Activities of acid and alkaline phosphatases were examined in spermatozoa isolated from 177 semen samples differing in sperm counts. Alkaline phosphatase was also determined in seminal fluid. The enzymes were assayed using disodium p-nitrophenyl phosphate as substrate and were studied with respect to susceptibility to various concentrations of tartrate (acid) and to heat (alkaline). Electrophoretic separation of alkaline phosphatase from seminal fluid was performed using an Helena apparatus. The results showed that acid phosphatase activity in spermatozoa decreased with increase in sperm densities and that elevation of tartrate from 0.028 to 0.17 M usually correlated an inhibition of the enzyme from 72% to 78% (mean values). Alkaline phosphatase was very low in sperm and generally below the sensitivity of the method used. Activity of alkaline phosphatase in seminal fluid showed a tendency to increase with the increase in sperm counts, but the significance of differences between groups was not statistically valid. Exposure of seminal fluid to 55 degrees C for 16 min resulted in enzyme inactivation of about 90% and in this respect the alkaline phosphatase resembles the enzyme of bone origin. The electrophoretic pattern, however, did not confirm this view and the type of alkaline phosphatase in seminal fluid is not clear.
This procedure describes the preparation of platelets from whole blood of healthy donors and pregnancy-induced hypertensive (PIH) patients by a rapid, one-step density gradient centrifugation, and the direct immunofluorescence staining of obtained platelets (CD63). Platelets are relatively fragile structures. Consequently, for the investigation of their biochemical properties it is recommended to isolate them by a simple method that does not damage their functional parameters and induce their activation. During platelet activation, several changes occur at the platelet surface. CD63 is the receptor for a lysosomal glycoprotein expressed in activated platelets. Currently, flow cytometry (fluorescence-activated cell sorting) is the most sensitive method to detect increased surface exposure of activation antigens on the platelet surface. The present technical note describes that compared with other whole blood flow cytometric techniques, our one-step density-gradient centrifugation method using OptiPrep can also prevent artificial, sample manipulation-related platelet activation.
Sialic acid levels were determined in spermatozoa and seminal plasma of 47 semen samples divided into two groups according to sperm counts. Group 1, up to 40 x IOh spermhl seminal fluid and group 2, above 40 x 10' sperm/ml seminal fluid. The content of sialic acid in spermatozoa ranged from 4.4-28.1 pg/lOX spermatozoa and from 70-95 mg/100 ml seminal plasma. Sialic acid was significantly higher in sperm and lower in seminal plasma of group 1 as compared to group 2. The lower content of sialic acid in seminal plasma might have a deteriorating effect on structural integrity of sperm.
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