Prostaglandins: Gas Chromatography

Metabolism of free (nonesteriRed) arachidonic acid (5,8,11,14-cis-eicosatetraenoic acid) via the cyclooxygenase (COX) pathway produces a cascade of biologically active compounds collectively known as prostaglandins (PGs). These include prostaglandin D2 (PGD2), prostaglandin E2 (PGE2), prostaglandin F2 (PGF2), prostacyclin (PGI2) and thromboxane A2 (TXA2) (Figure 1). The analysis of PGs and related compounds in isolated cells and organs has proved relatively easy. By contrast, measurement of PGs in plasma presents a more challenging analytical task. This is because: (1) a more complex mixture of chemically related compounds exists in plasma; (2) PGs exert their effects locally, i.e. within a short distance of their site of production, and are consequently rapidly metabolized; and (3) ex vivo generation of PGs may occur. PG metabolites are generally present in biological Suids, including plasma and urine, in higher concentrations than their precursors. These metabolites are not formed during processing and therefore the problem of generating artefactual results does not arise. It has been suggested that the measurement of PGmetabolites in urine may represent a more reliable approach for assessing in vivo generation of PGs than does quantiRcation of primary products in plasma. Recently, it has been shown that free-radical peroxidation of polyunsaturated fatty acids (PUFAs) by a mechanism independent of the COXpathway also produces novel families of PGF-like compounds. In contrast to the COX-derived products, COX-independent products are present mainly esteriRed to phospholipids and with the side chains across the cyclopentane ring in the cis orientation. Figure 2 illustrates the structural differences between COX-dependent and COX-independent 9 ,11 PGF2 . COX-independently derived PGF-like compounds are collectively known as isoprostanes. The measurement of isoprostanes has received considerable attention because they have proved to be a reliable marker of oxidative stress. Determination of the different families of isoprostanes also allows the assessment of the peroxidation of individual PUFAs. Accumulating data indicate that the metabolic fate of non-COX-derived PGF-like compounds is similar to that of the COX-derived products. PGs and related compounds can be assessed by several methods including bioassay, radioimmunoassay (RIA), high performance liquid chromatography (HPLC) linked to RIA or UV or mass spectrometric (MS) detection and gas chromatography}mass spectrometry (GC-MS). GC-MS is the preferred method for the quantiRcation and structural validation of PGs, combining the high resolution of GC separation employing fused silica capillary columns with the speciRcity and sensitivity of mass spectrometry.