RESUMEN
In the human body, the catecholamine norepinephrine is mainly metabolized to 3,4-dihydroxyphenylglycol (DHPG) which therefore serves as an important biomarker for norepinephrine's metabolism. Most data on DHPG concentrations in human plasma and urine has been generated by using HPLC-ECD or GC-MS technologies. Here, we describe a stable-isotope dilution GC-MS/MS method for the quantitative determination of DHPG in human urine using trideutero-DHPG (d(3)-DHPG) as internal standard and a two-step derivatization process with pentafluorobenzyl bromide (PFB-Br) and N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA). Two pentafluorobenzyl (PFB) trimethylsilyl (TMS) derivatives were obtained and identified, i.e., two isomeric DHPG-PFB-(TMS)(3) derivatives and the later eluting DHPG-tetrafluorobenzyl-(TMS)(2) derivative, i.e., DHPG-TFB-(TMS)(2). To our knowledge the DHPG-TFB-(TMS)(2) derivative and the underlying reaction have not been reported previously. In this reaction both vicinal aromatic hydroxyl groups of DHPG react with PFB-Br to form a heterocyclic seven-membered [1,4]dioxepin compound. The DHPG-TFB-(TMS)(2) derivative was used for quantitative GC-MS/MS analysis in the electron-capturing negative-ion chemical ionization mode by selected-reaction monitoring of m/z 351 from m/z 401 for DHPG and of m/z 352 from m/z 404 for d(3)-DHPG. Validation experiments on human urine samples spiked with DHPG in a narrow (0-33 nM) and a wide range (0-901 nM) revealed high recovery (86-104%) and low imprecision (RSD; 0.01-2.8%). LOD and relative LLOQ (rLLOQ) values of the method for DHPG were determined to be 76 amol and 9.4%, respectively. In urine of 28 patients suffering from chronic inflammatory rheumatic diseases, DHPG was measured at a mean concentration of 238 nM (38.3 µg/g creatinine). The DHPG concentration in the respective control group of 40 healthy subjects was measured to be 328 nM (39.2 µg/g creatinine). Given the unique derivatization reaction and collision-induced dissociation, and the straightforwardness the present method is highly specific, accurate, precise, and should be useful in clinical settings.
Asunto(s)
Catecoles/orina , Fluorobencenos/química , Cromatografía de Gases y Espectrometría de Masas/métodos , Metoxihidroxifenilglicol/análogos & derivados , Fiebre Reumática/orina , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Modelos Lineales , Masculino , Metoxihidroxifenilglicol/orina , Persona de Mediana Edad , Espectrometría de Masas en Tándem/métodos , Adulto JovenRESUMEN
Anandamide (arachidonoyl ethanol amide, AEA) is an endocannabinoid, acting on CB1 and CB2 receptors. Elevated plasma AEA concentrations in humans have been associated amongst others with obesity, psychological disorders and miscarriage. The occurrence in human plasma of ethanol amides of other unsaturated and saturated fatty acids, including oleic acid and palmitic acid, has also been reported. Most data available on anandamide and other fatty acid ethanol amides (FAEA) until now have been generated by using the LC-MS/MS methodology. Here, we describe a stable-isotope dilution GC-MS/MS method for the quantitative determination of AEA, oleic acid ethanol amide (OEA) and palmitic acid ethanol amide (PEA) in human plasma using their stable-isotope labeled analogs as internal standards. Other FAEA were found in plasma and their concentration was estimated. The present method involves a single solvent extraction of FAEA and their internal standards from plasma (50-1000 microl) with toluene, derivatization to the pentafluorobenzamide pentafluoropropionyl derivatives (FAEA-PFBz-PFP), and simultaneous quantification by selected reaction monitoring of the carboxylate anions produced by collision-induced dissociation of the parent ions [M-PFBz](-). The present method was fully validated for anandamide. Thus, accuracy and imprecision of the method were within the range of 100+/-20% and less than 20%, respectively, in the range investigated (0-4 nM). Mean overall recovery was 90+/-3%. The LOQ and LOD values of the method were determined to be 0.25 nM of added AEA in plasma samples and 400 amol of injected AEA-PFBz-PFP derivative, respectively. In plasma of 16 healthy individuals AEA concentration was measured to be 1.35+/-0.32 nM. This finding is concordant to literature AEA plasma concentrations as measured by LC-MS/MS. The plasma concentrations of OEA, PEA and other FAEA are higher than that of AEA. This GC-MS/MS method is straightforward, accurate, precise, highly specific for FAEA and useful in basic and clinical research.
