A highly sensitive LC-tandem MS assay for the measurement in plasma and in urine of salbutamol administered by nebulization during mechanical ventilation in healthy volunteers.

The new-generation nebulizers are commonly used for the administration of salbutamol in mechanically ventilated patients. The different modes of administration and new devices have not been compared. We developed a liquid chromatography-tandem mass spectrometry method for the determination of concentrations as low as 0.05 ng/mL of salbutamol, corresponding to the desired plasma concentration after inhalation. Salbutamol quantification was performed by reverse-phase HPLC. Analyte quantification was performed by electrospray ionization-triple quadrupole mass spectrometry using selected reaction monitoring detection ESI in the positive mode. The method was validated over concentrations ranging from 0.05 to 100 ng/mL in plasma and from 0.18 to 135 ng/mL in urine. The method is precise, with mean inter-day coefficient of variation (CV%) within 3.1-8.3% in plasma and 1.3-3.9% in urine, as well as accurate. The proposed method was found to reach the required sensitivity for the evaluation of different nebulizers as well as nebulization modes. The present assay was applied to examine whether salbutamol urine levels, normalized with the creatinine levels, correlated with the plasma concentrations. A suitable, convenient and noninvasive method of monitoring patients receiving salbutamol by mechanical ventilation could be implemented. Copyright © 2011 John Wiley & Sons, Ltd.

Variations of the carcinoembryonic antigen level in the plasma of patients with gynecologie cancers during therapy

In 63 patients with histologically proved gynecologie carcinoma, circulating carcinoembryonic antigen (CEA) was determined by radioimmunoassay before and at two intervals after treatment. Thirty-one patients of 63 had CEA values over 2.5 ng. per milliliter before treatment. In general, the CEA levels were low compared to those found in endodermal carcinoma. The percentage of elevated CEA values was slightly higher in cases of carcinoma of the cervix and corpus uteri than in those of carcinoma of the ovary. All patients with CEA levels greater than 2.5 ng. per milliliter treated by complete surgical resection of tumor showed a drop of CEA levels to below 2.5 ng. per milliliter seven weeks after operation. In contrast, patients with palliative therapy showed no change in CEA values. About half of the patients treated with a complete course of internal and external radiotherapy showed a drop of CEA levels to below 2.5 ng. per milliliter, whereas the other patients showed fluctuating CEA values. No correlation between clinical status and evolution of CEA levels in these patients could be drawn at the present time. The CEA test seems to be of little value for the earl of diagnosis of gynecologie carcinoma but appears to be interesting for the evaluation of therapy and the follow-up of patients with diagnosed cases.

Coagulation Factor Xa Activates Thrombin and Signals Ischemic Neuronal Death Via Par-1 and JNK in Ischemic Neural Tissue

Background: The coagulation factor thrombin mediates ischemic neuronal deathand, at a low concentration, induces tolerance to ischemia.We investigated its modeof activation in ischemic neural tissue using an in vitro approach to distinguish therole of circulating coagulation factors from endogenous cerebral mechanisms. Wealso studied the signalling pathway downstream of thrombin in ischemia and afterthrombin preconditioning.Methods: Rat organotypic hippocampal slice cultures to 30 minute oxygen (5%)and glucose (1 mmol/L) deprivation (OGD).Results: Selective factor Xa (FXa) inhibition by fondaparinux during and afterOGD significantly reduced neuronal death in the CA1 after 48 hours. Thrombinactivity was increased in the medium 24 hours after OGD and this increasewas prevented by fondaparinux suggesting that FXa catalyzes the conversion ofprothrombin to thrombin in neural tissue after ischemia in vitro. Treatment withSCH79797, a selective antagonist of the thrombin receptor protease activatedreceptor-1 (PAR-1), significantly decreased neuronal cell death indicating thatthrombin signals ischemic damage via PAR-1. The JNK pathway plays an importantrole in cerebral ischemia and we observed activation of the JNK substrate,c-Jun in our model. Both the FXa inhibitor, fondaparinux and the PAR-1 antagonistSCH79797, decreased the level of phospho-c-Jun Ser73. After thrombin preconditioningc-Jun was activated by phosphorylation in the nuclei of neurons of the CA1.Treatment with a synthetic thrombin receptor agonist resulted in the same c-Junactivation profile and protection against subsequent OGD indicating that thrombinalso signals via PAR-1 and c-Jun in cell protection.Conclusion: These results indicate that FXa activates thrombin in cerebral ischemia,leading via PAR-1 to the activation of the JNK pathway resulting in neuronal death.Thrombin induced tolerance also involves PAR-1 and JNK, revealing commonfeatures in cell death and survival signalling.

