Measurement of Prostaglandins and Other Eicosanoids

William F. Stenson1

1 Washington University School of Medicine, St. Louis, Missouri
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 7.33
DOI:  10.1002/0471142735.im0733s33
Online Posting Date:  May, 2001
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Abstract

Eicosanoids are arachidonic acid metabolites formed through the cyclooxygenase or one of the lipoxygenase metabolic pathways. They include the prostaglandins (PG), leukotrienes (LT), thromboxanes (Tx), peptidoleukotrienes, and 5‐hydroxyeicosatetraenoic acid (5‐HETE). This unit describes procedures for measuring individual eicosanoids using several commercial kits. These include kits for an enzyme immunoasssay (ELISA) and a radioimmunoassay (RIA). A method is also described that can be used to analyze the full range of eicosanoids produced in a biological system, and a support protocol presents an acetone/chloroform extraction method for purifying eicosanoids prior to assay. Another support protocol describes a different extraction protocol in which samples are run over an octadecylsilyl silica column.

     
 
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Table of Contents

  • Basic Protocol 1: Measurement of Eicosanoids by ELISA
  • Basic Protocol 2: Measurement of Eicosanoids by RIA
  • Basic Protocol 3: Global Assessment of Eicosanoid Synthesis in Vitro
  • Support Protocol 1: Direct Extraction of Eicosanoids into Organic Solvents
  • Support Protocol 2: Extraction of Eicosanoids by Octadecysilyl Column Chromatography
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Measurement of Eicosanoids by ELISA

  Materials
  • Samples containing analyte to be measured (e.g., cell culture media, urine), extracted as necessary (see protocol 4 and protocol 5)
  • Eicosanoid enzyme immunoassay (ELISA) kit (see Table 7.33.1); a typical kit includes:
    •  Multiwell assay plate coated with goat polyclonal antibody against mouse serum
    •  Standard containing known amount of analyte (to generate the standard curve)
    •  Dilution buffer (ELISA buffer)
    •  Analyte‐enzyme complex (analyte covalently bound to horseradish peroxidase or acetylcholinesterase)
    •  Primary antibody (mouse monoclonal antibody against the analyte)
    •  Wash buffer
    •  Substrate solution
  • Plate reader for enzyme‐linked immunoassays (ELISAs)

Basic Protocol 2: Measurement of Eicosanoids by RIA

  Materials
  • Sample containing analyte to be measured (e.g., cell culture media, urine), extracted as necessary (see protocol 4 and protocol 5)
  • Eicosanoid RIA kit (see Table 7.33.2); a typical kit contains:
    •  Assay buffer
    •  Standard containing known amount of unlabeled analyte (for the standard curve)
    •  Radiolabeled (3H or 125I) analyte (tracer)
    •  Primary antibody
    •  A means of separating the antibody‐bound analyte from the free analyte (e.g., secondary antibody or polyethylene glycol for precipitation of primary antibody)
  • Liquid scintillation cocktail (for 3H only)
  • 12 × 75–mm disposable polystyrene or polypropylene assay tubes

Basic Protocol 3: Global Assessment of Eicosanoid Synthesis in Vitro

  Materials
  • I407 epithelial cell line (ATCC‐CCL6) grown in DMEM in 25‐cm2 tissue culture flasks
  • Dulbecco's minimum essential medium (DMEM; appendix 2A)
  • [3H]Arachidonic acid (Amersham or NEN Life Sciences)
  • Unlabeled arachidonic acid (Sigma)
  • 25 µg/ml prostaglandin B 2 (PGB 2) in ethanol
  • Nitrogen source
  • Methanol, HPLC grade
  • Solvent A: 10,000:1 (v/v) HPLC‐grade water/acetic acid
  • Solvent B: 10,000:1 (v/v) HPLC‐grade acetonitrile/acetic acid
  • HPLC equipped with two pumps and a 4.6 × 100–mm octadecylsilyl (ODS) Hypersil analytical column (Hewlett Packard)
  • UV spectrophotometer
  • Flowthrough radioactivity detector (e.g., Flo‐One, Packard)
  • Additional reagents and equipment for acetone/chloroform extraction (see protocol 4)

Support Protocol 1: Direct Extraction of Eicosanoids into Organic Solvents

  Materials
  • Sample to be extracted
  • [3H]Eicosanoid as an internal standard (tracer)
  • Acetone
  • 0.5 to 2.0 M citric or formic acid
  • n‐Hexane or petroleum ether
  • Chloroform
  • Appropriate assay buffer for ELISA or RIA (optional; see protocol 1 or protocol 2)
  • Silanized glass test tubes

Support Protocol 2: Extraction of Eicosanoids by Octadecysilyl Column Chromatography

  Materials
  • Samples to be extracted
  • [3H]Eicosanoid (internal standard)
  • 15%, 80%, and 100% (v/v) ethanol
  • 0.1 M potassium phosphate buffer, pH 3
  • Petroleum ether
  • Methylformate
  • Appropriate assay buffer for ELISA or RIA (see protocol 1 or protocol 2)
  • Silanized glass test tube
  • Disposable C18 column (e.g., Sep‐Pak from Waters or Baker‐Bond from Baker)
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Figures

Videos

Literature Cited

Literature Cited
   Eckmann, L., Stenson, W.F., Savidge, T.C., Lowe, D.C., Barrett, K.E., Fierer, J., Smith, J.R., and Kagnoff, M.F. 1997. Role of intestinal epithelial cells in the host secretory response to infection with invasive bacteria. J. Clin. Invest. 100:296‐309.
   Holtzman, M.J., Turk, J., and Shornick, L.P. 1992. Identification of a pharmacologically distinct prostaglandin H synthase in cultured epithelial cells. J. Biol. Chem. 267:21438‐21445.
   Maxey, K.M., Middipati, K.R., and Birkmeier, J. 1992. Interference in enzyme immunoassays. J. Clin. Immunoassay 15:116‐120.
   Powell, W.S. 1982. Rapid extraction of arachidonic acid metabolites from biological samples using octadecylsilyl silica. Methods Enzymol. 86:467‐476.
   Pradelles, P., Grassi, J., and Maclouf, J. 1985. Enzyme immunoassays of eicosanoids using acetylcholinesterase as a label: An alternative to radioimmunoassay. Anal. Chem. 57:1170‐1173.
   Salmon, J.A. and Flowers, R.J. 1982. Extraction and thin layer chromatography of arachidonic acid metabolites. Methods Enzymol. 86:477‐492.
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