In vitro Research of the Concentration Dependence of Effect of Adrenaline on Platelets Aggregation.

PURPOSE: Paper intended to present experimental evidences that adrenaline has a direct effect of inducing platelets aggregation in the concentration range 1-8µM. MATERIAL/METHODS: Platelet rich plasma from patients of Colentina Clinical Hospital, following an informed consent. The platelet rich plasma (PRP) was prepared by centrifuging the anticoagulated sample at 200 G for 10 minutes. Aggregation was evaluated by optical aggregometry, classical method of Born, using Helena PACKS-4 Aggregometer. RESULTS: The curves transmission light-time followed the structure: a lag-time, a first phase aggregation, more or less linear, defined by a "Slope 1", a second wave of aggregation defined by "slope 2" and a "saturation" phase. Slope 1 increases with the concentration of adrenaline. The second slopes of the aggregation curves, maximum aggregation and areas under curves depended linear on adrenaline concentration. CONCLUSIONS: Adrenaline, in concentrations in the 1-8µM, induce aggregation of human platelets from platelet rich plasma. Linear regression models for slope and area were practically identical suggesting a rather unique than biphasic mechanism of action of adrenaline during the time course of aggregation.

Application of Biopharmaceutical and Clinical Metrics in Analyzing Modification of Platelets Aggregation Curves by Calcium Ion.

In context of Evidence Based Medicine concept, Good Clinical Practice rules specify that "data generated should be reliable and robust".Reliability and robustness are further translated in requirements concerning statistical and clinical significance of results. Paper presents main aspectsconnected with comparison of evolutions of endpoints as function of different parameters like time, administered dose, proportion of active components etc., leading to problems of comparison of curves, with direct application to comparison of platelets aggregation curves in presence of different concentrations of ionic calcium. Theoretical part presents comparison of curves in biopharmacyusing f2 metric and area under curve metric, and comparison of survival curves in clinical studies.Platelet aggregation test was performed using Bornturbidimetric light transmission method using Helena PACKS-4 Aggregometer. Blood samples were collected from patients in internal medicine ward of Colentina Clinical Hospital. Platelet rich plasma (PRP) was obtained by centrifugation at 200G. Washed platelets where extracted by centrifugation of PRP at 2700G. The supernatant was replaced with sodium chloride 0.9%. Platelets aggregation was induced by adding different concentrations of calcium gluconate into cuvettes which contained washed platelets. After digitalization, curves were compared using similarity factor f2and areas under curves. Paper puts in evidence that both type of comparison, after mathematical and statistical evaluation, have to define a clinical threshold for clinical significance. In case of f2, in dissolution studies the threshold is 10%, in case of bioequivalence based on area under curves threshold is 20%. Establishment of the threshold for significant clinical difference in comparison of aggregation curves is not only a problem of statistics.Graphical representation of data suggested significant differences between curves obtained with different concentrations of calcium ion. Application of both f2 method and log-rank test let to conclusion that differences were statistical significant. Representation of aria under curves as function of calcium concentration put in evidence an approximate linear dependence. In spite of apparently objective character of mathematical approach, the problem of comparison of aggregation curves remains practically unsolved since we do not know the threshold between clinical significant and non-significant results.

Sodium Ion Effect on Separation Of Butyrylcholinesterase from Plasma by Ion-Exchange Chromatography.

Purpose of the paper was the optimization of mobile phase in natrium chloride gradient in the chromatographic separation of butyrylcholinesterase from human plasma. MATERIALS/METHODS: Butyrylcholinesterase (BuChE) was isolated from human plasma using a diethylamminoethyl-cellulose column, by elution with 0.02M acetate buffer pH=4.0, gradually increasing NaCl percentage from 10% to 80%. The procedure lasted approximately 28 days. RESULTS: Absorbance of the successive collected fractions at 280 nm presented a maximum at 60 % NaCl concentration. Activity of obtained BuChE was maximum at the same concentration. Another observed effect of NaCl was the decrease of resistance of the column to flow of the elution fluid. In the absence of NaCl the flow rate was 7 mL/h. Increasing of NaCl concentration induced a continuous increase of the flow to a value of 21 mL/h at 60% NaCl solution. After this concentration the flow remained practically constant. The effect on the ionic exchange is essentially an effect on chromatographic partition coefficient, leading, as a rule, to a peak having a Gaussian form. Fitting separately of ascending and descending parts of the apparent peak, led to practically the same exponential coefficient. CONCLUSIONS: Separation of proteins and particularly of BuChE on a chromatographic DEAE Cellulose column can be considered as a method for separation and purification of BuChE from human plasma. Optimum concentration of NaCl is 60 %. Exponential fittings in the neighbourhood of maximum indicated a prevalence of effects of NaCl on the chromatographic partition face to effects on gel-sol equilibrium of stationary phase.