Asunto(s)
Amidas/química , Ácidos Araquidónicos/química , Moduladores de Receptores de Cannabinoides/química , Endocannabinoides , Cromatografía de Gases y Espectrometría de Masas/métodos , Alcamidas Poliinsaturadas/química , Espectrometría de Masas en Tándem/métodos , Adulto , Amidas/sangre , Ácidos Araquidónicos/sangre , Moduladores de Receptores de Cannabinoides/sangre , Femenino , Humanos , Masculino , Persona de Mediana Edad , Alcamidas Poliinsaturadas/sangreRESUMEN
Interferon-gamma (IFNgamma)-mediated indoleamine 2,3-dioxygenase (IDO) expression, important in innate immunity, immune suppression, and tolerance, can be counteracted by ferrous iron (FeSO(4)). Elevation of intracellular iron levels during stimulation with IFNgamma impeded IFNgamma-induced IDO mRNA and protein expression in HEp-2 cells. Decreased IDO expression was accompanied by decreased tryptophan degradation. Accordingly, IFNgamma-mediated suppressing effects on Chlamydia trachomatis (CT) infection were reduced or even abolished in the presence of FeSO(4). Conversely, lowering intracellular iron levels by deferoxamine (DFO) did not increase IFNgamma-induced IDO expression but potentiated Chlamydia-suppressing effects by lowering intracellular iron availability. Additionally, DFO led to a CT-induced IDO expression in HEp-2 cells not treated with IFNgamma. In summary, this study demonstrates that iron acts as a regulatory element for modulating IDO expression, in addition to its function as an essential element for chlamydial growth. This may represent an important control mechanism of IDO expression at the transcriptional level.
Asunto(s)
Infecciones por Chlamydia/enzimología , Chlamydia trachomatis/fisiología , Regulación Enzimológica de la Expresión Génica , Indolamina-Pirrol 2,3,-Dioxigenasa/genética , Interferón gamma/inmunología , Iones/metabolismo , Línea Celular , Infecciones por Chlamydia/genética , Infecciones por Chlamydia/inmunología , Infecciones por Chlamydia/metabolismo , Humanos , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Interferón gamma/genética , Triptófano/metabolismoRESUMEN
The post-traumatic changes of leukotrienes LTC4, LTD4, LTE4, and LTB4 in cerebrospinal fluid of rats from 10 min to 7 days were investigated after controlled cortical impact in relation to brain edema and cellular inflammatory response. LTC4 increased five-fold at 4 h, normalized at 24 h, and showed another four-fold increase at 7 days. The same pattern was observed for LTD4 and LTE4. LTB4 however, behaved differently: concentrations were lower and levels peaked two-fold at 24 h. Edema in the injured hemisphere increased continuously up to 24 h without change contralaterally. Leukocyte infiltration, macrophage presence and microglia activation were most prominent at 24 h, 7 days and 24 h respectively. Leukotriene changes in CSF seem to reflect those in the affected tissue, with a time delay and in lower concentrations, and were not linearly correlated to brain edema. The initially high leukotriene levels are rather likely to contribute to the cytotoxic edema than to enhance a vasogenic edema component. The profile of LTB4 was parallel to the time course of leukocyte infiltration, indicating initiation of infiltration as well as prolonged production by leukocytes themselves. The second leukotriene peak at 7 days is likely to follow a different pathway and might be related to a production in macrophages or activated glia.