Development of a dual isotopes technique and two-stage column chromatography to characterize the prostaglandin F 2α, E 2 and thromboxane B 2 formed by the guinea Pig lung

Abstract

A sensitive dual isotopes ([ 14C]-arachidonic acid, ([ 3H]-PGF 2α, and [ 3H]-PGF 2) technique coupled with two-stage column chromatographic systems was developed to characterize the prostaglandins synthesized by guinea pig lung. The assay consisted of incubating 600 g supernatant fraction of lung homogenate (75–600 μg protein) with 7.88 μM of [ 14C]-arachidonic acid, 1–2 nM [ 3H]-PGE 2 and [ 3H]-PGF 2α as internal standards, 5 mM glutathione, 5 mM epinephrine and 100 mM Tris HCl buffer (pH 8.2) in a total volume of 0.5 ml. The reaction was terminated by 0.25 ml of 1 N HCl and the mixtures extracted with 1.5 ml ethyl acetate. PGF 2α and PGE 2-like materials (“PGE 2”) in the ethyl acetate extract were separated from arachidonic acid by chromatography on two-stage silicic acid columns. Reverse isotope dilution technique revealed that [ 14C]-PCF 2α synthesized from [ 14C]-arachidonic acid by the lung enzyme was 100% pure. Moreover, the amount of PGF 2α synthesis as measured by this method was in accord with the amount obtained by radioimmunoassay. In contrast, only 15% of the [ 14C]-“PGE 2” was PGE 2; 85% of it was a KOH resistant and NaBH 4-reducible product that has been tentatively identified as thromboxane B 2. The kinetics of PGF 2α and “PGE 2” synthesis by guinea pig lung enzyme have been studied under various experimental conditions employing this method. The two stage open silicic acid column chromatography as described in the present report offers several advantages such as economy of time and expense and capacity to process multiple samples on a routine basis. Furthermore, this method also offers the advantage of correction for procedural losses of prostaglandins using internal recovery standards. This method should have broad applicability to study the prostaglandin synthesizing ability of various tissues especially in those laboratories that cannot afford to employ sophisticated instruments needed for prostaglandin analysis.