Inhibition of the renin-angiotensin system does not reduce platelet activity at rest or during stress in hypertension.

OBJECTIVES: We investigated the influence of angiotensin receptor blockade and angiotensin-converting enzyme inhibition on stress-induced platelet activation in hypertensive patients. Secondary aims were effects on inflammation, coagulation, and endothelial function. METHODS: Following a 4-week placebo period, 25 hypertensive patients entered a double-blind, crossover study comparing enalapril (20 mg once daily) and losartan (100 mg once daily) treatment (each for 8 weeks). Patients were studied at rest and after a standardized exercise test. RESULTS: Mean arterial pressure was reduced from 119 ± 2 to 104 ± 2 (enalapril) and 106 ± 2 (losartan) mmHg (both P <0.001). Plasma angiotensin II decreased from 2.4 ± 0.4 to 0.5 ± 0.1 pmol/l with enalapril, and increased to 7.2 ± 1.3 pmol/l with losartan (both P <0.001). Exercise-evoked platelet activation, as evidenced by increased numbers of P-selectin-positive platelets (P <0.01), elevated circulating platelet-platelet aggregates (P <0.01) and soluble P-selectin levels (P <0.001), and increased platelet responsiveness to adenosine diphosphate and thrombin (both P <0.05). Neither drug influenced these markers of platelet activation at rest or following exercise. Markers of inflammation (high-sensitivity C reactive protein, interleukin-6, tissue necrosis factor-α), coagulation (tissue plasminogen activator antigen, prothrombin fragment F1+2), and endothelial function (von Willebrand factor, soluble vascular cellular adhesion molecule-1, and intercellular adhesion molecule-1) were also uninfluenced by treatment. CONCLUSION: Enalapril and losartan failed to reduce platelet activity both at rest and during exercise in hypertensive patients. Markers of inflammation, coagulation, and endothelial function were similarly unaffected. Inhibition of the renin-angiotensin system promotes its beneficial effects in hypertension through mechanisms other than platelet inhibition.

A novel method for quantification of sulfolane (a metabolite of busulfan) in plasma by gas chromatography-tandem mass spectrometry.

The role of busulfan (Bu) metabolites in the adverse events seen during hematopoietic stem cell transplantation and in drug interactions is not explored. Lack of availability of established analytical methods limits our understanding in this area. The present work describes a novel gas chromatography-tandem mass spectrometric assay for the analysis of sulfolane (Su) in plasma of patients receiving high-dose Bu. Su and Bu were extracted from a single 100 μL plasma sample by liquid-liquid extraction. Bu was separately derivatized with 2,3,5,6-tetrafluorothiophenolfluorinated agent. Mass spectrometric detection of the analytes was performed in the selected reaction monitoring mode on a triple quadrupole instrument after electronic impact ionization. Bu and Su were analyzed with separate chromatographic programs, lasting 5 min each. The assay for Su was found to be linear in the concentration range of 20-400 ng/mL. The method has satisfactory sensitivity (lower limit of quantification, 20 ng/mL) and precision (relative standard deviation less than 15 %) for all the concentrations tested with a good trueness (100 ± 5 %). This method was applied to measure Su from pediatric patients with samples collected 4 h after dose 1 (n = 46), before dose 7 (n = 56), and after dose 9 (n = 54) infusions of Bu. Su (mean ± SD) was detectable in plasma of patients 4 h after dose 1, and higher levels were observed after dose 9 (249.9 ± 123.4 ng/mL). This method may be used in clinical studies investigating the role of Su on adverse events and drug interactions associated with Bu therapy.

