Profiling of Platelet Polyamines by a Novel Hplc Xtraction Technique

Abstract

Purified Human Blood Platelets were incubated in the presence of Tris – buffer, 6 – diamino – hexane at 37°C for 5mintues. Polyamines were subsequently extracted by sonification and pH adjusted to 4 with IM KOH. Extracted polyamines were derivatised with danslchloride and separated by high performance liquid chromatography (Hplc) at a flow rate of 1ml min using 70% phosphate buffer and 30% acetonitrile in methanol. The most adundant polyamines extracted with 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) finally Cadeverine (0.100 ± 0.03nmol 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 thesepolyamines in purified resting platelets may be important in further understanding of platelet function and thus further helps us to understand diseases in which platelets has long been implicated.