A simple and rapid label-free fluorimetric biosensor for protamine detection based on glutathione-capped CdTe quantum dots aggregation.

A novel fluorescent biosensor is developed, based on glutathione-capped CdTe quantum dots aggregation, for the determination of trace amount of an important drug, protamine. In this method with increasing the protamine concentration, the fluorescence of the quantum dots was quenched due to their aggregation. Different parameters affect the sensitivity, such as pH and the amount of the quantum dots, were optimized. Using the new optical biosensor, under the optimized conditions, protamine could be measured in the range of 2.0-200 ng mL(-1) with a detection limit of 1.0 ng mL(-)(1). The relative standard deviation for five replicates determination of 30.0 ng mL(-)(1) protamine was 1.26%. The influence of common interfering species on the protamine detection was studied. The results showed that the biosensor is highly selective and sensitive for the detection of protamine. The optical biosensor was successfully used for the determination of protamine in real samples.

Polyion selective polymeric membrane-based pulstrode as a detector in flow-injection analysis.

A method for the detection of polyions using fully reversible polyion selective polymeric membrane type pulstrodes as detectors in a flow-injection analysis (FIA) system is examined. The detection electrode consists of a plasticized polymeric membrane doped with 10 wt % of tridodecylmethylammonium-dinonylnaphthalene sulfonate (TDMA/DNNS) ion-exchanger salt. The pulse sequence used involves a short (1 s) galvanostatic pulse, an open-circuit pulse (0.5 s) during which the EMF of the cell is measured, and a longer (15 s) potentiostatic pulse to return the membrane to its original chemical composition. It is shown that total pulse sequence times can be optimized to yield reproducible real-time detection of injected samples of protamine and heparin at up to 20 samples/h. Further, it is shown that the same membrane detector can be employed for FIA detection of both polycations at levels ≥10 µg/mL and polyanions at levels of ≥40 µg/mL by changing the direction of the galvanostatic pulse. The methodology described may also be applicable in the detection of polyionic species at low levels in other flowing configurations, such as in liquid chromatography and capillary electrophoresis.

Establishment of a replacement batch for heparin sodium biological reference preparation.

An international collaborative study was organised to replace the current European Pharmacopoeia biological reference preparation for heparin sodium. The project was organised by the European Directorate for the Quality of Medicines & HealthCare in the frame of its Biological Standardisation Programme. A suitable candidate batch representative of the quality of heparin products currently marketed in Europe was donated to the EDQM and included in a collaborative study involving 19 laboratories from 10 European countries, the Americas, Australia and the Council of Europe. Laboratories were requested to perform their routine assays following the prescriptions of the Ph. Eur. for the assay and the identification of unfractionated heparin and for the assay of protamine. The results made it possible to demonstrate that the candidate batch was suitable for its intended use and it was therefore established by the European Pharmacopoeia Commission as the Ph. Eur. heparin sodium BRP batch 3 in June 2007.

[Antiheparin activity of the aneurysm wall and parietal thrombus and blood plasma of patients with aortic aneurysm]. / Aktywnosc antyheparynowa sciany i skrzepliny przysciennej tetniaka oraz osocza krwi chorych z tetniakiem aorty.

Aneurysm dilatation is filled by parietal thrombus. Its magnitude is determined by the ratio of coagulative to anticoagulative activities above all. The aim of the study was to assess antiheparin activity, contents of heparin neutralizing chemical compounds and glycosaminoglycans with anticoagulative activity as well in the aneurysm wall, parietal thrombus filling the aneurysm dilatation and blood plasma/blood serum of patients with aortic aneurysm. The studied material consisted of aneurysm walls and parietal thrombi extracts, as well as of blood plasma/serum of patients with aortic aneurysm. Extracts of normal aorta from organ donors and blood plasma/serum of healthy subjects were the control material. Antiheparin and antithrombin III activities, as well as contents of heparin neutralizing compounds and heparin-like acting glycosaminoglycans were evaluated in the investigated material with chemical methods. The aneurysm wall, parietal thrombus filling the aneurysm dilatation and blood plasma of patients with aortic aneurysm show high antiheparin activity and increased content of heparin neutralizing compounds. However differences in glycosaminoglycans content were not demonstrated. That can predispose to formation and enlargement of parietal thrombus in aneurysm dilatation.

Automated protamine dose assay in heparin reversal management after cardiopulmonary by pass.