Determination of trimipramine and its demethylated and hydroxylated metabolites in plasma by gas chromatography-mass spectrometry.

A gas chromatographic-mass spectrometric (GC-MS) method has been developed, for the determination of trimipramine (TRI), desmethyltrimipramine (DTRI), didesmethyltrimipramine (DDTRI), 2-hydroxytrimipramine (2-OH-TRI) and 2-hydroxydesmethyltrimipramine (2-OH-DTRI). The method includes two derivatization steps with trifluoroacetic acid anhydride and N-methyl-N-(tert.-butyldimethyl silyl)trifluoroacetamide and the use of an SE-54 capillary silica column. The limits of quantitation were found to be 2 ng/ml for DTRI and 4 ng/ml for all other substances. Besides, methods have been optimized for the hydrolysis of the glucuronic acid conjugated metabolites. This specific detection method is useful, as polymedication is a usual practice in clinical situations, and its sensitivity allows its use for single-dose pharmacokinetic studies.

Peri-operative management of antiplatelet therapy in patients with coronary artery disease. Joint position paper by members of the working group on Perioperative Haemostasis of the Society on Thrombosis and Haemostasis Research (GTH), the working group on Perioperative Coagulation of the Austrian Society for Anesthesiology, Resuscitation and Intensive Care (ÖGARI) and the Working Group Thrombosis of the European Society for Cardiology (ESC).

An increasing number of patients suffering from cardiovascular disease, especially coronary artery disease (CAD), are treated with aspirin and/or clopidogrel for the prevention of major adverse events. Unfortunately, there are no specific, widely accepted recommendations for the perioperative management of patients receiving antiplatelet therapy. Therefore, members of the Perioperative Haemostasis Group of the Society on Thrombosis and Haemostasis Research (GTH), the Perioperative Coagulation Group of the Austrian Society for Anesthesiology, Reanimation and Intensive Care (ÖGARI) and the Working Group Thrombosis of the European Society of Cardiology (ESC) have created this consensus position paper to provide clear recommendations on the perioperative use of anti-platelet agents (specifically with semi-urgent and urgent surgery), strongly supporting a multidisciplinary approach to optimize the treatment of individual patients with coronary artery disease who need major cardiac and non-cardiac surgery. With planned surgery, drug eluting stents (DES) should not be used unless surgery can be delayed for ≥12 months after DES implantation. If surgery cannot be delayed, surgical revascularisation, bare-metal stents or pure balloon angioplasty should be considered. During ongoing antiplatelet therapy, elective surgery should be delayed for the recommended duration of treatment. In patients with semi-urgent surgery, the decision to prematurely stop one or both antiplatelet agents (at least 5 days pre-operatively) has to be taken after multidisciplinary consultation, evaluating the individual thrombotic and bleeding risk. Urgently needed surgery has to take place under full antiplatelet therapy despite the increased bleeding risk. A multidisciplinary approach for optimal antithrombotic and haemostatic patient management is thus mandatory.

Sensitive bioassay for determination of fluconazole concentrations in plasma using a Candida albicans mutant hypersusceptible to azoles.

