RESUMEN
The lipid peroxidation product 4-hydroxynonenal (HNE) is a biomarker of oxidative stress which is essentially involved in the pathophysiology of many diseases. The analysis of HNE is challenging because this aldehyde is extremely reactive and thus unstable. Hence, we adopted a gas chromatography-mass spectrometry (GC-MS) method based on derivatization of HNE with pentafluorobenzyl-hydroxylamine-HCl followed by trimethylsilylation to trimethylsilyl ethers. Ions representative for a negative ion chemical ionization mode were recorded at m/z = 152 for HNE and at m/z = 162 for the deuterated analogon (HNE-d11) as internal standard. This excellent stable and precise GC-MS method was carefully validated for HNE, and showed good linearity (r(2) = 0.998), and high specificity and sensitivity. Within-day precisions were 4.4-6.1% and between-day precisions were 5.2-10.2%. Accuracies were between 99% and 104% for the whole calibration range (2.5-250 nmol/L) of HNE. To examine the versatility of this modified GC-MS method, we analyzed HNE in ethylenediaminetetraacetic acid (EDTA) plasma in a well-defined collective of migraine patients; recently published. The results underline our former observations that women with migraine are afflicted with increased levels of HNE. Patients with thyroidal dysfunction showed no significant HNE alterations. This was confirmed by normal HNE EDTA plasma levels in hyper- und hypothyroid Sprague-Dawley rats. Taken together, the GC-MS method presented herein is of excellent quality to record oxidative stress-related bioactive HNE levels. This is important for a reorientation of oxidative stress analytics in other human diseases first of atherosclerosis and cancer.
Asunto(s)
Aldehídos/sangre , Cromatografía de Gases y Espectrometría de Masas/métodos , Adulto , Aldehídos/química , Animales , Biomarcadores , Estudios de Casos y Controles , Femenino , Humanos , Hidroxilaminas/química , Hipertiroidismo/sangre , Hipotiroidismo/sangre , Peroxidación de Lípido , Trastornos Migrañosos/sangre , Ratas , Ratas Sprague-Dawley , Sensibilidad y Especificidad , Compuestos de Trimetilsililo/análisisRESUMEN
Malondialdehyde (MDA) is considered to be a biomarker for enzymatic degradation and lipid peroxidation of polyunsaturated fatty acids. Usually, MDA determination from different biological materials is performed by reaction with thiobarbituric acid (TBA) followed by high-performance liquid chromatography (HPLC) analysis and fluorometric detection. As this method lacks specificity and sensitivity, we developed a gas chromatography-mass spectrometry (GC-MS) method based on derivatization of MDA with 2,4-dinitrophenylhydrazine. Representative ions in negative ion chemical ionization (NICI) mode were recorded at m/z 204 for MDA and at m/z 206 for the deuterated analogon (MDA-d2) as internal standard. This stable and precise GC-MS method showed good linearity (r² = 0.999) and higher specificity and sensitivity than the HPLC method and was validated for both total MDA (t-MDA) and free MDA (f-MDA). Within-day precisions were 1.8-5.4%, between-day precisions were 4.8-9.2%; and accuracies were between 99% and 101% for the whole calibration range (0.156-5.0 µmol/L for t-MDA and 0.039-0.625 µmol/L for f-MDA). Although comparison of t-MDA levels from GC-MS and HPLC results using Passing-Bablok regression analysis as well as Bland-Altman plot showed a correlation of the data, a tendency to increased results for the HPLC values was detectable, due to possible formation of unspecific products of the TBA reaction.
