes/genética , Genotipo , Humanos , Fenilpropanolamina/orina , Adulto JovenRESUMEN
Ulcerative colitis (UC) is characterized by erosions of the intestinal mucosa. The number of patients with UC has recently been increasing rapidly. Since the diagnosis of UC is complex and difficult, a simple, rapid, noninvasive technique for diagnosing UC is needed urgently. The expression of cytochrome P450 (P450 or CYP) species in mouse liver is known to be changed dependent on the disease. Various components such as P450 substrates and P450 metabolites in the blood may possibly change with the UC-specific way in mouse. In this study, in order to evaluate UC-specific components in UC mouse serum, we analyzed the influence of serum derived from UC mice on the results of fluorescent P450 inhibition assays based on 12 human P450 enzymes, such as CYP1A1, CYP2C8, CYP2E1,CYP3A4, CYP1A2, CYP2D6, CYP2A13, CYP2B6, CYP2C9, CYP2C18, CYP2C19, and CYP3A5. At first, in order to induce UC, mice received 4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS) dissolved in their drinking water for 7 days. Next, these 12 human P450 enzymes were expressed in E. coli cells. Then, P450 fluorescent competition reaction was performed using these P450 enzymes and serum of UC mice. We found that the metabolism of fluorescent substrates by CYP2B6, CYP2C19, CYP2E1, and CYP1A1 in the presence of serum obtained from DSS-treated mice was activated by 42%, 37%, 37%, and 23%, respectively, relative to that associated with sera from control mice. A receiver operating characteristic (ROC) curve analysis was carried out with the 31 samples of UC mice and healthy mice. Area under the ROC curve (AUC) value was calculated from ROC curve. AUC value of CYP2E1 and CYP2C19 showed 0.921 and 0.892, respectively. Therefore, it was shown that CYP2C19 and CYP2E1 could be used as biomarkers for evaluating ulcerative colitis. From these results, it is suggested that these simple fluorescent P450 inhibition assays have potential as a new diagnostic procedure for UC in mouse. This study is the first report on a simple non-invasive method for evaluating UC using P450 enzyme and serum interaction.
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Colitis Ulcerosa/patología , Sistema Enzimático del Citocromo P-450/metabolismo , Colorantes Fluorescentes/metabolismo , Animales , Área Bajo la Curva , Biomarcadores/sangre , Colitis Ulcerosa/inducido químicamente , Citocromo P-450 CYP2C19/sangre , Citocromo P-450 CYP2C19/genética , Citocromo P-450 CYP2C19/metabolismo , Citocromo P-450 CYP2E1/sangre , Citocromo P-450 CYP2E1/genética , Citocromo P-450 CYP2E1/metabolismo , Sistema Enzimático del Citocromo P-450/sangre , Sistema Enzimático del Citocromo P-450/genética , Sulfato de Dextran/toxicidad , Progresión de la Enfermedad , Femenino , Colorantes Fluorescentes/química , Humanos , Hígado/metabolismo , Ratones , Ratones Endogámicos C57BL , Curva ROC , Reacción en Cadena en Tiempo Real de la Polimerasa , Especificidad por SustratoRESUMEN
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.