The antifungal agent fluconazole (FLC) is widely used in clinical practice. Monitoring FLC levels is useful in complicated clinical settings and in experimental infection models. A bioassay using Candida pseudotropicalis, a simple and cost-effective method, is validated only for FLC levels ranging from 5 to 40 mg/liter. An extension of the analytical range is needed to cover most yeast MICs. A new bioassay in RPMI agar containing methylene blue was developed using C. albicans DSY1024, a mutant rendered hypersusceptible to FLC constructed by the deletion of the multidrug efflux transporter genes CDR1, CDR2, CaMDR1, and FLU1. Reproducible standard curves were obtained with FLC concentrations in plasma ranging from 1 to 100 mg/liter (quadratic regression coefficient &gt; 0.997). The absolute sensitivity was 0.026 microg of FLC. The method was internally validated according to current guidelines for analytical method validation. Both accuracy and precision lied in the required +/-15% range. FLC levels measured by bioassay and by high-performance liquid chromatography (HPLC) performed with 62 plasma samples from humans and rats showed a strong correlation (coefficients, 0.979 and 0.995, respectively; percent deviations of bioassay from HPLC values, 0.44% +/- 15.31% and 2.66% +/- 7.54%, respectively). In summary, this newly developed bioassay is sensitive, simple, rapid, and inexpensive. It allows nonspecialized laboratories to determine FLC levels in plasma to within the clinically relevant concentration range and represents a useful tool for experimental treatment models.

Genome-wide association study identifies loci influencing concentrations of liver enzymes in plasma.

Concentrations of liver enzymes in plasma are widely used as indicators of liver disease. We carried out a genome-wide association study in 61,089 individuals, identifying 42 loci associated with concentrations of liver enzymes in plasma, of which 32 are new associations (P = 10(-8) to P = 10(-190)). We used functional genomic approaches including metabonomic profiling and gene expression analyses to identify probable candidate genes at these regions. We identified 69 candidate genes, including genes involved in biliary transport (ATP8B1 and ABCB11), glucose, carbohydrate and lipid metabolism (FADS1, FADS2, GCKR, JMJD1C, HNF1A, MLXIPL, PNPLA3, PPP1R3B, SLC2A2 and TRIB1), glycoprotein biosynthesis and cell surface glycobiology (ABO, ASGR1, FUT2, GPLD1 and ST3GAL4), inflammation and immunity (CD276, CDH6, GCKR, HNF1A, HPR, ITGA1, RORA and STAT4) and glutathione metabolism (GSTT1, GSTT2 and GGT), as well as several genes of uncertain or unknown function (including ABHD12, EFHD1, EFNA1, EPHA2, MICAL3 and ZNF827). Our results provide new insight into genetic mechanisms and pathways influencing markers of liver function.

Antibacterial burst-release from minimal Ag-containing plasma polymer coatings.

Biomaterials releasing silver (Ag) are of interest because of their ability to inhibit pathogenic bacteria including antibiotic-resistant strains. In order to investigate the potential of nanometre-thick Ag polymer (Ag/amino-hydrocarbon) nanocomposite plasma coatings, we studied a comprehensive range of factors such as the plasma deposition process and Ag cation release as well as the antibacterial and cytocompatible properties. The nanocomposite coatings released most bound Ag within the first day of immersion in water yielding an antibacterial burst. The release kinetics correlated with the inhibitory effects on the pathogens Pseudomonas aeruginosa or Staphylococcus aureus and on animal cells that were in contact with these coatings. We identified a unique range of Ag content that provided an effective antibacterial peak release, followed by cytocompatible conditions soon thereafter. The control of the in situ growth conditions for Ag nanoparticles in the polymer matrix offers the possibility to produce customized coatings that initially release sufficient quantities of Ag ions to produce a strong adjacent antibacterial effect, and at the same time exhibit a rapidly decaying Ag content to provide surface cytocompatibility within hours/days. This approach seems to be favourable with respect to implant surfaces and possible Ag-resistance/tolerance built-up.

Determination of the enantiomers of thioridazine, thioridazine 2-sulfone, and of the isomeric pairs of thioridazine 2-sulfoxide and thioridazine 5-sulfoxide in human plasma.

