Purification and quantification of arachidonate metabolites: critical evaluation of methods and interpretation of results.

Abstract

S INCE THE INITIAL description of prostaglandins in 1935, a vast body of knowledge has accumulated on the physiology and chemistry of arachidonate metabolites and how they relate to perinatal medicine. In addition to contributing to the initiation of labor in the mother, arachidonate metabolites are involved in closure and/or maintenance of ductus arteriosus patency; they help effect changes in pulmonary vascular tone, and they act as mediators in lung injury. Moreover, a protective effect of endogenous prostaglandins in the gastrointestinal (GI) tract has been speculated on, and caveats have been raised regarding inhibition of their synthesis with indomethacin. Almost every organ system appears to be linked to the arachidonate acid pathway. While much has been learned about how arachidonic acid metabolism relates to perinatal medicine in the past two decades, difficulties in measuring prostaglandins and leukotrienes in tissues, blood, and other biological fluids have delayed the progress of research (see also chapter by Mitchell). Since radioimmunoassay (RIA) was introduced into the field of prostaglandin measurement in the early 197Os, a large amount of RIA work has been published in the perinatal area. The concentrations recorded in the same body fluids have varied enormously among the different reports. Some of the early studies that used RIA to analyze prostaglandins in the peripheral circulation reported concentrations in control subjects in the ng/mL range. More recent RIA studies have indicated much lower concentrations, ie, in the pg/mL range (Table 1). Even though RIA is a highly sensitive technique, its specificity has been questioned. For example, RIA studies by Seiss and Dray l l at the Pasteur Institute, using a range of sample preparation techniques, indicated marked decreases in plasma 6-keto-prostaglandin F,, (6-keto-PGF,,) concentrations in humans with increasing sample cleanup, even with corrections for recovery (Table 2). In our laboratory, we have also found that the prostaglandin concentrations in human milk measured by RIA are much lower after purification with octadecylsilyl extraction and high-performance liquid chromatography (HPLC) (Table 3). ‘* The decrease in reported concentrations of arachidonic acid metabolites over the past several years is presumably due to improved techniques that have increased the specificity of RIA, such as the use of more specific antibodies and improved chromatographic clean-up (eg, silicic acid chromatography, thin-layer chromatography [TLC], and HPLC). Other techniques such as those using gas chromatography/mass spectrometry (GUMS) have also been highly beneficial in accurately quantitating prostaglandin metabolites. However, only recently has GUMS been shown to be effective in the low pg/mL range, where many of the arachidonic acid metabolites are currently thought to exist in the peripheral circulation. In this report, we will review some of the major techniques that have been used to purify and quantitate arachidonic acid metabolites. Readers who are interested in more specific details are referred to references 13 and 14 and to those cited in the text following.