Measuring substrate binding and affinity of purified membrane transport proteins using the scintillation proximity assay

The scintillation proximity assay (SPA) is a rapid radioligand binding assay. Upon binding of radioactively labeled ligands (here L-[(3)H]arginine or D-[(3)H]glucose) to acceptor proteins immobilized on fluoromicrospheres (containing the scintillant), a light signal is stimulated and measured. The application of SPA to purified, detergent-solubilized membrane transport proteins allows substrate-binding properties to be assessed (e.g., substrate specificity and affinity), usually within 1 d. Notably, the SPA makes it possible to study specific transporters without interference from other cellular components, such as endogenous transporters. Reconstitution of the target transporter into proteoliposomes is not required. The SPA procedure allows high sample throughput and simple sample handling without the need for washing or separation steps: components are mixed in one well and the signal is measured directly after incubation. Therefore, the SPA is an excellent tool for high-throughput screening experiments, e.g., to search for substrates and inhibitors, and it has also recently become an attractive tool for drug discovery.

Cardiac transplantation with hearts from donors after circulatory declaration of death: haemodynamic and biochemical parameters at procurement predict recovery following cardioplegic storage in a rat model

OBJECTIVES: Donation after circulatory declaration of death (DCDD) could significantly improve the number of cardiac grafts for transplantation. Graft evaluation is particularly important in the setting of DCDD given that conditions of cardio-circulatory arrest and warm ischaemia differ, leading to variable tissue injury. The aim of this study was to identify, at the time of heart procurement, means to predict contractile recovery following cardioplegic storage and reperfusion using an isolated rat heart model. Identification of reliable approaches to evaluate cardiac grafts is key in the development of protocols for heart transplantation with DCDD. METHODS: Hearts isolated from anaesthetized male Wistar rats (n = 34) were exposed to various perfusion protocols. To simulate DCDD conditions, rats were exsanguinated and maintained at 37°C for 15-25 min (warm ischaemia). Isolated hearts were perfused with modified Krebs-Henseleit buffer for 10 min (unloaded), arrested with cardioplegia, stored for 3 h at 4°C and then reperfused for 120 min (unloaded for 60 min, then loaded for 60 min). Left ventricular (LV) function was assessed using an intraventricular micro-tip pressure catheter. Statistical significance was determined using the non-parametric Spearman rho correlation analysis. RESULTS: After 120 min of reperfusion, recovery of LV work measured as developed pressure (DP)-heart rate (HR) product ranged from 0 to 15 ± 6.1 mmHg beats min(-1) 10(-3) following warm ischaemia of 15-25 min. Several haemodynamic parameters measured during early, unloaded perfusion at the time of heart procurement, including HR and the peak systolic pressure-HR product, correlated significantly with contractile recovery after cardioplegic storage and 120 min of reperfusion (P < 0.001). Coronary flow, oxygen consumption and lactate dehydrogenase release also correlated significantly with contractile recovery following cardioplegic storage and 120 min of reperfusion (P < 0.05). CONCLUSIONS: Haemodynamic and biochemical parameters measured at the time of organ procurement could serve as predictive indicators of contractile recovery. We believe that evaluation of graft suitability is feasible prior to transplantation with DCDD, and may, consequently, increase donor heart availability.

Practical sources of error in measuring pulmonary artery occlusion pressure: a study in participants of a special intensivist training program of The Scandinavian Society of Anaesthesiology and Intensive Care Medicine (SSAI)