Thioridazine is a commonly prescribed phenothiazine drug administered as a racemate and it is believed that its antipsychotic effect is mainly associated with (R)-thioridazine. A method based on high-performance liquid chromatography has been developed for the determination of the enantiomers of thioridazine and thioridazine 2-sulfone (THD 2-SO2 or sulforidazine) and of the enantiomers of the diastereoisomeric pairs of thioridazine 2-sulfoxide (THD 2-SO or mesoridazine) and thioridazine 5-sulfoxide (THD 5-SO) in the plasma of thioridazine-treated patients. The method involves sequential achiral and chiral HPLC. The limits of quantitation for total (R) + (S) concentrations were found to be 15 ng/ml for thioridazine and 5 ng/ml for its metabolites. The limits for the determination of the (R)/(S) ratios were found to be 60 ng/ml for racemic THD and 10 ng/ml for racemic THD 2-SO, THD 2-SO2, THD 5-SO (FE) and THD 5-SO (SE). The method has been used to determine the concentrations of the enantiomers of thioridazine and of its metabolites in the plasma of a patient treated with 100 mg of racemic thioridazine hydrochloride per os per day for 14 days. The results show a high enantioselectivity in the metabolism of this drug: the (R)/(S) ratios for THD, THD 2-SO (FE), THD 2-SO (SE), THD 2-SO2, THD 5-SO (FE) and THD 5-SO (SE) were found to be 3.90, 1.22, 6.10, 4.10, 0.09 and 28.0, respectively.

Valganciclovir in adult solid organ transplant recipients: pharmacokinetic and pharmacodynamic characteristics and clinical interpretation of plasma concentration measurements.

Valganciclovir and ganciclovir are widely used for the prevention of cytomegalovirus (CMV) infection in solid organ transplant recipients, with a major impact on patients' morbidity and mortality. Oral valganciclovir, the ester prodrug of ganciclovir, has been developed to enhance the oral bioavailability of ganciclovir. It crosses the gastrointestinal barrier through peptide transporters and is then hydrolysed into ganciclovir. This review aims to describe the current knowledge of the pharmacokinetic and pharmacodynamic characteristics of this agent, and to address the issue of therapeutic drug monitoring. Based on currently available literature, ganciclovir pharmacokinetics in adult solid organ transplant recipients receiving oral valganciclovir are characterized by bioavailability of 66 +/- 10% (mean +/- SD), a maximum plasma concentration of 3.1 +/- 0.8 mg/L after a dose of 450 mg and of 6.6 +/- 1.9 mg/L after a dose of 900 mg, a time to reach the maximum plasma concentration of 3.0 +/- 1.0 hours, area under the plasma concentration-time curve values of 29.1 +/- 5.3 mg.h/L and 51.9 +/- 18.3 mg.h/L (after 450 mg and 900 mg, respectively), apparent clearance of 12.4 +/- 3.8 L/h, an elimination half-life of 5.3 +/- 1.5 hours and an apparent terminal volume of distribution of 101 +/- 36 L. The apparent clearance is highly correlated with renal function, hence the dosage needs to be adjusted in proportion to the glomerular filtration rate. Unexplained interpatient variability is limited (18% in apparent clearance and 28% in the apparent central volume of distribution). There is no indication of erratic or limited absorption in given subgroups of patients; however, this may be of concern in patients with severe malabsorption. The in vitro pharmacodynamics of ganciclovir reveal a mean concentration producing 50% inhibition (IC(50)) among CMV clinical strains of 0.7 mg/L (range 0.2-1.9 mg/L). Systemic exposure of ganciclovir appears to be moderately correlated with clinical antiviral activity and haematotoxicity during CMV prophylaxis in high-risk transplant recipients. Low ganciclovir plasma concentrations have been associated with treatment failure and high concentrations with haematotoxicity and neurotoxicity, but no formal therapeutic or toxic ranges have been validated. The pharmacokinetic parameters of ganciclovir after valganciclovir administration (bioavailability, apparent clearance and volume of distribution) are fairly predictable in adult transplant patients, with little interpatient variability beyond the effect of renal function and bodyweight. Thus ganciclovir exposure can probably be controlled with sufficient accuracy by thorough valganciclovir dosage adjustment according to patient characteristics. In addition, the therapeutic margin of ganciclovir is loosely defined. The usefulness of systematic therapeutic drug monitoring in adult transplant patients therefore appears questionable; however, studies are still needed to extend knowledge to particular subgroups of patients or dosage regimens.

