Eric Van De Velde

Quantification of seven β-lactam antibiotics and two β-lactamase inhibitors in human plasma using a validated UPLC-MS/MS method

There is an increasing interest in monitoring plasma concentrations of β-lactam antibiotics. The objective of this work was to develop and validate a rapid ultra-performance liquid chromatographic method with tandem mass spectrometric detection (UPLC-MS/MS) for simultaneous quantification of amoxicillin, ampicillin, cefuroxime, cefazolin, ceftazidime, meropenem, piperacillin, clavulanic acid and tazobactam. Sample clean-up included protein precipitation with acetonitrile and back-extraction of acetonitrile with dichloromethane. Six deuterated β-lactam antibiotics were used as internal standards. Chromatographic separation was performed on a Waters ACQUITY UPLC system using a BEH C(18) column (1.7 μm, 100 mm×2.1 mm) applying a binary gradient elution of water and acetonitrile both containing 0.1% formic acid. The total run time was 5.5 min. The developed method was validated in terms of precision, accuracy, linearity, matrix effect and recovery. The assay has now been successfully used to determine concentrations of amoxicillin/clavulanic acid, cefuroxime and meropenem in plasma samples from intensive care patients.

Effect of age on β-receptors, Gsα- and Giα-proteins in rat heart

Abstract β-adrenoceptors, Gsα- and Giα-proteins were investigated in a crude plasma membrane preparation from ventricles of young (2–4 months) and senescent (22–24 months) Wistar rats. Receptor density, ligand affinity and β1/β2-receptor ratio were independent of the age of the rats. The percentage of βreceptors coupled to G-proteins increased with age. An age-related increase in the level of Gsα (124%) was paralleled by an increase in the ratio between the high and low molecular weight form of Gsα. The level of Giα-protein almost doubled (170%) upon aging. We conclude that the age-related differences are small at the level of the β-adrenoceptor molecule, but that the increase in Giα-proteins could be responsible for the age-related reduction in myocardial inotropic and chronotropic responses. Moreover, we suggest that the changes in degree of high affinity coupling between β-receptor and Gs-protein are possibly linked to alterations in the ratio between the Gs-molecular weight subtypes.

Effect of aging on properties and function of β-adrenoceptors in rat lung

Abstract β-Adrenoceptor density and ligand affinity, basal adenylate cyclase activity and cAMP synthesis upon stimulation with forskolin, fluoride, guanine nucleotides (GTP or guanylyl imidodiphosphate (GppNHp) or isoproterenol in the presence of the nucleotides were studied in membranes prepared from lungs of young (aged 2–3 months) and of old (aged 24–25 months) male Wistar rats. There was a significant (P

The β-adrenergic transduction system in kidneys from young and senescent rats

Abstract β-Adrenoceptor density, ligand affinity, high-affinity agonist binding, basal adenylate cyclase activity and cAMP synthesis upon stimulation with either forskolin, F−, guanine nucleotides (GTP or GppNHp) or isoproterenol in the presence of the nucleotides were studied in membranes prepared from kidneys of young (2–3 month) and old (24–25 month) male Wistar rats. There is a significant (P

Glycan microheterogeneity of α1-acid glycoprotein in sera and dialysates of patients on continuous ambulatory peritoneal dialysis

Abstract Total concentration and concanavalin (Con A) dependent microheterogeneity of α1-acid glycoprotein (AGP) were studied in sera of eight chronic renal failure patients before the start of continuous ambulatory peritoneal dialysis (CAPD), and in sera and dialysate fluids for up to 6 months of CAPD. The glycan heterogeneity of AGP in the samples was expressed as a reactivity coefficient, i.e. the ratio of the Con A-reactive AGP components to the Con A non-reactive AGP component. Concentrations of AGP in serum and reactivity coefficients were markedly elevated in non-dialysed uraemic patients. AGP concentrations in serum increased further during the first week on CAPD, and then gradually decreased to pre-dialysis values, which were reached after 1 to 6 mth. The reactivity coefficients did not change significantly during the CAPD treatment. Dialysate AGP concentrations were low in comparison to those in serum, and there was a good correlation between the reactivity coefficients in the dialysate fluids and those in the corresponding sera. The effect of peritonitis was evaluated in a separate group of eight CAPD patients. Serum and dialysate AGP concentrations were significantly higher during than after peritonitis while the corresponding reactivity coefficients were only slightly elevated.

