Ralf Kaiser

Detection and validated quantification of the phosphodiesterase type 5 inhibitors sildenafil, vardenafil, tadalafil, and 2 of their metabolites in human blood plasma by LC-MS/MS--application to forensic and therapeutic drug monitoring cases.

Introduction: Phosphodiesterase type 5 inhibitors such as sildenafil, vardenafil, and tadalafil are a class of drugs used primarily in the treatment of erectile dysfunction. Sildenafil and tadalafil are also approved for the treatment of pulmonary hypertension. The aim of this study was to develop and validate a procedure for the detection and quantification of these 3 drugs and some of their metabolites in human blood plasma. Methods: After liquid–liquid extraction of 0.5 mL of blood plasma using diethyl ether–ethyl acetate (1:1), the analytes sildenafil, norsildenafil, vardenafil, norvardenafil, and tadalafil were separated using a Shimadzu Prominence High-Performance Liquid Chromatography System (C18 separation column, gradient elution, and a total flow of 0.5 mL/min). They were detected using an AB Sciex 3200 Q-Trap LC-MS-MS System (electrospray ionization and multiple reaction monitoring mode). The method was fully validated according to international guidelines. Results: The assay was found to be selective for the tested compounds. It was linear from 5 to 1000 ng/mL for sildenafil, from 2 to 700 ng/mL for norsildenafil, from 0.5 to 350 ng/mL for vardenafil, from 0.5 to 200 ng/mL for norvardenafil, and from 5 to 1000 ng/mL for tadalafil. The recoveries were generally more than 50%. Matrix effects were not observed. Accuracy, repeatability, and intermediate precision were within the required limits (<15% or <20% near the limit of quantification). No instability was observed after repeated freezing and thawing or in processed samples. Conclusions: A liquid chromatography–tandem mass spectrometry assay for the determination of sildenafil, norsildenafil, vardenafil, norvardenafil, and tadalafil in human blood plasma was developed and validated. It has proven to be selective, linear, accurate, and precise for all studied drugs. The method has also proven to be applicable for forensic cases and for therapeutic drug monitoring.

Ischaemia-induced up-regulation of Toll-like receptor 2 in circulating monocytes in cardiogenic shock.

AIMSnTo investigate the role of Toll-like receptor 2 (TLR2) in uncomplicated acute myocardial infarction (AMI) and in cardiogenic shock (CS).nnnMETHODS AND RESULTSnIn patients with uncomplicated AMI (n = 20), CS (n = 30) and in age-matched healthy controls (HC; n = 20), TLR2 expression on monocytes was assessed by flow cytometry. Tumour necrosis factor alpha (TNFα) and interleukin-6 (IL6) expression in monocytes was analysed by intracellular cytokine staining. TLR2 expression was increased in patients with AMI compared with HC [mean fluorescence intensity (MFI) 111.1 ± 8.2 vs. 66.9 ± 1.5, P < 0.001]. In patients with CS, TLR2 expression was further increased (132.8 ± 5.6 MFI, P = 0.009 vs. AMI). This was accompanied by an increased expression of the proinflammatory cytokines TNFα (4.3 ± 1.6% in AMI vs. 20.5 ± 5.9% in CS, P = 0.004) and IL6 (6.3 ± 1.6% in AMI vs. 20.6 ± 6.2% in CS, P = 0.032). Furthermore, in all patients with myocardial infarction (AMI + CS; n = 50), a strong correlation between the monocytic TLR2 expression and the symptom to reperfusion time (r(2)= 0.706, P < 0.001) was found, implying tissue hypoxia dependency. Symptom to reperfusion time is a main factor to influence TLR2 expression but not the presence of CS. TLR2 expression of mononuclear cells exposed in vitro to hypoxia was assessed by flow cytometry and western blot. In vitro measurements showed a hypoxia-mediated monocytic TLR2 expression up-regulation.nnnCONCLUSIONnWe demonstrate TLR2 up-regulation and increased proinflammatory cytokine expression in circulating monocytes in AMI/CS depending on disease severity, implying an important role of TLR2 expression in ischaemic injury.