Asunto(s)
Cromatografía de Gases y Espectrometría de Masas , Malondialdehído/sangre , Adulto , Cromatografía Líquida de Alta Presión , HumanosRESUMEN
BACKGROUND AND PURPOSE: Oxidative stress is discussed to be implicated in the pathophysiology of migraine. However, data are in part controversial and the possible underlying mechanisms remain elusive to date. The aim of this study was to investigate the oxidative stress status of female patients with migraine and its implications on migraine-related metabolic alterations. METHODS: Oxidative stress markers malondialdehyde (MDA), 4-hydroxy-2-nonenal (HNE), carbonylated proteins, parameters of associated nitric oxide stress, inflammation, lipid- and glucose-metabolism were determined in the interictal phase in female patients with migraine and controls. RESULTS: We found significantly increased HNE levels in female migraineurs compared with controls. Logistic regression analyses of HNE revealed an odds ratio for migraine of 4.55. HNE showed significant correlations with the nitric oxide pathway, the insulin- and the lipid-metabolism. CONCLUSIONS: We show here that increased oxidative stress is associated with migraine and contributes to migraine-related metabolic risk like nitrosative stress, an atherogenic lipid profile and hyperinsulinemia. Our data suggest that oxidative stress may represent a key event in the pathophysiology of migraine and a suitable therapeutic target.
Asunto(s)
Metabolismo de los Lípidos/fisiología , Trastornos Migrañosos/metabolismo , Estrés Oxidativo/fisiología , Adulto , Estudios de Cohortes , Femenino , Humanos , Inflamación/epidemiología , Inflamación/metabolismo , Inflamación/fisiopatología , Persona de Mediana Edad , Trastornos Migrañosos/epidemiología , Trastornos Migrañosos/fisiopatología , Factores de Riesgo , Caracteres SexualesRESUMEN
Nitric oxide (NO) has been implicated in migraine attacks, but the role of NO in migraine remains unclear. We here hypothesize that increased NO in the headache-free period is associated with migraine. One hundred and thirty probands participated in this study. Various parameters of the NO pathway, such as nitrate, nitrite, arginine, citrulline, nitrosylated proteins, asymmetric dimethylarginine, symmetrical dimethylarginine, expression of endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase and two polymorphisms of eNOS were investigated. We found significant increased nitrate and decreased nitrite levels in migraineurs in the headache-free period. Nitrate and nitrite levels showed a significant inverse correlation. Logistic regression revealed an odds ratio of 3.6 for migraine. Other parameters of the NO pathway were neither altered in migraineurs nor correlated with nitrate. We show here that migraine patients suffer under sustained increased nitrosative stress in the headache-free period, which is associated with a 3.6-fold higher risk for migraine.
Asunto(s)
Migraña con Aura , Óxido Nítrico/sangre , Estrés Fisiológico/fisiología , Adulto , Anciano , Amidohidrolasas/sangre , Arginina/análogos & derivados , Arginina/sangre , Femenino , Humanos , Masculino , Migraña con Aura/epidemiología , Migraña con Aura/genética , Migraña con Aura/metabolismo , Nitratos/sangre , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo III/genética , Nitritos/sangre , Polimorfismo Genético , Factores de RiesgoRESUMEN
There is growing evidence that alterations in the insulin and glucose metabolism may be involved in the pathogenesis of migraine. Nitric oxide (NO) stress has been associated with migraine. However, the role of NO on the insulin and glucose metabolism in migraineurs has remained elusive to date. The aim of the present study was to investigate the insulin and glucose metabolism in migraineurs and to determine possible interactions with the NO pathway. One hundred and twenty non-obese probands participated in this study, including 48 migraineurs and 72 healthy volunteers. Various parameters of the NO pathway, glucose metabolism as well as body measurement parameters were determined. We found a highly significantly increased insulin and Homeostasis Model Assessment (HOMA)-index in migraine patients, whereas fasting glucose was decreased. Logistic regression revealed an odds ratio of 5.67 for migraine, when comparing the lowest with the highest quartile of HOMA. Multivariate analysis showed that HOMA, waist-to-length ratio and nitrite as parameters of NO stress were highly significantly correlated. We show here that hyperinsulinaemia is associated with migraine and, furthermore, is correlated with increased NO stress. These findings represent a new pathophysiological mechanism that may be of clinical relevance.
