Figure 1
Validation of a new LX extraction method and description of a novel urinary tetraene. A, Urine aliquots (3 ml) were spiked with increasing concentrations of authentic LXA4 (from 0–2 ng/ml) and extracted using either the protocol described in the instructions to the LXA4 enzyme-linked immunosorbent assay (ELISA) kit (▪) or the new technique (□). In parallel, the same concentrations of LXA4 were used to construct a standard curve. Samples and standards were subjected to ELISA measurements. iLXA4 levels in standard and in urine were plotted for regression analysis. Results, corrected for PGB2 recovery, are from a single experiment representative of two with duplicates. B, Urine (5 ml) was extracted and suspended with 100 μl of methanol. 5 μl of this suspension was injected into a dual pump reversed phase high-performance liquid chromatography (RP-HPLC) gradient system equipped with a photodiode array detector. The column waters symmetry C18, 3 μm, 2.1 × 150 mm was eluted with methanol/water/acetic acid (65/35/0.01%; v:v:v). Retention times of authentic LXA4, LXB4, and their all-trans isomers are indicated by arrows. The ultraviolet (UV) spectrum of urinary material eluting beneath the peak at 4.3 minutes is shown in the inset.
