The signaling pathway for aldosterone-induced mitochondrial production of superoxide anion in the myocardium.

Mineralocorticoid receptor (MR) antagonists decrease morbidity and mortality in heart failure patients for whom oxidative stress is usual; however, the underlying mechanism for this protection is unclear. Since aldosterone stimulates reactive oxygen species (ROS) production in several tissues, we explored its effect and the intracellular pathway involved in the rat myocardium. Aldosterone dose-dependently increased O2(-) production in myocardial slices. At 10 nmol/L, aldosterone increased O2(-) to 165 ± 8.8% of control, an effect prevented not only by the MR antagonists eplerenone and spironolactone (107 ± 7.8 and 103 ± 5.3%, respectively) but also by AG1478 (105 ± 8.0%), antagonist of the EGF receptor (EGFR). Similar results were obtained by silencing MR expression through the direct intramyocardial injection of a lentivirus coding for a siRNA against the MR. The aldosterone effect on O2(-) production was mimicked by the mKATP channel opener diazoxide and blocked by preventing its opening with 5-HD and glibenclamide, implicating the mitochondria as the source of O2(-). Inhibiting the respiratory chain with rotenone or mitochondrial permeability transition (MPT) with cyclosporine A or bongkrekic acid also canceled aldosterone-induced O2(-) production. In addition, aldosterone effect depended on NADPH oxidase and phosphoinositide 3-kinase activation, as apocynin and wortmannin, respectively, inhibited it. EGF (0.1 µg/mL) similarly increased O2(-), although in this case MR antagonists had no effect, suggesting that EGFR transactivation occurred downstream from MR activation. Inhibition of mKATP channels, the respiratory chain, or MPT did not prevent Akt phosphorylation, supporting that it happened upstream of the mitochondria. Importantly, cardiomyocytes were confirmed as a source of aldosterone induced mitochondrial ROS production in experiments performed in isolated cardiac myocytes. These results allow us to speculate that the beneficial effects of MR antagonists in heart failure may be related to a decrease in oxidative stress.

Simple methodology for the therapeutic drug monitoring of the tyrosine kinase inhibitors dasatinib and imatinib.

A simple HPLC method has been developed to measure imatinib and N-desmethylimatinib (norimatinib) in plasma or serum at concentrations attained during therapy. Adaptation of this method to LC-MS/MS also allows dasatinib assay. A small sample volume (100 µL HPLC-UV, 50 µL LC-MS/MS) is required and analysis time is <5 min in each case. Detection was by UV (270 nm) or selective reaction monitoring (two transitions per analyte) tandem mass spectrometry. Assay calibration was linear (0.05-10 mg/L imatinib, 0.01-2.0 mg/L norimatinib and 1-200 µg/L dasatinib), with acceptable accuracy (86-114%) and precision (<14% RSD) for both methods. A comparison between whole blood and plasma confirmed that plasma is the preferred sample for imatinib and norimatinib assay. For dasatinib, although whole blood concentrations were slightly higher, plasma is still the preferred sample. Despite considerable variation in the (median, range) plasma imatinib and norimatinib concentrations in patient samples [1.66 (0.02-4.96) and 0.32 (0.01-0.99) mg/L, respectively, N = 104], plasma imatinib was >1 mg/L (suggested target for response) in all but one sample from patients achieving complete molecular response. As to dasatinib, the median (range) plasma dasatinib concentration was 13 (2-143) µg/L (N = 33). More observations are needed to properly assess the potential role of therapeutic drug monitoring in guiding treatment with dasatinib.

An automated method for the simultaneous measurement of azole antifungal drugs in human plasma or serum using turbulent flow liquid chromatography-tandem mass spectrometry.