Asunto(s)
Hiperinsulinismo/complicaciones , Trastornos Migrañosos/complicaciones , Trastornos Migrañosos/metabolismo , Óxido Nítrico/metabolismo , Estrés Oxidativo/fisiología , Adulto , Glucemia , Ensayo de Inmunoadsorción Enzimática , Femenino , Glucosa/metabolismo , Humanos , Insulina/metabolismo , Masculino , Nitratos/sangre , Óxido Nítrico Sintasa de Tipo II/biosíntesis , Óxido Nítrico Sintasa de Tipo III/biosíntesis , Nitritos/sangre , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
OBJECTIVE: There is growing evidence that nitric oxide (NO) is critically involved in obesity and its clinical consequences like cardiovascular disease, hypertension and diabetes. We hypothesize that NO is already involved in the pathophysiology of juvenile obesity. We here determined the role of NO, its metabolites arginine and citrulline in obese and normal weight children. DESIGN: We investigated 57 obese and 57 normal weight age- and gender-matched juveniles. Various clinical parameters as well as body measurements and intima media thickness were determined. RESULTS: Obese juveniles revealed highly significant alterations in the NO pathway. NOX and citrulline were decreased in obese compared to normal weight juveniles and negatively correlated with body weight. Arginine was increased in obese juveniles and positively correlated with body weight. We found a significant negative correlation between NOX and oxidized low-density lipoprotein. Analysis of gamma-aminobutyric acid (GABA) revealed correlations with the NO pathway as NOX and citrulline were negatively correlated with GABA and arginine showed a positive correlation. CONCLUSION: We show here that NO and its metabolites arginine and citrulline are already involved in juvenile obesity that may contribute to atherogenesis via reduced bioavailability of NO. Moreover, we identify GABA as a new parameter in the mechanism of obesity-related NO reduction.
Asunto(s)
Arginina/metabolismo , Enfermedades Cardiovasculares/etiología , Citrulina/metabolismo , Resistencia a la Insulina/fisiología , Óxido Nítrico/fisiología , Obesidad/metabolismo , Adolescente , Enfermedades Cardiovasculares/metabolismo , Enfermedades Cardiovasculares/prevención & control , Femenino , Humanos , Masculino , Óxido Nítrico/deficiencia , Obesidad/complicacionesRESUMEN
OBJECTIVE: Statins inhibit the rate-limiting step in cholesterol biosynthesis, the conversion of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) to mevalonate by HMG-CoA reductase. Statins are usually taken in the evening as the HMG-CoA reductase activity is high during the night. This recommendation might not apply if statins are given as extended-release (ER) formulations. The present study investigated the influence of time of intake of fluvastatin 80 mg ER on cholesterol biosynthesis. Main objectives were to measure the change in 24-hour urinary mevalonic acid excretion, to determine plasma concentrations of mevalonic acid and fluvastatin and to monitor triglycerides, total cholesterol, HDL-cholesterol and LDL-cholesterol. METHODS: This was a randomized, 2-period crossover study in 26 hypercholesterolemic patients who received a single daily dose of fluvastatin both in the morning and in the evening. RESULTS: At baseline, the amount of mevalonic acid was 204.9 +/- 68.1 microg/g creatinine. After a single dose of fluvastatin mean urine values of mevalonate were significantly reduced to 129.8 +/- 66.2 micro/g (evening) and to 118.7 +/-34.3 microg/g (morning; n.s. between groups), thus representing a reduction of about 39%. Compared to baseline, plasma mevalonate concentrations were decreased by fluvastatin resulting in similar 24-hour profiles after the morning and the evening dosage. The pharmacokinetics of fluvastatin were similar in both periods of the study, with higher plasma concentrations for several hours following the evening dosage. CONCLUSION: This study demonstrates that fluvastatin ER is equally effective in inhibiting cholesterol biosynthesis when given once daily in the morning and once daily in the evening.