Effect of hypoxia on integrin-mediated adhesion of endothelial progenitor cells

Homing of endothelial progenitor cells (EPCs) is crucial for neoangiogenesis, which might be negatively affected by hypoxia. We investigated the influence of hypoxia on fibronectin binding integrins for migration and cell‐matrix‐adhesion. AMP‐activated kinase (AMPK) and integrin‐linked kinase (ILK) were examined as possible effectors of hypoxia.Human EPCs were expanded on fibronectin (FN) and integrin expression was profiled by flow cytometry. Cell‐matrix‐adhesion‐ and migration‐assays on FN were performed to examine the influence of hypoxia and AMPK‐activation. Regulation of AMPK and ILK was shown by Western blot analysis. We demonstrate the presence of integrin β1, β2 and α5 on EPCs. Adhesion to FN is reduced by blocking β1 and α5 (49% and 2% of control, P < 0.05) whereas α4‐blockade has no effect. Corresponding effects were shown for migration. Hypoxia and AMPK‐activation decrease adhesion on FN. Although total AMPK‐expression remains unchanged, phospho‐AMPK increases eightfold.The EPCs require α5 for adhesion on FN. Hypoxia and AMPK‐activation decrease adhesion. As α5 is the major adhesive factor for EPCs on FN, this suggests a link between AMPK and α5‐integrins. We found novel evidence for a connection between hypoxia, AMPK‐activity and integrin activity. This might affect the fate of EPCs in ischaemic tissue.

The role of circulating thrombospondin-1 in patients with precapillary pulmonary hypertension

BackgroundThe vasoconstrictive protein TSP-1 is released from endothelial cells upon increased shear stress and hypoxia. Both conditions are prevalent in pulmonary hypertension (PH). TSP-1 damages the local microcirculation by disrupting pathways, which are essential for specific medical therapeutics. Furthermore, TSP-1 induces excessive fibrosis and smooth muscle proliferation - a common finding in advanced PH - via TGF-ß and might promote disease progression. The prognostic impact of circulating TSP-1, influence on hemodynamic parameters and interaction with other biomarkers in patients with PH is incompletely understood.This study examines prospectively circulating TSP-1 in association with hemodynamic parameters, clinical variables and mortality.MethodsCirculating TSP-1 was measured prospectively in 93 patients with precapillary PH undergoing right heart catheterization and in 19 subjects without PH. TSP-1 levels were determined by ELISA and examined in the context of hemodynamic variables. For evaluation of survival, patients were monitored for adverse events on a 3-monthly basis and contacted at the end of the study after 5xa0years. In addition, levels of big-endothelin and humoral cofactors of TSP-1 release were measured.ResultsPatients with PH had significantly increased TSP-1 levels compared to controls without PH (1114u2009±u2009136xa0ng/mL vs. 82.1u2009±u200915.8xa0ng/mL, pu2009<u20090.05). Levels were correlated with mean pulmonary artery pressure (PAPm, ru2009=u2009−0.58, pu2009<u20090.001) and pulmonary vascular resistance (PVR, ru2009=u20090.33, pu2009=u20090.002). Survivors had lower TSP-levels as non-survivors and all cause mortality associated with TSP-1 plasma levels above 2051xa0ng/mL (pu2009=u20090.0002, HR 1.49).ConclusionsHigh plasma levels of TSP-1 are associated with increased PAPm, increased PVR and decreased survival. Due to its interaction with therapeutic pathways, studies are warranted to clarify the impact of TSP-1 on of specific medications for PH.

Prognostic impact of renal function in precapillary pulmonary hypertension.

Impairment of renal function is associated with adverse outcome in various diseases. Patients with pulmonary hypertension (PH) show diminished cardiac function and organ perfusion. The aim of this study was to investigate the associations between renal function and both haemodynamic parameters and long‐term survival in patients with PH.

Association of elevated plasma viscosity with small vessel occlusion in ischemic cerebral disease.

INTRODUCTIONnElevated plasma viscosity (PV) is observed in patients with vascular risk factors, such as diabetes mellitus or arterial hypertension. In this study we investigated the association of plasma viscosity and the different clinical and radiological entities of cerebral ischemia.nnnMETHODSnPV of 465 consecutively admitted patients with clinical symptoms of acute cerebral ischemia without radiological signs of bleeding was measured. Data is expressed as median [range] unless stated otherwise. p<0.05 was considered statistically significant.nnnRESULTSnPatients with acute cerebral ischemia (TIA or Stroke) showed increased PV (TIA 1.27mPas [1.07-1.53], stroke 1.27mPas [1.07-1.56]) compared to patients without cerebral ischemia (Mimics) (1.23mPas [1.06-1.42]). The group with radiologically proven small vessel disease (SVD) had a significantly higher mean values of PV (1.29mPas [1.06-1.54]) compared to those with signs of large vessel disease or cardioembolic events (1.22mPas [1.07-1.56], p<0.001). Patients with chronic heart failure (p=0.007), arterial hypertension (p<0.001) and diabetes mellitus (p=0.002) had higher PV compared to patients without these cardiovascular risk factors. Hyperlipidemia or nicotine abuse showed no relation to PV.nnnCONCLUSIONnElevated PV is not only associated TIA and Stroke but is also found in patients with radiological signs of cerebral SVD. High levels of PV could be an underestimated risk for TIA and Stroke and participate in the complex pathophysiology of SVD. Prospective observational and interventional studies are warranted for further evaluation of PV in neurological ischemic diseases.