Azole antifungal drugs are important in the prophylaxis and treatment of invasive aspergillosis. Therapeutic drug monitoring may be indicated to (1) monitor adherence, (2) guide dosage and (3) minimise the risk of drug-drug interactions and dose-related toxicity. TurboFlow(TM) technology offers online, automated sample preparation. An Aria Transcend(TM) TLX-II coupled with a TSQ Vantage(TM) MS was used. Centrifuged samples (25 µL) were mixed with internal standard solution (975 µL) and 30 µL injected directly onto a C18-P-XL TurboFlow column. Analytes were focussed onto a Phenomenex Gemini Phenyl analytical column and eluted using a methanol/water gradient (flow-rate, 0.8 mL/min). Analytes were monitored in selected reaction monitoring mode (two transitions per analyte, positive mode APCI). Calibration ranges were as follows: itraconazole, hydroxyitraconazole, and posaconazole 0.05-5.0 mg/L; voriconazole and fluconazole 0.1-10 mg/L. Total analysis time was 12 min. TurboFlow column recovery was >77% for all analytes. Calibration was linear (R (2) > 0.99) for all analytes. Inter- and intra-assay imprecision (% RSD) was <8% and accuracy (nominal internal quality control values) 90-105% for all analytes. The limit of detection was 0.01 mg/L for all analytes. No matrix effects were observed. This method is simple, robust and suitable for measuring these compounds at concentrations attained during therapy.

Basic drug analysis by strong cation-exchange liquid chromatography-tandem mass spectrometry: simultaneous analysis of amisulpride, and of metamfetamine and amfetamine in serum/plasma.

In the HPLC of basic drugs and metabolites, good efficiency and peak shape can often be attained using strong cation-exchange packings with isocratic 100% methanol eluents containing an ionic modifier at an appropriate pH* and ionic strength. Solvent extracts can be analysed directly, and use of ammonium acetate as modifier facilitates the use of atmospheric pressure chemical ionization (APCI)-tandem mass spectrometry, selected reaction monitoring mode. For the analysis of amisulpride and of metamfetamine/amfetamine in plasma (200 µL) after single oral doses in man, a column packed with Waters Spherisorb S5SCX (5 µm average particle size, 100 × 2.1 mm i.d.) was used with methanolic ammonium acetate (40 mmol/L, pH* 6.0, flow rate 0.5 mL/min) as eluent (35°C). Deuterated internal standards were used for each analyte. Detection was by positive-mode APCI. Responses for all analytes were linear over the calibration ranges. Intra-assay precision (RSD) was 2-18%, and inter-assay precision was 2-12%. The limit of detection was 0.5 µg/L for all analytes. No significant matrix effects or isobaric interferences were noted. The total analysis time was 7 min. Similar methodology can be applied to a wide range of basic analytes using MS/MS detection.

Suspected clozapine poisoning in the UK/Eire, 1992-2003.

OBJECTIVE: Toxicological analyses are often performed to investigate suspected poisoning, but the interpretation of results may not be straightforward. We studied suspected poisoning cases 1992-2003 where blood clozapine and N-desmethylclozapine (norclozapine) were measured in order to assess the relationship of these parameters to outcome. METHODS: Samples were referred from clinicians, pathologists/coroners, or via the Clozaril Patient Monitoring Service (CPMS, Novartis). Information was gathered from clinical, post-mortem, or coroners' reports. RESULTS: There were seven fatal [five male, two female; median (range) age 28 (24-41) year] and five non-fatal [four male, one female; median age 35 (26-41) year] clozapine overdoses. The median post-mortem blood clozapine and norclozapine concentrations were 8.2 (3.7-12) and 1.9 (1.4-2.4)mg/L, respectively [median clozapine:norclozapine ratio 4.4 (2.9-5.1)]. The median plasma clozapine and norclozapine concentrations (first or only sample) were 3.9 (1.7-7.0) and 0.40 (0.30-0.70)mg/L, respectively [median clozapine:norclozapine ratio 7.6 (5.3-18)] in the remainder. These overdoses were in patients who were poorly or non-adherent to clozapine, or who had taken tablets prescribed for someone else. In 54 further people who died whilst receiving clozapine [38 male, 16 female; median age 41 (22-70) year], the median post-mortem blood clozapine and norclozapine concentrations were 1.9 (0-7.7, n = 43) and 1.4 (0-6.0, n = 39)mg/L, respectively [median clozapine:norclozapine ratio 1.5 (0.4-7.6, n = 38)]. The median post-mortem increase in blood clozapine and norclozapine as compared to the most recent ante-mortem measurement was 489 (98-5,350)% and 371 (139-831)%, respectively [median sample time before death 14 (0-30, n = 21) days]. CONCLUSION: Clozapine poisoning cannot be diagnosed on the basis of blood clozapine and norclozapine concentrations alone. The analysis of ante-mortem blood specimens collected originally for white cell count monitoring and the blood clozapine:norclozapine ratio may provide additional interpretative information.