Asunto(s)
Ácidos Grasos Monoinsaturados/administración & dosificación , Ácidos Grasos Monoinsaturados/uso terapéutico , Inhibidores de Hidroximetilglutaril-CoA Reductasas/administración & dosificación , Inhibidores de Hidroximetilglutaril-CoA Reductasas/uso terapéutico , Hiperlipidemias/tratamiento farmacológico , Indoles/administración & dosificación , Indoles/uso terapéutico , Ácido Mevalónico/orina , Adulto , Biomarcadores , Colesterol/metabolismo , HDL-Colesterol/sangre , LDL-Colesterol/sangre , Estudios Cruzados , Preparaciones de Acción Retardada , Ácidos Grasos Monoinsaturados/farmacocinética , Femenino , Fluvastatina , Humanos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacocinética , Hiperlipidemias/sangre , Hiperlipidemias/orina , Indoles/farmacocinética , Masculino , Ácido Mevalónico/sangre , Persona de Mediana Edad , Factores de Tiempo , Triglicéridos/sangreRESUMEN
This review focuses on the advances in electron capture mass spectrometry. Electron-capture (EC) is a sensitive ionisation technique for mass spectrometry providing selectivity towards electrophoric compounds. Recent advances in instrumentation have led to a more widespread application of this method in biomedical and pharmaceutical analysis. After a brief introduction to EC-mass spectrometry (MS), potential targets for EC-MS analysis are defined and enhancement of sensitivity by electrophoric derivatisation is discussed. A wide range of applications is selected, including prostanoid analysis in biomedical systems (with the oxidative stress indicators isoprostanes) and the trace level analysis of endogenous low-molecular weight compounds. Application to the trace level gas chromatography-negative ion chemical ionization MS (GC-NICI-MS) analysis of complex glucuronides is also demonstrated, as well as a wide range of drugs analysed in human blood. The review should point out the versatility and unique sensitivity of the technique, making it useful for basic research in medicinal chemistry, as well as clinical diagnosis, pharmaceutical and toxicological applications.
Asunto(s)
Espectrometría de Masas/métodos , Cromatografía Liquida , Electrones , Cromatografía de Gases y Espectrometría de Masas/instrumentación , Cromatografía de Gases y Espectrometría de Masas/métodos , Hidrocarburos Fluorados/análisis , Espectrometría de Masas/instrumentación , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
Prostaglandins (PGs) play an important role in bone remodeling because eicosanoids are local mediators of bone metabolism, which can induce physiological and pathological responses of bone tissue. Biosynthesis of PGs is catalyzed by constitutively expressed PG endoperoxide G/H synthase (PGHS) 1 and by the inducible isoform PGHS-2. In MC3T3-E1 osteoblast-like cells, expression of PGHS-2 was shown by mechanical forces, cytokines, growth factors, and hormones. Recently, endothelin (ET) 1-stimulated PGHS-2 mRNA expression was described, leading to a burst in prostaglandin E2 (PGE2) production. In this study, we investigated ET-1-induced signal transduction pathway(s) involved in the PGHS-2 mRNA production. Time course of PGHS-2 mRNA expression reaching the maximum within 45 minutes is in good agreement with the concept of an immediate early gene product. Inhibition of phospholipase C (PLC), phospholipase D (PLD), phosphatidylinositol-3 kinase (PI-3-kinase), and protein kinase C (PKC) had no influence on PGHS-2 synthesis. Using specific blockers of tyrosine kinases indicated involvement of p38 MAPK but not p42/44 MAPK. By preloading cells with exoenzyme C3, we were able to show requirement of the Rho family of G proteins for p38 MAPK phosphorylation and PGHS-2 mRNA synthesis, whereas pertussis toxin (PTX) and cholera toxin (CTX) had no remarkable effect.