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Sistema Enzimático del Citocromo P-450 , Expresión Génica , Medicago truncatula/enzimología , Oxigenasas , Proteínas de Plantas , Sistema Enzimático del Citocromo P-450/sangre , Sistema Enzimático del Citocromo P-450/química , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/aislamiento & purificación , Escherichia coli/genética , Escherichia coli/metabolismo , Medicago truncatula/genética , Oxigenasas/biosíntesis , Oxigenasas/química , Oxigenasas/genética , Oxigenasas/aislamiento & purificación , Proteínas de Plantas/biosíntesis , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/aislamiento & purificación , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
Vitamin D deficiency is common in patients with chronic obstructive pulmonary disease (COPD), yet a comprehensive analysis of environmental and genetic determinants of serum 25-hydroxyvitamin D (25[OH]D) concentration in patients with this condition is lacking. We conducted a multi-centre cross-sectional study in 278 COPD patients aged 41-92 years in London, UK. Details of potential environmental determinants of vitamin D status and COPD symptom control and severity were collected by questionnaire, and blood samples were taken for analysis of serum 25(OH)D concentration and DNA extraction. All participants performed spirometry and underwent measurement of weight and height. Quadriceps muscle strength (QS) was measured in 134 participants, and sputum induction with enumeration of lower airway eosinophil and neutrophil counts was performed for 44 participants. Thirty-seven single nucleotide polymorphisms (SNP) in 11 genes in the vitamin D pathway (DBP, DHCR7, CYP2R1, CYP27B1, CYP24A1, CYP27A1, CYP3A4, LRP2, CUBN, RXRA, and VDR) were typed using Taqman allelic discrimination assays. Linear regression was used to identify environmental and genetic factors independently associated with serum 25(OH)D concentration and to determine whether vitamin D status or genetic factors independently associated with % predicted forced expiratory volume in one second (FEV1), % predicted forced vital capacity (FVC), the ratio of FEV1 to FVC (FEV1:FVC), daily inhaled corticosteroid (ICS) dose, respiratory quality of life (QoL), QS, and the percentage of eosinophils and neutrophils in induced sputum. Mean serum 25(OH)D concentration was 45.4nmol/L (SD 25.3); 171/278 (61.5%) participants were vitamin D deficient (serum 25[OH]D concentration <50nmol/L). Lower vitamin D status was independently associated with higher body mass index (P=0.001), lower socio-economic position (P=0.037), lack of vitamin D supplement consumption (P<0.001), sampling in Winter or Spring (P for trend=0.006) and lack of a recent sunny holiday (P=0.002). Vitamin D deficiency associated with reduced % predicted FEV1 (P for trend=0.060) and % predicted FVC (P for trend=0.003), but it did not associate with FEV1:FVC, ICS dose, QoL, QS, or the percentage of eosinophils or neutrophils in induced sputum. After correction for multiple comparisons testing, genetic variation in the vitamin D pathway was not found to associate with serum 25(OH)D concentration or clinical correlates of COPD severity. Vitamin D deficiency was common in this group of COPD patients in the UK, and it associated independently with reduced % predicted FEV1 and FVC. However, genetic variation in the vitamin D pathway was not associated with vitamin D status or severity of COPD.
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Enfermedad Pulmonar Obstructiva Crónica/epidemiología , Deficiencia de Vitamina D/epidemiología , Vitamina D/análogos & derivados , Adulto , Anciano , Anciano de 80 o más Años , Índice de Masa Corporal , Estudios Transversales , Sistema Enzimático del Citocromo P-450/sangre , Sistema Enzimático del Citocromo P-450/genética , Proteínas de Unión al ADN/sangre , Proteínas de Unión al ADN/genética , Eosinófilos/metabolismo , Eosinófilos/patología , Femenino , Regulación de la Expresión Génica , Humanos , Londres/epidemiología , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/sangre , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/genética , Masculino , Persona de Mediana Edad , Neutrófilos/metabolismo , Neutrófilos/patología , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/sangre , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/genética , Prevalencia , Enfermedad Pulmonar Obstructiva Crónica/sangre , Enfermedad Pulmonar Obstructiva Crónica/complicaciones , Enfermedad Pulmonar Obstructiva Crónica/genética , Grupos Raciales , Receptores de Calcitriol/sangre , Receptores de Calcitriol/genética , Receptores de Superficie Celular/sangre , Receptores de Superficie Celular/genética , Pruebas de Función Respiratoria , Índice de Severidad de la Enfermedad , Factores de Transcripción/sangre , Factores de Transcripción/genética , Vitamina D/sangre , Deficiencia de Vitamina D/sangre , Deficiencia de Vitamina D/complicaciones , Deficiencia de Vitamina D/genéticaRESUMEN
Vitamin D deficiency is common in children with asthma, and it associates with poor asthma control, reduced forced expiratory volume in one second (FEV1) and increased requirement for inhaled corticosteroids (ICS). Cross-sectional studies investigating the prevalence, determinants and clinical correlates of vitamin D deficiency in adults with asthma are lacking. We conducted a multi-centre cross-sectional study in 297 adults with a medical record diagnosis of ICS-treated asthma living in London, UK. Details of potential environmental determinants of vitamin D status, asthma control and medication use were collected by questionnaire; blood samples were taken for analysis of serum 25(OH)D concentration and DNA extraction, and participants underwent measurement of weight, height and fractional exhaled nitric oxide concentration (FeNO), spirometry and sputum induction for determination of lower airway eosinophil counts (n=35 sub-group). Thirty-five single nucleotide polymorphisms (SNP) in 11 vitamin D pathway genes (DBP, DHCR7, RXRA, CYP2R1, CYP27B1, CYP24A1, CYP3A4 CYP27A1, LRP2, CUBN, VDR) were typed using Taqman allelic discrimination assays. Linear regression was used to identify environmental and genetic factors independently associated with serum 25(OH)D concentration, and to determine whether vitamin D status was independently associated with Asthma Control Test (ACT) score, ICS dose, FeNO, forced vital capacity (FVC), FEV1 or lower airway eosinophilia. Mean serum 25(OH)D concentration was 50.6nmol/L (SD 24.9); 162/297 (54.5%) participants were vitamin D deficient (serum 25(OH)D concentration <50nmol/L). Lower vitamin D status was associated with higher body mass index (P=0.014), non-White ethnicity (P=0.036), unemployment (P for trend=0.012), lack of vitamin D supplement use (P<0.001), sampling in Winter or Spring (P for trend <0.001) and lack of a recent sunny holiday abroad (P=0.030), but not with potential genetic determinants. Vitamin D status was not found to associate with any marker of asthma control investigated. Vitamin D deficiency is common among UK adults with ICS-treated asthma, and classical environmental determinants of serum 25(OH)D operate in this population. However, in contrast to studies conducted in children, we found no association between vitamin D status and markers of asthma severity or control.
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Asma/epidemiología , Deficiencia de Vitamina D/epidemiología , Vitamina D/análogos & derivados , Administración por Inhalación , Adolescente , Corticoesteroides/uso terapéutico , Adulto , Anciano , Antiasmáticos/uso terapéutico , Asma/sangre , Asma/complicaciones , Asma/tratamiento farmacológico , Índice de Masa Corporal , Estudios Transversales , Sistema Enzimático del Citocromo P-450/sangre , Sistema Enzimático del Citocromo P-450/genética , Proteínas de Unión al ADN/sangre , Proteínas de Unión al ADN/genética , Eosinófilos/metabolismo , Eosinófilos/patología , Femenino , Regulación de la Expresión Génica , Humanos , Londres/epidemiología , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/sangre , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/genética , Masculino , Persona de Mediana Edad , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/sangre , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/genética , Grupos Raciales , Receptores de Calcitriol/sangre , Receptores de Calcitriol/genética , Receptores de Superficie Celular/sangre , Receptores de Superficie Celular/genética , Pruebas de Función Respiratoria , Índice de Severidad de la Enfermedad , Factores de Transcripción/sangre , Factores de Transcripción/genética , Vitamina D/sangre , Deficiencia de Vitamina D/sangre , Deficiencia de Vitamina D/complicaciones , Deficiencia de Vitamina D/tratamiento farmacológicoRESUMEN