BACKGROUND: Physiological data obtained with the pulmonary artery catheter (PAC) are susceptible to errors in measurement and interpretation. Little attention has been paid to the relevance of errors in hemodynamic measurements performed in the intensive care unit (ICU). The aim of this study was to assess the errors related to the technical aspects (zeroing and reference level) and actual measurement (curve interpretation) of the pulmonary artery occlusion pressure (PAOP). METHODS: Forty-seven participants in a special ICU training program and 22 ICU nurses were tested without pre-announcement. All participants had previously been exposed to the clinical use of the method. The first task was to set up a pressure measurement system for PAC (zeroing and reference level) and the second to measure the PAOP. RESULTS: The median difference from the reference mid-axillary zero level was - 3 cm (-8 to + 9 cm) for physicians and -1 cm (-5 to + 1 cm) for nurses. The median difference from the reference PAOP was 0 mmHg (-3 to 5 mmHg) for physicians and 1 mmHg (-1 to 15 mmHg) for nurses. When PAOP values were adjusted for the differences from the reference transducer level, the median differences from the reference PAOP values were 2 mmHg (-6 to 9 mmHg) for physicians and 2 mmHg (-6 to 16 mmHg) for nurses. CONCLUSIONS: Measurement of the PAOP is susceptible to substantial error as a result of practical mistakes. Comparison of results between ICUs or practitioners is therefore not possible.

Second international collaborative study evaluating performance characteristics of methods measuring the von Willebrand factor cleaving protease (ADAMTS-13)

BACKGROUND: Over the last 4 years ADAMTS-13 measurement underwent dramatic progress with newer and simpler methods. AIMS: Blind evaluation of newer methods for their performance characteristics. DESIGN: The literature was searched for new methods and the authors invited to join the evaluation. Participants were provided with a set of 60 coded frozen plasmas that were prepared centrally by dilutions of one ADAMTS-13-deficient plasma (arbitrarily set at 0%) into one normal-pooled plasma (set at 100%). There were six different test plasmas ranging from 100% to 0%. Each plasma was tested 'blind' 10 times by each method and results expressed as percentage vs. the local and the common standard provided by the organizer. RESULTS: There were eight functional and three antigen assays. Linearity of observed-vs.-expected ADAMTS-13 levels assessed as r2 ranged from 0.931 to 0.998. Between-run reproducibility expressed as the (mean) CV for repeated measurements was below 10% for three methods, 10-15% for five methods and up to 20% for the remaining three. F-values (analysis of variance) calculated to assess the capacity to distinguish between ADAMTS-13 levels (the higher the F-value, the better the capacity) ranged from 3965 to 137. Between-method variability (CV) amounted to 24.8% when calculated vs. the local and to 20.5% when calculated vs. the common standard. Comparative analysis showed that functional assays employing modified von Willebrand factor peptides as substrate for ADAMTS-13 offer the best performance characteristics. CONCLUSIONS: New assays for ADAMTS-13 have the potential to make the investigation/management of patients with thrombotic microangiopathies much easier than in the past.

Duplex sonographic criteria for measuring carotid stenoses

PURPOSE: The aim of this retrospective study was to determine optimal duplex sonographic criteria for use in our institution for diagnosing severe carotid stenoses and to correlate those findings with angiographic measurements obtained by the European Carotid Surgery Trial (ECST), North American Symptomatic Carotid Endarterectomy Trial (NASCET), and Common Carotid (CC) methods of grading carotid stenoses. METHODS: We analyzed the angiographic data using the ECST, NASCET, and CC methods and compared the results with the duplex sonographic findings. We then calculated the sensitivity, specificity, positive and negative predictive values, and accuracy of the duplex sonographic method. Taking these parameters into account, the optimal intrastenotic peak systolic velocity (PSV) and end diastolic velocity (EDV) were derived for diagnosing severe stenoses according to the 3 angiographic methods. RESULTS: Optimal PSV and EDV values for diagnosing a 70% or greater stenosis in our laboratory were as follows: with the NASCET method of angiographic grading of stenoses, PSV 220 cm/second or greater and EDV 80 cm/second or greater, and with the ECST and CC methods, PSV 190 cm/second or greater, and EDV 65 cm/second or greater. The optimal PSV and EDV for diagnosing a stenosis of 80% or greater with the ECST grading method were 215 cm/second or greater and 90 cm/second or greater, respectively. CONCLUSIONS: Duplex sonography is a sensitive and accurate tool for evaluating severe carotid stenoses. Optimal PSVs and EDVs vary according to the angiographic method used to grade the stenosis. They are similar for stenoses 70% or greater with the NASCET method and for stenoses 80% or greater with the ECST method.