Characterization of the β-adrenergic transduction system in spleen mononuclear leukocyte membranes of young and senescent rats

The effect of aging on some of the properties of the beta-adrenergic transduction system was determined in a membrane fraction of spleen mononuclear leukocytes from young (2-3 months) and old (24-25 months) rats. Receptor density was unchanged and the percentage of receptors in the high affinity configuration for isoproterenol was reduced with increasing age. Adenylate cyclase activity, either unstimulated or stimulated with forskolin, GTP or isoproterenol was not affected by aging. This suggests the presence of compensatory mechanisms in the beta-adrenergic signal transduction system of rat spleen mononuclear cells in order to ensure equal cAMP production for equal agonist stimulation.

Properties of the ventricular adrenergic signal transduction system during ontogeny of spontaneous hypertension in rats

The purpose of this study was to characterize adrenergic receptors and associated G proteins in ventricles of spontaneously hypertensive rats (SHRs) at different stages of development. The beta- and alpha1-adrenoceptor densities and subtype distribution, and beta-adrenoceptor-G protein coupling were studied by radioligand binding, and levels of G(Salpha), G(ialpha), and G(q/11alpha) protein species were determined by Western blotting in SHRs and Wistar-Kyoto (WKY) control rats aged 3.5 weeks, 3 months, and 8 months. In 3.5-week-old SHRs, both the beta-adrenoceptor density and the percentage of agonist high-affinity binding sites were higher than in age-matched WKY rats. The beta1/beta2-subtype distribution, the alpha1-adrenoceptor density, and the alpha1B/alpha1A-subtype distribution were similar in rats of both strains at all ages. Although essentially no differences in G(salpha) levels between SHRs and WKY rats were detected, higher G(ialpha) and lower Gq/1alpha concentrations were found in 3.5-week-old SHRs. In 3-month-old SHRs, increased levels of Gq/11alpha proteins were observed. In 8-month-old SHRs, none of the parameters was different from those of controls. We conclude that the differences in properties of the adrenergic signal transduction system between SHRs and WKY rats are exclusively observable before and at the onset of the overt hypertension. Moreover, the hypertensive genotype apparently affects G proteins more readily than adrenoceptors.

Release of dopamine, noradrenaline and dopamine B-hydroxylase from mouse neuroblastoma☆

The murine C1300 neuroblastoma tumor was found to secrete dopamine, noradrenaline and dopamine B-hydroxylase into the circulation of tumor-bearing A/J mice. The plasma levels of dopamine, noradrenaline and dopamine B-hydroxylase increased with the size of the tumor, and the increase in noradrenaline paralleled the increase in dopamine B-hydroxylase (r = 0.86). The vesicular storage of dopamine and noradrenaline in the tumor was evidenced by a decrease of the tissue content of dopamine and noradrenaline 24 hours after the administration of reserpine (5 micrograms/g) respectively to 17.6% and 7.8% of control values. A similar observation could be made for the levels of dopamine and noradrenaline in the plasma of reserpinized C1300 mice. The total activity of dopamine B-hydroxylase in the tumor and in plasma was unaffected by the reserpine treatment. Chronic administration of 6-hydroxydopamine (100 micrograms/g for 8 days) had no effect on the tissue contents of dopamine, noradrenaline or dopamine B-hydroxylase. The release of catecholamines and dopamine B-hydroxylase from the C1300 neuroblastoma was studied in vitro on superfused tumor slices. Stimulation of these slices with 56 mM KC1 or with 5.10(-5) M tyramine failed to induce the release of endogenous dopamine, noradrenaline or dopamine B-hydroxylase above the basal outflow levels. These results are suggestive for a non-exocytotic release of catecholamines and dopamine B-hydroxylase from the neuroblastoma tumor.