The tyrosine kinase inhibitors sorafenib and imatinib are important in the treatment of a range of cancers but adverse effects in some patients necessitate dosage modifications. CYP3A4 has a major role in the oxidation of sorafenib to its N-oxide and N-hydroxymethyl metabolites and also acts in concert with CYP2C8 to mediate imatinib N-demethylation. CYP3A4 expression and function are impaired in patients with advanced liver disease, whereas the functions of CYP2C enzymes are relatively preserved. We evaluated the biotransformation of sorafenib and imatinib in well-characterized microsomal fractions from 17 control subjects and 19 individuals with hepatic cirrhosis of varying severity. The principal findings were that liver disease impaired the microsomal oxidation of sorafenib to its major metabolites to 40-44% of control (P<0.01), whereas the N-demethylation of imatinib was relatively unimpaired. The impairments in sorafenib biotransformation were correlated with decreased serum albumin concentrations and increased serum bilirubin concentrations in patients with liver disease, but not with the overall grade of liver disease according to the Child-Pugh system. In contrast, there was no relationship between imatinib N-demethylation and clinicopathologic factors in liver disease patients. These findings were accounted for in terms of the differential roles of CYPs 3A4 and 2C8 in the intrinsic clearance of the drugs. CYP3A4 has the major role in the intrinsic clearance of sorafenib but plays a secondary role to CYP2C8 in the intrinsic clearance of imatinib. In agreement with these findings CYP2C protein expression and CYP2C8-mediated paclitaxel 6α-hydroxylation were unimpaired in cirrhotic livers. This information could be adapted in individualized approaches such as in vivo CYP3A4 phenotyping to optimize sorafenib safety and efficacy in cancer patients with liver dysfunction.
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Sistema Enzimático del Citocromo P-450/sangre , Mesilato de Imatinib/sangre , Cirrosis Hepática/sangre , Tasa de Depuración Metabólica/fisiología , Microsomas Hepáticos/enzimología , Niacinamida/análogos & derivados , Compuestos de Fenilurea/sangre , Adolescente , Adulto , Anciano , Niño , Preescolar , Inhibidores del Citocromo P-450 CYP3A/sangre , Inhibidores del Citocromo P-450 CYP3A/farmacología , Inhibidores del Citocromo P-450 CYP3A/uso terapéutico , Femenino , Humanos , Mesilato de Imatinib/farmacología , Mesilato de Imatinib/uso terapéutico , Lactante , Recién Nacido , Cirrosis Hepática/tratamiento farmacológico , Masculino , Tasa de Depuración Metabólica/efectos de los fármacos , Microsomas Hepáticos/efectos de los fármacos , Persona de Mediana Edad , Niacinamida/sangre , Niacinamida/farmacología , Niacinamida/uso terapéutico , Compuestos de Fenilurea/farmacología , Compuestos de Fenilurea/uso terapéutico , Inhibidores de Proteínas Quinasas/sangre , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Sorafenib , Adulto JovenRESUMEN
BACKGROUND: Cerebral Small Vessel Disease (SVD) can cause cognitive impairment, disability and dementia. While it is still unclear about the pathogenesis of SVD, several risk factors of SVD have been identified, and studies suggested that hypertension may play a critical role in SVD. Furthermore, studies have demonstrated that CYP2J2 isoform, 50 G>T variant, is associated with increasing the risk of ischemic stroke. Thus, we hypothesized that CYP2J2 50 G>T variant is associated with increased risk of cerebral SVD. METHODS: Thus, in this case-control study, we evaluated the association of CYP2J2 polymorphisms with the susceptibility to cerebral SVD in a population of Chinese Han adults. RESULTS: We found that CYP2J2 50 G>T genotype was significantly higher in SVD patients compared to healthy control group. Furthermore, 50 G>T genotype of CYP2J2 was associated with a significantly higher risk of SVD. Additionally, this polymorphism was significantly associated with WMH volume and a number of impaired cognitive domains in SVD patients. CONCLUSION: In conclusion, our study demonstrated that CYP2J2 50 G>T polymorphism is associated with increased risk of cerebral SVD in Han Chinese.