BACKGROUND: To evaluate the impact of automated Protamine Dose Assay (PDA) performed with Hemochron 8000 (International Technodyne Company, Edison, NJ) on the management of heparin reversal after cardiopulmonary bypass (CPB). PDA was compared with empirical protamine to heparin ratio with regard to calculation of the protamine dose, and the sensitivity of PDA and ACT to residual circulating heparin after protamine administration was investigated too. DESIGN: prospective and randomized study. SETTING: cardiac surgical center of a General Hospital. PARTICIPANTS: 50 patients undergoing elective cardiac surgery with CPB. INTERVENTIONS: after CPB patients randomly received protamine according to our standard empirical ratio of 1 mg. protamine/100 U. heparin (group S, 24 patients), or to PDA result (group T, 26 patients) based on protamine titration method of determining circulating heparin. After protamine administration ACT and PDA were performed to assess heparin reversal and detect residual circulating heparin. Based on the PDA result, additional protamine was administered in both groups when required. MEASUREMENTS: in both groups basal and post-heparin ACT values, protamine doses, ACT and PDA after protamine administration were measured. RESULTS: The protamine dose was significantly lower (30%) in patients treated according to PDA. In 20% of patients showing normal ACT PDA revealed still circulating heparin, and additional protamine was required. In all other cases ACT and PDA both confirmed heparin reversal. CONCLUSIONS: PDA allowed us to administer a significantly lower amount of protamine. This can reduce incidence of adverse effects of over- and under-infusion of protamine. PDA also proved to be more sensitive than ACT in detecting residual circulating heparin after protamine administration.

Determination of protamine sulphate in drug formulations using high performance liquid chromatography.

Protamine sulphate, which has the property of neutralising heparin, is determined in pharmaceutical formulations using spectrophotometric (BP 1995) or biochemical methods (USP XXIII 1995). Accuracy of these methods is not very high. We applied the HPLC technique for the assay of protamine sulphate in a gel formulation. The assay was carried out on a diol-type column with a mobile phase containing 8% acetonitrile in 0.15% trifluoroacetic acid at pH 2.5. The flow rate was 1.0 ml min(-1). The results obtained show that HPLC can be used for the determination of protamine sulphate in pharmaceutical preparations. The method is rapid and more accurate than those described until now.

Evaluation of a new protamine titration method to assay heparin in whole blood and plasma.

When unfractionated heparin is used for therapeutic anticoagulation, the heparin effect must be monitored to avoid thrombotic or hemorrhagic complications. The ability of a factor Xa inhibition (XaI) assay was compared with that of a low-level heparin protamine titration (LLHPT) assay to measure the concentration of heparin after heparin was added in vitro to specimens of plasma and whole blood. Heparin effect on the activated partial thromboplastin time also was assessed in the same specimens. The XaI and LLHPT assays had comparable precision and provided linear results over a wide range of heparin concentrations. Both assays slightly underestimated the total amount of heparin added to the specimens. The most rapid test was the whole blood LLHPT assay; this test therefore may be useful for bedside monitoring of heparin. A significant disadvantage of the LLHPT assay was the large sample size required to perform it. These results provide in vitro evidence that the XaI and LLHPT assays can provide equally precise monitoring of heparin concentration.

Prototype of a new protamine sulphate titration kit for use in cardiopulmonary bypass surgery.

A new protamine sulphate titration kit has been developed and tested in patients undergoing cardiac bypass surgery. When a prototype of the kit was used, protamine sulphate dosage was significantly reduced (P less than 0.01) without a significant increase in 24-hour postoperative blood loss (P greater than 0.05).

Neutralization of heparin by basic proteins of tumor cells: studies in vitro with protein rich in 14C-arginine.

14C-arginine rich basic protein isolated from the cytoplasm of Ehrlich ascites tumor cells neutralizes the anticoagulant of activity heparin. The action of this protein is greater than H3 histone rich in arginine derived from calf thymus.

The determination of the heparin neutralising capacity of protamine using acridine orange fluorescence.

A new method to estimate the heparin neutralising capacity of protamine has been investigated. The technique has been applied to two commercial heparin preparations tested against the W.H.O. 1st International Reference preparation of protamine. The technique is based on the fluorescence of acridine orange dye and the selective binding of heparin with protamine. A titration procedure has been devised and the two variations of the assay are compared and evaluated. The titration is rapid, accurate and convenient.