Thiocyanate supplementation decreases atherosclerotic plaque in mice expressing human myeloperoxidase.

Elevated levels of the heme enzyme myeloperoxidase (MPO) are associated with adverse cardiovascular outcomes. MPO predominantly catalyzes formation of the oxidants hypochlorous acid (HOCl) from Cl(-), and hypothiocyanous acid (HOSCN) from SCN(-), with these anions acting as competitive substrates. HOSCN is a less powerful and more specific oxidant than HOCl, and selectively targets thiols; such damage is largely reversible, unlike much HOCl-induced damage. We hypothesized that increased plasma SCN(-), and hence HOSCN formation instead of HOCl, may decrease artery wall damage. This was examined using high-fat fed atherosclerosis-prone LDLR(-/-) mice transgenic for human MPO, with and without SCN(-) (10 mM) added to drinking water. Serum samples, collected fortnightly, were analyzed for cholesterol, triglycerides, thiols, MPO, and SCN(-); study-long exposure was calculated by area under the curve (AUC). Mean serum SCN(-) concentrations were elevated in the supplemented mice (200-320 µM) relative to controls (< 120 µM). Normalized aortic root plaque areas at sacrifice were 26% lower in the SCN(-)-supplemented mice compared with controls (P = 0.0417), but plaque morphology was not appreciably altered. Serum MPO levels steadily increased in mice on the high-fat diet, however, comparison of SCN(-)-supplemented versus control mice showed no significant changes in MPO protein, cholesterol, or triglyceride levels; thiol levels were decreased in supplemented mice at one time-point. Plaque areas increased with higher cholesterol AUC (r = 0.4742; P = 0.0468), and decreased with increasing SCN(-) AUC (r = - 0.5693; P = 0.0134). These data suggest that increased serum SCN(-) levels, which can be achieved in humans by dietary manipulation, may decrease atherosclerosis burden.

Measurement of total mirtazapine and normirtazapine in plasma/serum by liquid chromatography with fluorescence detection.

A simple high performance liquid chromatography (HPLC) method for the measurement of the new antidepressant mirtazapine and its N-demethyl metabolite, normirtazapine, in human plasma or serum during low dose mirtazapine therapy has been developed. A Waters Spherisorb S5 SCX column was used with ammonium perchlorate (50 mmol/l) in methanol/water (95 + 5 (v/v)), apparent pH 6.7, as eluent, and fluorescence detection. Only small volumes of sample (0.2 ml) and extraction solvent are used. An interference study found no significant co-elution with drug or metabolite, although paroxetine co-elutes with the internal standard. The recovery of mirtazapine and normirtazapine (mean +/- S.D.) was 79 +/- 2, and 64 +/- 3%, respectively. The LOD was estimated as 0.5 microg/l, LLOQ was 1 microg/l, with a linear response over the concentration range 4-1000 microg/l (both analytes). The analytes were stable in serum for at least 10 months when stored at -20 degrees C. Intra- and inter-day accuracy were in the range 91-107 and 93-103%, respectively. In clinical samples (n = 14, median mirtazapine dose 45 mg per day, range 15-45 mg per day) the median (range) mirtazapine and normirtazapine concentrations were 26 (8-40) and 21 (8-32) microg/l, respectively.

