Profilling of Platelet Polymines by a Novel HPLC Extraction Technique

Abstract

Purified Human Blood Platelets were incubated in the presence of Tris-buffer, 6-diamino-hexane at 37°C for 5minutes. Polyamines were subsequently extracted by sonification and pH adjusted to 4 with IM KOH. Extracted polyamines were derivatised with dansylchloride and separated by high performance liquid chromatography (Hplc) at a flow rate of 1 ml/min using 70% phosphate buffer and 30% acetonitrile in methanol. The most abundant polyamines extracted were Spermidine with a mean value of 0.190 ± 0.03nmol mg protein, followed by Putrescine (0.153 ± 0.02nmol mg protein), Spermine (0.12 ± 0.02nmol mg protein) and finally Cadeverine (0.100 +_ 0.03 nmol/mg protein). For the first time we have been able to establish a novel approach to the extraction and separation of important biogenic polyamines in human blood platelets using high performance liquid chromatography without subjecting them to hydrolysis or any rigorous chemical modification prior to their analysis. We suggest that the presence and extent of these polyamines in purified resting platelets may be important in further understanding of platelet function and thus further help us to understand disease processes in which platelets has long been implicated.