Asunto(s)
Endotelina-1/farmacología , Isoenzimas/biosíntesis , Sistema de Señalización de MAP Quinasas/fisiología , Proteínas Quinasas Activadas por Mitógenos/fisiología , Osteoblastos/enzimología , Prostaglandina-Endoperóxido Sintasas/biosíntesis , Proteínas de Unión al GTP rho/fisiología , ADP Ribosa Transferasas/farmacología , Animales , Toxinas Botulínicas/farmacología , Línea Celular , Toxina del Cólera/farmacología , Ciclooxigenasa 2 , Dinoprostona/biosíntesis , Inducción Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Isoenzimas/genética , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Toxina del Pertussis/farmacología , Fosforilación/efectos de los fármacos , Prostaglandina-Endoperóxido Sintasas/genética , Procesamiento Proteico-Postraduccional/efectos de los fármacos , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Acetato de Tetradecanoilforbol/farmacología , Transcripción Genética/efectos de los fármacos , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
A sensitive and specific method for the quantitative determination of morphine in human plasma is presented. Morphine was extracted from plasma by solid phase extraction on C18 and converted to its pentafluorobenzyl carbonate trimethylsilyl derivative. The derivatives were analysed without further purification. Using gas chromatography/negative ion chemical ionisation mass spectrometry, a useful diagnostic fragment ion at m/z 356 is obtained at high relative abundance. Deuterated morphine was used as internal standard. Calibration graphs were linear within the range 1.25 to 320 nmol/L. Intra-day precision was 3.82% (15 nmol/L), 2.85% (75 nmol/L) and 4.13% (225 nmol/L), inter-day variability was found to be 1.77% (15 nmol/L), 4.95% (75 nmol/L) and 9.88% (225 nmol/L). Inter-day accuracy showed deviations of 2.18% (15 nmol/L), -0.72% (75 nmol/L) and -0.13% (225 nmol/L). The method is rugged and robust and has been applied to the batch analysis of morphine during pharmacokinetic profiling of the drug.
Asunto(s)
Carbonatos/química , Fluorobencenos/química , Derivados de la Morfina/sangre , Morfina/sangre , Calibración , Cromatografía de Gases y Espectrometría de Masas , Humanos , Indicadores y Reactivos , Morfina/farmacocinética , Reproducibilidad de los ResultadosRESUMEN
A sensitive and specific method for the quantitative determination of paroxetine in human plasma is presented. After solvent extraction from plasma with hexane/ethyl acetate (1 : 1) at alkaline pH and derivatization to the pentafluorobenzyl carbamate derivative, paroxetine was measured by gas chromatography-negative ion chemical ionization mass spectrometry. The carboxylate anion at m/z 372 was obtained at high relative abundance. [2H6]-labeled paroxetine was used as an internal standard and its rapid and facile preparation from the unlabeled compound is described. Calibration graphs were linear within a range of 0.094-12.000 ng x ml(-1) using 1 ml of plasma and 0.469-60 ng x ml(-1) using 200 microl of plasma. Intra-day precision was 1.47% (0.375 ng x ml(-1)), 3.16% (3 ng x ml(-1)) and 1.37% (9 ng x ml(-1)) for the low-level method, and 3.37% (1.875 ng x ml(-1)), 2.72% (15 ng x ml(-1)) and 2.22% (45 ng x ml(-1)) for the high-level method. Inter-day precision was 1.65% (0.375 ng x ml(-1)), 2.13% (3 ng x ml(-1)) and 1.66% (9 ng x ml(-1)) for the low-level method, and 1.10% (1.875 ng x ml(-1)), 1.56% (15 ng x ml(-1)) and 1.90% (45 ng x ml(-1)) for the high-level method. At the limit of quantification (0.094 ng x ml(-1)), intra-day precision was 4.30% (low-level method) and 2.56% (high-level method), and inter-day precision was 3.23% (low-level method) and 3.00% (high-level method). The method is rugged, rapid and robust and has been applied to the batch analysis of paroxetine during pharmacokinetic profiling of the drug.
