Abstract
Leukotriene B4 (LTB4) is a potent chemoattractant for neutrophils and is thought to play a role in a variety of inflammatory responses in humans. The metabolism of LTB4 in vitro is complex with several competing pathways of biotransformation, but metabolism in vivo, especially for normal human subjects, is poorly understood. As part of a Phase I Clinical Trial of human tolerance to LTB4, four human subjects were injected with 150 nmol/kg LTB4 with one additional subject as placebo control. The urine of the subjects was collected in two separate pools (0-6 and 7-24 h), and aliquots from these urine collections were analyzed using high performance liquid chromatography, UV spectroscopy, and negative ion electrospray ionization tandem mass spectrometry for metabolites of LTB4. In the current investigation, 11 different metabolites of LTB4 were identified in the urine from those subjects injected with LTB4, and none were present in the urine from the placebo-injected subject. The unconjugated LTB4 metabolites found in urine were structurally characterized as 18-carboxy-LTB4, 10,11-dihydro-18-carboxy-LTB4, 20-carboxy-LTB4, and 10,11-dihydro-20-carboxy-LTB4. Several glucuronide-conjugated metabolites of LTB4 were characterized including 17-, 18-, 19-, and 20-hydroxy-LTB4, 10-hydroxy-4,6,12-octadecatrienoic acid, LTB4, and 10,11-dihydro-LTB4. The amount of LTB4 glucuronide (16.7-29.4 pmol/ml) and 20-carboxy-LTB4 (18.9-30.6 pmol/ml) present in the urine of subjects injected with LTB4 was determined using an isotope dilution mass spectrometric assay before and after treatment of the urine samples with beta-glucuronidase. The urinary metabolites of LTB4 identified in this investigation were excreted in low amounts, yet it is possible that one or more of these metabolites could be used to assess LTB4 biosynthesis following activation of the 5-lipoxygenase pathway in vivo.
Highlights
Leukotriene B4 (LTB4) is a potent chemoattractant for neutrophils and is thought to play a role in a variety of inflammatory responses in humans
Once -oxidation has occurred, 20-COOH-LTB4 can be further metabolized by -oxidation into 18-carboxy-LTB4 (18-COOH-LTB4) and 16-COOH-LTB3 [13, 14] which requires formation of the CoA ester at the -terminal carboxyl moiety
The solventextracted components were subjected to normal phase high performance liquid chromatographic (HPLC) using a silica column with gradient elution maximized for the separation of LTB4, 20-OH-LTB4, and 20-COOH-LTB4
Summary
Materials—The free acid of LTB4 for the injection into human subjects was obtained from Cascade Biochem, Ltd. (Reading, UK), in solution at a concentration of 2 mg/ml in ethanol/water (90:10, v/v). (Reading, UK), in solution at a concentration of 2 mg/ml in ethanol/water (90:10, v/v) The purity of this LTB4 (ϳ96%) was assessed by reversed phase HPLC with UV monitoring at 235 and 270 nm. Preparation of LTB4 for Injection—In order to prepare LTB4 for injection into human subjects, ethanolic LTB4 was converted into the sodium salt by the addition of 1 eq of aqueous sodium hydroxide. The urine samples were acidified to pH 3.7–3.8 using formic acid and subjected to solid phase extraction using 1-g cartridges (Waters, Milford, MA). The pH of the mixture was adjusted to 3.8 using formic acid, and the leukotrienes present in the solid phase-extracted urine were extracted using 1:1 (v/v) hexane/ethyl acetate. Urinary metabolites of LTB4 excreted by human subjects injected with LTB4 and identified by UV spectroscopy, chromatographic retention times, and mass spectrometry
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