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Spectrophotometric monitoring of lipoxygenase and cyclooxygenase pathway activity using ionophore stimulated whole blood

  • Mediators of Acute Inflammation
  • Published:
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Abstract

We have employed clotting human blood stimulated with ionophore to develop a system for measuring cyclooxygenase, 5-lipoxygenase, and 12-lipoxygenase pathway products released into the serum fraction. In a single chromatographic run, 5-HETE, 12-HETE, 12-OH-heptadecatrienoic acid (HHT), LTB4, 20-OH-LTB4, and 20-COOH-LTB4 are quantitated by UV monitoring after separation by HPLC. The kinetics of product formation/release of all fatty acid products into the serum show an apparent lag of approximately 2 min, after which time the amounts of HHT, 5-HETE, and LTB4, respectively, plateau at 10 min while 12-HETE increases over a 60 min period. The system is responsive to standard cyclooxygenase and lipoxygenase inhibitors, and is of value of evaluating prospective blockers of AA metabolism in a whole blood setting.

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Abbreviations

AA:

arachidonic acid

ACN:

acetonitrile

CO:

cyclooxygenase

DMSO:

dimethyl sulfoxide

HETE:

hydroxyeicosatetraenoic acid

HPLC:

high performance liquid chromatography

IC50 :

concentration causing 50% inhibition

LO:

lipoxygenase

LTB4 :

leukotriene B4

MeOH:

methanol

NDGA:

nordihyroguaiaretic acid

NSAID:

nonsteroidal anti-inflammatory drug

PGB2 :

prostaglandin B2

TEA:

triethylamine

TFA:

trifluoroacetic acid

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Sweeney, F.J., Eskra, J.D., Ernest, M.J. et al. Spectrophotometric monitoring of lipoxygenase and cyclooxygenase pathway activity using ionophore stimulated whole blood. Agents and Actions 21, 393–396 (1987). https://doi.org/10.1007/BF01966526

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  • DOI: https://doi.org/10.1007/BF01966526

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