Asunto(s)
Paroxetina/sangre , Calibración , Deuterio , Cromatografía de Gases y Espectrometría de Masas/métodos , Humanos , Tasa de Depuración Metabólica , Estructura Molecular , Paroxetina/química , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , SolventesRESUMEN
The chemistry of the lemon-scented oil gland secretion of Collohmannia gigantea, a middle-derivative mixonomatan oribatid mite, was investigated by gas chromatography - mass spectrometry. Gas chromatographic profiles of whole body extracts of C. gigantea revealed two distinct chromatographic zones, the first containing a set of six volatile compounds, comprising the lemon-scented monoterpene aldehydes neral and geranial, the scented monoterpene ester neryl formate, a distinctly scented aromatic aldehyde (2-hydroxy-6-methyl-benzaldehyde = 2,6-HMBD), and the two non-scented hydrocarbons, tridecane and pentadecane. All six components appeared to be present in steady relative proportions in scenting mites only, indicating their unity within the scented secretion. In contrast, the components of the second chromatographic zone were less volatile and found in both, scenting and nonscenting mites. Chemically, they represent a set of fatty acids of already known cuticular origin. The secretion bouquet of the first chromatographic zone was linked with oil glands by histochemical means: large amounts of aldehydes were present only in oil gland reservoirs, not in any other region of the mite body. While chemical profiles of oil gland secretions of several dozen astigmatid mites are known, only one other oribatid oil gland composition, from a desmonomatan species, has been elucidated, being almost the same as that of C. gigantea. Moreover, all components of these two secretions are widely distributed amongst astigmatid mite species and may also be common in a restricted set of middle-derivative oribatids. These findings are consistent with the idea of astigmatid mite origin from a mixonomatan-desmonomatan group.
Asunto(s)
Aldehídos/metabolismo , Glándulas Exocrinas/metabolismo , Ácaros/metabolismo , Aceites Volátiles/metabolismo , Terpenos/metabolismo , Aldehídos/análisis , Animales , Glándulas Exocrinas/química , Femenino , Cromatografía de Gases y Espectrometría de Masas , Histocitoquímica , Masculino , Aceites Volátiles/análisis , Terpenos/análisisRESUMEN
A sensitive and specific method for the determination of methylphenidate in human plasma is presented. Methylphenidate was extracted from plasma by solvent extraction with hexane at pH 9.3 and derivatized to its heptafluorobutyrate derivative. The derivative was measured by gas chromatography/negative ion chemical ionization mass spectrometry without any further purification. Using this detection mode, a diagnostically useful fragment ion at m/z 369 was obtained at high relative abundance. (18)O(2)-labelled methylphenidate was used as an internal standard and its rapid and facile preparation from the unlabeled compound is described. Calibration graphs were linear within the range 0.14-18.25 ng ml(-1). The inter-assay precision was 8.7% (0.14 ng ml(-1)) and 3.1% (4.56 ng ml(-1)) and the intra-assay variability was 1.3% (0.14 ng ml(-1)) and 0.4% (4.56 ng ml(-1)). Accuracy determinations showed deviations of +0.7% (0.14 ng ml(-1)) and -2.5% (4.56 ng ml(-1)). The method is rugged, rapid and robust and has been applied to the batch analysis of methylphenidate during pharmacokinetic profiling of the drug.
Asunto(s)
Estimulantes del Sistema Nervioso Central/sangre , Metilfenidato/sangre , Estimulantes del Sistema Nervioso Central/farmacocinética , Doping en los Deportes , Cromatografía de Gases y Espectrometría de Masas , Humanos , Indicadores y Reactivos , Metilfenidato/farmacocinética , Control de Calidad , Reproducibilidad de los Resultados , Solventes , Detección de Abuso de Sustancias/métodosRESUMEN
Vitamin K prophylaxis usually is administered orally or intramuscularly, but in neonatal intensive care oral administration might not be feasible and intramuscular administration is not general practice in very small infants. No data are available about plasma levels after intravenous administration of vitamin K to neonates. Therefore, we investigated plasma levels in 18 infants: 14 preterms with a birthweight of 1785+/-648 g and 4 sick newborns with a birth-weight of 3167+/-510 g after administration of a single dose of 0.3+/-0.1 mg/kg phylloquinone (vitamin K(1)) (Konakion MM((R)), Roche) intravenously after birth. Blood was collected 22.9+/-18.4 hours after intravenous administration of vitamin K(1). In 10 neonates a second sample was obtained 111.8+/-49.1 hours after the first vitamin K(1) administration. Gas chromatography-mass spectrometry (GC-MS) was used as the method for determination of vitamin K(1). The measured plasma concentration after intravenous administration of vitamin K(1) was 191.3+/-102.6 ng vitamin K in the first sample /mL in the first sample and 98.7+/-75.2 ng vitamin K(1)/mL in the second samples. These results are similar to those described in newborns after oral administration of 3 mg vitamin K(1) and after intramuscular administration of 1.5 mg vitamin K(1). In conclusion, the recommendation of the producer to give 0.4 mg/kg of vitamin K intravenously to neonates, in whom oral or intramuscular administration is not feasible, seems to be rational.
