RESUMO
The aim of this study was to develop a simple method for quantifying plasma levels of sildenafil and its metabolite by liquid chromatography with a C18 reverse-phase column and UV detection. For both compounds, linearity was assessed in the range from 10 and 1 000 ng · ml-1 and had correlation coefficients of r=0.995 and r=0.997 for sildenafil and its metabolite, respectively. The inter- and intra-day coefficients of variation was<5.3%. The limits of detection and quantification were 1 and 10 ng · ml-1. Drug levels were determined satisfactorily in two patients. A simple and reliable method was developed for use in children with Pulmonary Arterial Hypertension under treatment with sildenafil.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Inibidores da Fosfodiesterase 5/sangue , Piperazinas/sangue , Sulfonas/sangue , Criança , Humanos , Purinas/sangue , Citrato de SildenafilaRESUMO
Several aldehyde dehydrogenase (ALDH) complexes have been purified from the membranes of acetic acid bacteria. The enzyme structures and the chemical nature of the prosthetic groups associated with these enzymes remain a matter of debate. We report here on the molecular and catalytic properties of the membrane-bound ALDH complex of the diazotrophic bacterium Gluconacetobacter diazotrophicus. The purified ALDH complex is a heterodimer comprising two subunits of 79.7 and 50 kDa, respectively. Reversed-phase high-pressure liquid chromatography (HPLC) and electron paramagnetic resonance spectroscopy led us to demonstrate, for the first time, the unequivocal presence of a pyrroloquinoline quinone prosthetic group associated with an ALDH complex from acetic acid bacteria. In addition, heme b was detected by UV-visible light (UV-Vis) spectroscopy and confirmed by reversed-phase HPLC. The smaller subunit bears three cytochromes c. Aliphatic aldehydes, but not formaldehyde, were suitable substrates. Using ferricyanide as an electron acceptor, the enzyme showed an optimum pH of 3.5 that shifted to pH 7.0 when phenazine methosulfate plus 2,6-dichlorophenolindophenol were the electron acceptors. Acetaldehyde did not reduce measurable levels of the cytochrome b and c centers; however, the dithionite-reduced hemes were conveniently oxidized by ubiquinone-1; this finding suggests that cytochrome b and the cytochromes c constitute an intramolecular redox sequence that delivers electrons to the membrane ubiquinone.
Assuntos
Aldeído Desidrogenase/metabolismo , Citocromos b/metabolismo , Citocromos c/metabolismo , Gluconacetobacter/enzimologia , Cofator PQQ/química , Aldeído Desidrogenase/química , Aldeído Desidrogenase/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Membrana Celular , Citocromos b/química , Citocromos c/química , Regulação Bacteriana da Expressão Gênica/fisiologia , Regulação Enzimológica da Expressão Gênica/fisiologia , NADH NADPH Oxirredutases/metabolismo , OxirreduçãoRESUMO
AIMS: Gluconacetobacter xylinum is well known for its ability to produce large amounts of cellulose, however, little is known about its cell physiology. Our goal was to study the respiratory metabolism and components of the respiratory system of this bacterium in static cultures. To reach our goal, a medium formulation had to be designed to improve cell growth and cellulose production together with a novel method for the recovery of cells from cellulose pellicles. METHODS AND RESULTS: Successive modifications of a nutrient medium improved G. xylinum cell growth 4.5-fold under static culture conditions. A blender homogenization procedure for the releasing of cells from the cellulose matrix gave a high yield of cells recovered. Respiratory activities of purified cells were greatly stimulated by exogenous substrates and showed to be resistant to KCN. Unexpectedly, exogenous NADH was oxidized at high rates. Cytochromes a, b, c and d were identified after spectral analyses. CONCLUSIONS: Partial bioenergetic characterization of G. xylinum cells allowed us to propose a scheme for its respiratory system. In addition, the growth medium for biomass production and the procedure for the efficient recovery of cells from cellulose pellicles were significantly improved. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides the first-ever bioenergetic characterization of G. xylinum grown in static cultures. In addition, a novel methodology to obtain purified cells in suitable quantities for biochemical research is described.