High plasma thiocyanate levels are associated with enhanced myeloperoxidase-induced thiol oxidation and long-term survival in subjects following a first myocardial infarction.

Elevated levels of myeloperoxidase (MPO) are associated with poor cardiovascular outcomes. MPO uses H2O2 to generate oxidants including HOCl and HOSCN, from chloride and thiocyanate (SCN(-)) ions, respectively. SCN(-) is the preferred substrate. Elevation of this anion decreases HOCl generation and increases HOSCN formation, a thiol-specific oxidant. Such changes are of potential relevance to people with elevated SCN(-) levels, such as smokers. In this retrospective study, we examined whether elevated plasma MPO and SCN(-) levels increased thiol oxidation as a result of increased HOSCN formation, and impacted on long-term survival in 176 subjects (74 non-smokers, 46 smokers, and 56 previous smokers) hospitalized after a first myocardial infarction. Plasma thiols were not significantly altered in smokers compared to non-smokers or past smokers. However, significant positive correlations were detected between SCN(-) levels and MPO-induced thiol loss in the total population (r = 0.19, P = 0.020) and smokers alone (r = 0.58, P < 0.0001). Twelve-year all-cause mortality data indicate that above median MPO is significantly associated with higher mortality, but below-median MPO and above-median SCN(-) results in increased survival, compared to below-median SCN(-). Cox proportional hazard analysis showed a significant decrease in mortality for each 1 µM increase in SCN(-) (0.991; P = 0.040). Subject age was, as expected, a strong predictor of subject survival. Overall these data suggest that subjects with below-median MPO and above-median SCN(-) have better long-term survival, and that elevated plasma levels of SCN(-) might be protective in at least some populations.

Characterization of a microparticulate strong anion-exchanger in the HPLC of acidic drugs.

The use of Waters Spherisorb S5SAX for the HPLC of acidic compounds, including a number of non-steroidal anti-inflammatory drugs (NSAIDs), has been investigated. Adequate retention, separation, and peak efficiency and symmetry were obtained for most analytes on a 250 x 4.6 mm i.d. column using methanol containing ammonium perchlorate (10 mmol L(-1), pH 6.7 or pH 8.3) as eluent. The results of changes in (i) eluent pH (constant ionic strength); (ii) eluent ionic strength (constant pH); and (iii) adding water to the eluent (constant pH) were consistent with a retention mechanism dominated by ion-exchange with the bonded strong anion-exchange (SAX) moieties. However, there were some unexpected observations, including (i) a general decrease in retention at eluent pH values above 7.7; (ii) a marked increase in retention on adding 1% (v/v) water to the eluent; (iii) a subsequent marked decrease in retention on adding 5% (v/v) or more water; and (iv) decreased column activity with time. These observations may be due to (i) interaction between the charged SAX moieties and ionised surface silanols (with ionization increasing at higher eluent pH values) and (ii) influence of the solvation of silanols, analytes, SAX moiety, and counter-ion varying with both pH and water content. Nevertheless, the factors influencing separation of individual NSAIDs remain unclear especially as no relation between log k and pKa exists for these compounds. Hydrophobic interactions are unlikely to be important since basic and neutral compounds were hardly retained. Ease of accessibility of the counter-ion to the SAX moiety for analyte displacement may be a factor.

Micro-extraction techniques in analytical toxicology: short review.

