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The development of methodology for clinical measurement of 5-lipoxygenase pathway intermediates from human peripheral blood mononuclear cells.
- Source :
-
Journal of pharmaceutical and biomedical analysis [J Pharm Biomed Anal] 2008 Dec 15; Vol. 48 (5), pp. 1397-403. Date of Electronic Publication: 2008 Sep 30. - Publication Year :
- 2008
-
Abstract
- Recent studies have shown a correlation between 5-lipoxygenase (5-LO) pathway up-regulation and cardiovascular risk. Despite the existence of several assays for products of the 5-LO pathway, a reliable method for clinical determination of 5-LO activity remains to be established. In the present communication, we report conditions that allow measurement of 5-hydroxyeicosatetraenoic acid (5-HETE) and leukotriene B(4) (LTB(4)) in peripheral blood mononuclear cells (PBMCs) isolated from the blood of atherosclerosis patients before and after stimulation by the calcium ionophore, A23187. LTB(4), a potent mediator of inflammation-linked cardiovascular disease, was measured using an existing competitive enzyme immunoassay (EIA) kit after making specific methodological improvements that allowed PBMCs to be used in this format for the first time. LTB(4) was also measured by LC/MS/MS along with 5-HETE, a direct by-product of the action of 5-LO on arachidonic acid and a molecule for which no commercial EIA kit exists. The LC/MS/MS assay was validated over a range of 0.025-25ng/mL for LTB(4) and 0.1-25ng/mL for 5-HETE. The EIA method has a validated range covering 0.025-4ng/mL. When both assays were applied to analyze LTB(4) from stimulated PBMCs isolated from 25 subjects with various degrees of atherosclerosis, a high correlation was obtained (r=0.9426, Pearson's correlation coefficient). A high correlation was also observed between the levels of LTB(4) and 5-HETE measured by LC/MS/MS after ionophore stimulation (r=0.9159). Details are presented for optimized sample collection, processing, storage, and analysis in accordance with the logistical demands of clinical analysis.
- Subjects :
- Arachidonate 5-Lipoxygenase genetics
Calcimycin metabolism
Calcimycin pharmacology
Chromatography, Liquid
Humans
Hydroxyeicosatetraenoic Acids genetics
Hydroxyeicosatetraenoic Acids metabolism
Ionophores metabolism
Ionophores pharmacology
Leukotriene B4 analysis
Leukotriene B4 genetics
Leukotriene B4 metabolism
Reproducibility of Results
Tandem Mass Spectrometry
Temperature
Time Factors
Arachidonate 5-Lipoxygenase blood
Arachidonate 5-Lipoxygenase metabolism
Immunoenzyme Techniques methods
Leukocytes, Mononuclear enzymology
Subjects
Details
- Language :
- English
- ISSN :
- 0731-7085
- Volume :
- 48
- Issue :
- 5
- Database :
- MEDLINE
- Journal :
- Journal of pharmaceutical and biomedical analysis
- Publication Type :
- Academic Journal
- Accession number :
- 18996667
- Full Text :
- https://doi.org/10.1016/j.jpba.2008.09.034