Aspects on Measurements of Prostaglandins and Thromboxanes

The prostaglandins (PGs) and thromboxanes (TXs) belong to a rapidly growing family of compounds, which are all oxygenated metabolites of certain polyunsaturated fatty acids. The Figure shows some pathways in arachidonic acid metabolism in man. Many of these metabolites are extremely potent compounds, and consequently occur in the body in utterly small amounts (for review articles, see Samuelsson et al., 1975; 1978). Research in this increasingly complex field has often been hampered by the lack of sufficiently sensitive and specific methods for detection and quantitation of these products. Ever since the identification of the first few 'primary' prostaglandins was achieved around 1960, great efforts have been devoted to the development of suitable assay methods (Samuelsson et al., 1975,1978). The earliest assays were all based on the various biological activities of the PGs. Later, physical and chemical methods were found: ultraviolet spectrometry was used for certain compounds; gas-liquid chromatography with flame ionisation detection for others; an enzymatic method was based on the activity of a PG-metabolising enzyme, 15-hydroxyprostanoate dehydrogenase (Granstrom and Samuelsson, 1978). With these methods it was possible to detect a few nanomoles of many compounds; however, this sensitivity was far from sufficient in most biological studies. During the early 1970s several methods were developed which allowed the detection of picomole or even femtomole amounts of several PGs and TXs. These methods include gas-liquid chromatography with electron capture detection, quantitative mass spectrometry with deuterium-labelled carriers and multiple ion detection, bioassay with cascade superfusion techniques, and radioimmunoassay with the related methods viroimmunoassay and . competitive protein binding assay. Of all these methods, three different types have gained particularly widespread use: bioassay, gas chromatography -mass spectrometry, and radioimmunoassay. Most bioassays are based on the fact that many members of the PG family exhibit strong and characteristic effects on smooth muscle from various organs. Strips from different segments of the gastrointestinal tract, and later also of vessels, are probably the most commonly employed bioassay systems (Moncada et al., 1978). Other bioassays are based on the effects of PGs and TXs on platelet aggregation, on estrous cycle length, on blood pressure, and other parameters. The organ bath type of bioassay has now been widely replaced by the more sensitive superfusion technique (Gaddum, 1953). The specificity of the assay is greatly improved by use of the cascade technique, in which several assay organs, differing in their reactions to various PGs and TXs, are superfused by the same sample (Vane, 1969). These assay organs should preferably also be treated with specific antagonists to other biologically active substances which may occur together with the PGs and TXs. In the search for new, active compounds or in the assay of unknown substances, bioassay can probably never be surpassed by other techniques. The method is very sensitive, but, unless the sample is extensively purified before analysis, specificity can probably never be absolute in bioassay. Published quantitative bioassay data are often many times higher than those obtained by other methods, indicating that more than the intended compound was bioassayed. The most serious drawback of bioassay is the fact that biologically inactive substances cannot be detected with this method. Most end products of PG metabolism, such as the tetranor dioic acids excreted into urine, are completely devoid of biological activity. Also the major circulating metabolites must be quantitated by other methods. As will be discussed below, it is necessary to focus on the inactive end products instead of the potent parent compounds in many studies of the roles of PGs in the body. The gas chromatography-mass spectrometry methods are based on the addition of a known amount of the deuterium-labeled compound to a sample containing a small amount of the protium form (Green et al., 1978). In the final mass