High-pressure liquid chromatography of oxo-eicosanoids derived from arachidonic acid.
Eicosanoids are a large group of biologically active metabolites of arachidonic acid and related C20 fatty acids. Many of these compounds contain hydroxyl groups which can be converted to oxo groups by a variety of substrate-specific dehydrogenases. In many cases, this results in a reduction in potency, but in others, such as the oxidation of 5-hydroxyeicosatetraenoic acid to its oxo metabolite 5-oxo-eicosatetraenoic acid, there is a dramatic increase in biological activity. Thus, it is often very important to analyze the relative amounts of oxo- and hydroxy-eicosanoids formed by various cells and tissues. The present study was designed to compare the chromatographic behavior of oxo-eicosanoids and their hydroxy counterparts in commonly used mobile phases for reversed-phase and normal-phase HPLC. We examined three groups of eicosanoids: prostaglandins, leukotriene B4 and some of its metabolites, and monohydroxy-eicosanoids and their oxo metabolites. We found that in reversed-phase HPLC, the retention times of oxo-eicosanoids were longer than those of the corresponding hydroxy-eicosanoids in mobile phases containing acetonitrile as the major organic component, whereas the reverse was true for mobile phases containing methanol. Normal-phase HPLC using mobile phases containing hexane, isopropanol, and acetic acid gave excellent separation of oxo- and hydroxy-eicosanoids. Increasing the concentration of acetic acid in the mobile phase selectively reduced the retention times of oxo-eicosatetraenoic acids compared to monohydroxy-eicosatetraenoic acids, whereas the reverse was true for isopropanol. Differences in the chromatographic behavior of oxo- and hydroxy-eicosanoids can be useful clues in the structural characterization of these compounds, as illustrated by the chromatographic properties of a complex series of LTB4 metabolites formed by rat neutrophils.
Powell
W S
WS
Meakins-Christie Laboratories, McGill University, Montreal, Quebec, Canada. Bill@Meakins.LAN.McGill.CA
Wang
L
L
Khanapure
S P
SP
Manna
S
S
Rokach
J
J
D000818:Animals; D001095:Arachidonic Acids / Q000302:isolation & purification / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D015777:Eicosanoids / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D006893:Hydroxyeicosatetraenoic Acids / Q000302:isolation & purification / Q000378:metabolism; D066298:In Vitro Techniques; D007975:Leukotriene B4 / Q000302:isolation & purification / Q000378:metabolism; D015394:Molecular Structure; D009504:Neutrophils / Q000378:metabolism; D011453:Prostaglandins / Q000302:isolation & purification / Q000378:metabolism; D051381:Rats
D001095:Arachidonic Acids; D015777:Eicosanoids; D006893:Hydroxyeicosatetraenoic Acids; D011453:Prostaglandins; D007975:Leukotriene B4