Asunto(s)
Enfermedades del Recién Nacido/sangre , Recien Nacido Prematuro/sangre , Vitamina K 1/administración & dosificación , Femenino , Cromatografía de Gases y Espectrometría de Masas , Semivida , Humanos , Recién Nacido , Infusiones Intravenosas , Cuidado Intensivo Neonatal/normas , Masculino , Vitamina K 1/sangre , Vitamina K 1/farmacocinéticaRESUMEN
A sensitive and specific method for the quantitative determination of morphine in human plasma is presented. Morphine was extracted from plasma by solvent extraction with ethyl acetate and derivatized to its heptafluorobutyrate derivative. The derivatives were measured by gas chromatography-negative ion chemical ionization mass spectrometry without any further purification. Using this detection mode, a diagnostic useful fragment ion at m/z 637 is obtained at high relative abundance. Deuterated morphine was used as an internal standard. Calibration graphs were linear within a range of 0.78 ng/ml and 50 ng/ml. Inter-assay precision was 2.3% (2.85 ng/ml) and 1.4% (14.25 ng/ml), intra-assay variability was found to be 1.5% (3.71 ng/ml) and 0.5% (14.54 ng/ml). Accuracy showed deviations of -9.3% (2.85 ng/ml) and -4.2% (14.25 ng/ml). The method is rugged and robust and has been applied to the batch analysis of morphine during pharmacokinetic profiling of the drug.
Asunto(s)
Analgésicos Opioides/sangre , Cromatografía de Gases y Espectrometría de Masas/métodos , Morfina/sangre , Humanos , Reproducibilidad de los ResultadosRESUMEN
Prostaglandin E(2) production stimulated by various agents (arachidonic acid, prostaglandin F(2alpha), ionomycin, the calcium ionophore A23187, and melittin) was investigated after pretreatment of murine osteoblast-like MC3T3-E1 cells with the putative phospholipase C blocker, U73122. The aminosteroid dose dependently inhibited prostaglandin E(2) production induced by all agonists, except arachidonic acid. The results suggest an inhibitory role of U73122 on phospholipase A(2) activity or activation.
Asunto(s)
Dinoprostona/biosíntesis , Estrenos/farmacología , Osteoblastos/efectos de los fármacos , Pirrolidinonas/farmacología , Abortivos/farmacología , Animales , Antibacterianos/farmacología , Ácido Araquidónico/metabolismo , Calcimicina/farmacología , Calcio/metabolismo , Radioisótopos de Carbono , Línea Celular , Células Clonales , Dinoprost/farmacología , Activación Enzimática , Ionomicina/farmacología , Meliteno/farmacología , Ratones , Osteoblastos/metabolismo , Inhibidores de Fosfodiesterasa/farmacología , Fosfolipasas A/metabolismoRESUMEN
1. The present study was carried out to clarify the effect of the imidazole antimycotics econazole, bifonazole and clotrimazole on prostanoid biosynthesis. Osteoblast-like MC3T3-E1 cells stimulated by endothelin-1, melittin, ionomycin or arachidonic acid showed diminished prostaglandin E(2) (PGE(2)) production upon pretreatment with econazole. Following pretreatment with bifonazole, stimulation with ionomycin or arachidonic acid also resulted in decreased PGE(2) formation. Clotrimazole inhibited ionomycin but not arachidonic acid stimulated PGE(2) synthesis in MC3T3-E1 cells. 2. The results observed in osteoblast-like UMR-106 cells pretreated with econazole, bifonazole or clotrimazole and stimulated by arachidonic acid were similar with the exception of clotrimazole which was a more effective inhibitor of PGE(2) biosynthesis than in MC3T3-E1 cells. 3. Upon treatment with arachidonic acid thromboxane B(2) (TXB(2)) production in human platelets was abolished completely at concentrations of the three imidazole antimycotics higher than 5 microM (IC(50)<1 microM). 4. These data were confirmed by a direct assay using purified ram seminal vesicle prostaglandin H(2) synthase-1 (PGHS-1), which clearly showed inhibitory properties of econazole (IC(50) 4.7+/-2.3 microM), bifonazole (IC(50) 9.4+/-0.8 microM) and clotrimazole (IC(50) 4.4+/-0.6 microM). 5. Summarizing, these results indicate an inhibitory effect of econazole, bifonazole and clotrimazole on PGHS-1, varying in its potency dependent on the cell system used. In addition TXB(2) formation is affected at doses even lower than those needed to suppress PGE(2) biosynthesis.