Assessment of operability by means of CTPA and perfusion SPECT in patients with chronic thromboembolic pulmonary hypertension

Background Chronic thromboembolic pulmonary hypertension (CTEPH) can potentially be cured by pulmonary thrombendarterectomy (PEA), the criteria for differentiation between operable and non-operable patients are not standardized. Purpose To retrospectively evaluate the value of rigidly registered computed tomography pulmonary angiography (CTPA) and single photon emission CT (SPECT) in differentiating for PEA. Material and Methods Forty-nine patients with CTEPH (21 men; age, 58u2009±u200913 years) were evaluated by an interdisciplinary expert board using all available diagnostic information and their consensus statement as gold standard. For SPECT a lobe based perfusion score was visually assessed using the score of 0 (lack of perfusion) to 1 (normal perfusion) calculating percentage of vascular obstruction (PVO). By CTPA, vascular obstruction index (OI) of central, peripheral, and global PA-bed were determined. The accuracy of the alignment between CTPA and SPECT was determined by fusion score (FS) ranging from 1 (no alignment) to 5 (exact alignment). Angiography provided PA pressure (PAP), pulmonary vascular resistance (PVR), and PA wedge pressure (PAWP). Receiver operating characteristics (ROC) analysis was performed. Results Twenty-nine patients were considered surgically amenable, and 20 patients were inoperable. Mean PAP, PVR, and PAWP were 48u2009±u200911u2009mmHg, 868u2009±u2009461 dynes*sec*cm−5, and 11u2009±u20095u2009mmHg, without differences between surgical and non-surgical patients (Pu2009>u20090.5). In all patients accurate registration was reached (FSu2009=u20094.1u2009±u20090.7; range, 2–5). PVO and central OI separated PEA-amenable patients (Pu2009≤u20090.001) resulting in the area under the curve of 0.828 (cutoff for PVO: 37.8% with a sensitivity of 82% and specificity of 79%) and 0.755 (cutoff for central OI: 29% with a sensitivity and specificity of 86.2% and 79%) for operability. Conclusion An accurate interpretation of rigidly registered CTPA and perfusion SPECT may contribute to stratification of operability in patients with CTEPH.

Mid-regional pro-adrenomedullin: an indicator of the failing Fontan circuit in patients with univentricular hearts?

In patients after the Fontan procedure, assessment of a failing Fontan circuit is difficult. Natriuretic peptides failed to be reliable markers of functional status or systemic ventricular function in this patient cohort. The aim of the study was to assess the clinical utility of mid‐regional pro‐adrenomedullin (MR‐proADM) in patients after the Fontan procedure.

Associations of circulating natriuretic peptides with haemodynamics in precapillary pulmonary hypertension

BACKGROUNDnWhile N-terminal B-type natriuretic peptide (NT-proBNP) has been examined extensively in pulmonary hypertension (PH), limited data exists on the subtype A, C and D. The aim of this prospective pilot study was a head-to-head comparison of NPs in respect to haemodynamic parameters and the influence of renal function.nnnMETHODSnPlasma samples were drawn during routine right heart catheterization in 62 patients with precapillary PH and 20 control patients. MR-proANP measurements were performed on the automated Kryptor platform, NT-proBNP by CLIA, NT-proCNP and DNP levels by ELISA. Results are expressed as median [range] and tested non-parametrically. Non-parametric locally linear multiple regression was performed to determine the influence of renal function on NP levels. P-values <0.05 were considered significant.nnnRESULTSnPatients with PH had significantly higher MR-proANP and NT-proBNP levels. NT-proCNP showed a trend to higher levels, while DNP did not differ from control subjects. Both MR-proANP and NT-proBNP were associated with cardiac index (CI), right atrial pressure (RAP), mean pulmonary artery pressure (PAPm) and pulmonary vascular resistance index (PVRI). NT-proCNP was associated with RAP, while DNP showed no associations with haemodynamic variables. Associations of haemodynamic parameters with NPs were weakened in patients with in elevated serum creatinine and showed increased regression slopes.nnnCONCLUSIONnMR-proANP demonstrated equivalent associations with haemodynamics compared to NT-proBNP, but both markers depend on intact renal function. NT-proCNP was correlated with RAP and renal function, while DNP showed no associations. Larger studies should evaluate MR-proANP as candidate prognostic biomarker in PH.

Antidepressant effects of direct-acting antivirals against hepatitis C virus-Results from a pilot study

The new direct‐acting antiviral agents (DAA) have revolutionized the treatment of patients with chronic hepatitis C virus (HCV) infection. This study investigates to which extent DAA affect fatigue and mood and, if so, whether this results from changes to tryptophan (TRP) metabolism, as reflected by two critical biosynthetic pathways, serotonin (SRT) generation from TRP and TRP degradation through kynurenines (KYN) via indoleamine 2,3‐dioxygenase (IDO).