This paper discusses new developments in plasma micro-extraction techniques in the context of established micro-extraction and protein precipitation methodology. Simple liquid-liquid solvent extraction (LLE) of plasma with direct GC or HPLC analysis of the resulting extract has been used for many years. Butyl acetate and methyl t-butyl ether (MTBE) give efficient extraction of many drugs and metabolites from small volumes of plasma or whole blood at an appropriate pH, and form the upper layer, thus simplifying extract removal. Butyl acetate does not interfere with NPD, ECD or MS in GC, whilst MTBE has a relatively low UV cutoff (220 nm). Thus, HPLC eluents that use a high proportion of an organic component allow MTBE extracts to be analysed directly. 'Salting-out' and extractive derivatization using acetic anhydride or phenylboronic acid can be used with appropriate analytes. As regards protein precipitation, an important consideration is lowering the pH, although this is not feasible with acid-labile analytes. More recent developments include sold-phase micro-extraction (SPME) and liquid-phase micro-extraction (LPME). This latter technique especially may prove invaluable as analytes that cannot easily be extracted with LLE can be isolated simply at low cost with a minimum of apparatus.

Polyvinyl alcohol-coated perfluorocarbon supports for metal chelating affinity separation of a monoclonal antibody.

The preparation, characterisation and testing of stable non-porous coated perfluorocarbon supports functionalised with the metal chelate, iminodiacetic acid (IDA) is described. Polyvinyl alcohol (PVA), a neutral hydrophilic polymer was esterified with perfluorooctanoyl chloride and anchored to the surface of solid perfluorocarbon particles through multiple fluorophilic interactions. The PVA-coated particles were then activated by epoxidation and coupled with IDA. The presence of surface-attached chelates was clearly demonstrated by the binding and selective desorption of Zn2+ ions. Three particulate perfluorocarbons were selected as potential starting materials and the conditions for preparation of metal chelating adsorbents optimised with respect to ease of manufacture, ligand density and binding capacity towards a monoclonal antibody known to bind to commercial Zn(2+)-IDA supports. The choice of base particle strongly influenced the ligand densities and specific binding capacities towards the monoclonal antibody that could be achieved under optimal preparative conditions. Possible ways in which these metal chelating adsorbents may be employed to recover the monoclonal antibody directly from culture vessels are discussed.

Chronic administration of nifedipine induces up-regulation of functional calcium channels in rat myocardium.

Previous studies from our laboratory demonstrated the up-regulation of cardiac dihydropyridine (DHP) receptors in rabbits chronically treated with nifedipine (NIFE). The goal of the present study was to further examine the functionality of this increased number of receptors by analysing different steps of excitation contraction coupling mechanism in adult rats chronically treated with NIFE (a single 10-mg oral dose/kg/day for 28 days). Ca2+ channel density was assessed by specific binding at the DHP receptors with [methyl-(3)H]PN 200-110 in rat ventricular membranes. Chronic NIFE treatment produced up-regulation of Ca2+ channels, being the maximal binding capacities 222+/-19 fmol/mg protein (n=14) and 310+/-21 fmol/mg protein (n=11) in untreated and treated animals, respectively (P<0.05). The functional consequences of this up-regulation of Ca2+ channels were determined in isolated ventricular myocytes by measuring L-type Ca2+ currents (I(Ca)) with the whole-cell configuration of patch-clamp technique and by intracellular Ca2+ (Ca2+(i)) transients estimated by the Indo-1/AM fluorescence ratio (410/482) simultaneously monitored with cell shortening. Peak I(Ca) density recorded at 0 mV was 32% greater in myocytes isolated from the treated group than in those obtained from the untreated group (-10.43+/-0.73 pA/pF (n=13) vs-7.10+/-0.59 pA/pF (n=12) P<0.05). Ca2+(i) transient amplitude and cell shortening, explored at 1 and 2 mM extracellular calcium ([Ca]0) were significantly higher in ventricular myocytes obtained fom NIFE-treated rats than in myocytes isolated from untreated animals. At 2 mM [Ca]0, the values of Ca2+(i) transient and shortening were 460+/-61 nM and 11+/-1 % of resting length (L(0)) in myocytes from treated rats (n=9) and 212+/-22 nM and 5.3+/-0.5% of L(0) in myocytes from control rats (n=6, P<0.05). The results demonstrate an up-regulation of functionally-active cardiac Ca2+ channels after NIFE treatment, and offer a possible explanation for a "withdrawal effect" at myocardial level after the suppression of the treatment with this drug.