Asunto(s)
Inhibidores Enzimáticos del Citocromo P-450 , Inhibidores Enzimáticos/farmacología , Prostaglandinas/biosíntesis , Animales , Plaquetas/efectos de los fármacos , Plaquetas/metabolismo , Línea Celular , Clotrimazol/farmacología , Ciclooxigenasa 1 , Dinoprostona/biosíntesis , Relación Dosis-Respuesta a Droga , Econazol/farmacología , Humanos , Imidazoles/farmacología , Ionomicina/farmacología , Isoenzimas/efectos de los fármacos , Isoenzimas/metabolismo , Proteínas de la Membrana , Prostaglandina-Endoperóxido Sintasas/efectos de los fármacos , Prostaglandina-Endoperóxido Sintasas/metabolismo , Tromboxano B2/biosíntesis , Células Tumorales CultivadasRESUMEN
A colorimetric assay using the basic azo dye Janus green has been developed to assess cell numbers in anchorage-dependent cell cultures, with special regard to the enumeration of osteoblastic cells. Therefore, cells are fixed in ethanol and stained with a 0.2% solution of Janus green for 3 min, followed by a destaining step of 1 min in tap water. The addition of diluted hydrochloric acid easily and immediately leads to dye elution from stained cell layers into the acidic supernatant which consequently is transferred into 96-well plates and read on a microplate reader at 595 nm. Working under standardized conditions, Janus green uptake in several cell lines is shown to be linearly correlated with cell numbers over a broad range of cell densities, in MC3T3-E1 cells from about 3% up to more than 300% of confluency. Absolute sensitivity of the assay allows detection of less than 1000 cells/cm(2). In comparison to many other colorimetric assays, the Janus green technique is simple to perform, fast, precise, stable, cheap, and well suited for processing large quantities of samples. Moreover, it is applicable to any culture formate and size, from irregular formed carriers up to 96-multiwell plates.
Asunto(s)
Compuestos Azo/química , Recuento de Células/métodos , Colorimetría/métodos , Células 3T3 , Animales , Calibración , División Celular/fisiología , Línea Celular , Estudios de Evaluación como Asunto , Ratones , Control de Calidad , Reproducibilidad de los ResultadosRESUMEN
An improved method for the quantitative measurement of the angiotensin-converting enzyme inhibitor lisinopril in human plasma is presented. The assay is based on gas chromatography/negative ion chemical ionization mass spectrometry. The method involves solid phase extraction on C18 sorbent and derivatization to the pentafluorobenzyl diester trifluoroacetamide derivatives.
Asunto(s)
Inhibidores de la Enzima Convertidora de Angiotensina/sangre , Lisinopril/sangre , Inhibidores de la Enzima Convertidora de Angiotensina/farmacocinética , Cromatografía de Gases y Espectrometría de Masas , Humanos , Indicadores y Reactivos , Lisinopril/farmacocinéticaRESUMEN
A simple, highly accurate and precise method for the quantitative measurement of the angiotensin-converting enzyme inhibitor lisinopril in human plasma is presented. The assay is based on gas chromatography/negative ion chemical ionization mass spectrometry. The preparation of stable isotope labelled lisinopril for use as an internal standard is described. The method involves solid phase extraction on C18 sorbent and derivatization to the methyl diester-trifluoroacetamide derivatives. The detection limit was found to be 50 pg and a lower limit of quantification was reached down to 0.5 ng/mL plasma.