Prediction of free imatinib concentrations based on total plasma concentrations in patients with gastrointestinal stromal tumours.

AIM: Total imatinib concentrations are currently measured for the therapeutic drug monitoring of imatinib, whereas only free drug equilibrates with cells for pharmacological action. Due to technical and cost limitations, routine measurement of free concentrations is generally not performed. In this study, free and total imatinib concentrations were measured to establish a model allowing the confident prediction of imatinib free concentrations based on total concentrations and plasma proteins measurements. METHODS: One hundred and fifty total and free plasma concentrations of imatinib were measured in 49 patients with gastrointestinal stromal tumours. A population pharmacokinetic model was built up to characterize mean total and free concentrations with inter-patient and intrapatient variability, while taking into account α1 -acid glycoprotein (AGP) and human serum albumin (HSA) concentrations, in addition to other demographic and environmental covariates. RESULTS: A one compartment model with first order absorption was used to characterize total and free imatinib concentrations. Only AGP influenced imatinib total clearance. Imatinib free concentrations were best predicted using a non-linear binding model to AGP, with a dissociation constant Kd of 319 ng ml(-1) , assuming a 1:1 molar binding ratio. The addition of HSA in the equation did not improve the prediction of imatinib unbound concentrations. CONCLUSION: Although free concentration monitoring is probably more appropriate than total concentrations, it requires an additional ultrafiltration step and sensitive analytical technology, not always available in clinical laboratories. The model proposed might represent a convenient approach to estimate imatinib free concentrations. However, therapeutic ranges for free imatinib concentrations remain to be established before it enters into routine practice.

Increased plasma levels of N-terminal brain natriuretic peptide (NT-proBNP) in type 2 diabetic patients with vascular complications.

AIMS: The plasma levels of either brain natriuretic peptide (BNP) or the N-terminal fragment of the prohormone (NT-proBNP) have recently gained extreme importance as markers of myocardial dysfunction. Patients with type 2 diabetes are at high risk of developing cardiovascular complications. This study was aimed to assess whether plasma NT-proBNP levels are at similar levels in type 2 diabetics with or without overt cardiovascular diseases. METHODS: We assayed plasma NT-proBNP in 54 type 2 diabetics, 27 of whom had no overt macro- and/or microvascular complications, while the remaining ones had either or both. The same assay was carried out in 38 healthy control subjects age and sex matched as a group with the diabetics. RESULTS: Plasma NT-proBNP was higher in diabetics (median 121 pg/ml, interquartile range 50-240 pg/ml, ) than in those without complications (37 pg/ml, 21-54 pg/ml, P<0.01). Compared with the controls (55 pg/ml, 40-79 pg/ml), only diabetics with vascular complications had significantly increased plasma NT-proBNP levels (P<0.001). In the diabetics, coronary heart disease and nephropathy (defined according to urinary excretion of albumin) were each independently associated with elevated values of plasma NT-proBNP. CONCLUSIONS: In type 2 diabetes mellitus, patients with macro- and/or micro-vascular complications exhibit an elevation of plasma NT-proBNP levels compared to corresponding patients with no evidence of vascular disease. The excessive secretion of this peptide is independently associated with coronary artery disease and overt nephropathy. The measurement of circulating NT-proBNP concentration may therefore be useful to screen for the presence of macro- and/or microvascular disease.