Urinary excretion of lipoxin A4 and related compounds: Development of new extraction techniques for lipoxins

LX are tetraene-containing eicosanoids generated by lipoxygenase (LO) transformation of arachidonic acid (Serhan and Romano, 1995). LX possess potent anti-inflammatory activity in vivo, and temporal biosynthesis of LX, concurrent with spontaneous resolution, has been observed during exudate formation (Levy et al, 2001). Limited results are currently available on the involvement of LX in clinical settings. Recently, a rabbit anti-LXA4 antiserum has been raised to produce an enzyme-linked immunosorbent assay (ELISA) kit for LXA4 (Levy et al, 1993). Although specific and accurate with isolated cells, this kit has not been tested with complex biological matrix such as urine. Initial attempts to determine urinary excretion of LXA4 using the LXA4 ELISA kit were unsuccessful because of high unspecific absorbance readings. In this report, we show that the LXA4 extraction procedure indicated in the ELISA kit is inadequate for urinary measurements of immunoreactive (i)LXA4. We present the development of a new extraction technique, more selective for LX, that abolishes background contamination and minimizes the unspecific readings. Using this method, we show for the first time that urine from healthy subjects contain (i)LXA4 material and identify a urinary tetraene with the physical properties of a LXA4 metabolite. Although reliable methods have been previously established to quantitate LXA4 from whole blood (Brezinski et al, 1992), the present extraction technique, which optimizes for LXA4 recovery from human urine, represents a substantial achievement for LX investigation and may open a new avenue of clinical studies on LXA4.