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Quantification of 20-hydroxyeicosatetraenoic acid by colorimetric competitive enzyme linked immunosorbent assay

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Abstract

Analysis of 20-hydroxyeicosatetraenoic acid (20-HETE), a potent vasoconstrictor produced by the cytochrome P450 pathway, presently requires high-performance liquid chromatography (HPLC) and gas chromatography/ mass spectrometry (GC/MS). To simplify 20-HETE analysis, competitive ELIS As were developed using polyclonal anti-20-HETE coated ELISA plates to which free 20-HETE and 20-HETE conjugated to horseradish peroxidase (HRP) or alkaline phosphatase (AP) were added. Assays were developed with and without a proprietary enhancer solution which allows for the extraction-free measurement of 20-HETE in urine samples. The bound 20-HETE-HRP or 20-HETE-AP was detected using 3,3′,5,5,-tetramethylbenzidine and p-nitrophenyl phosphate, respectively. Sensitivities expressed as 80% B/B0, were 01 ng/ml for the HRP assay, and 0–5 ng/ml for the AP assay, withr 2 = 0.99 for both formats. Of the 17 lipids tested for cross-reactivity, arachidonic acid showed the highest (0.32%) followed by racemic 5-HETE (007%) and 8,9-dihydroxyeicosatrienoic acid (DHET) (004%). Preliminary validation experiments examining serum and urine concentrations of 20-HETE yield values that fall within the ranges established by GC/MS in the literature. These ELISAs provide simple and inexpensive methods for the analysis of 20-HETE in biological samples.

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Abbreviations

AP:

Alkaline phosphatase

ANG II:

angiotensin II

BCA:

bicinchoninic acid

BSA:

bovine serum albumin

CO:

carbon monoxide

CYP:

cytochrome P450

DCC:

N’N-dicyclohexylcarbodiimide

DHET:

dihydroxyeicosatrienoic

DMF:

dimethylformamide

EET:

epoxyeicosatrienoic acid

ELISA:

enzyme linked immunosorbent assay

GC/MS:

gas chromatogra-phy/mass spectroscopy; 20-HETE 20-hydroxyeicosatetraenoic acid;

HPLC:

high-performance liquid chromatography

HRP:

horse-radish peroxidase

IgG:

immunoglobulin G

NHS:

N-hydroxysuccinimide

NO:

nitric oxide

PBS:

phosphate buffered saline(Dulbecco’s, Ca+2- and Mg+2-free)

PMA:

phosphomolybdic acid

pNPP:

p-nitrophenyl phosphate

SAS:

saturated ammonium sulphate

Tg:

thyroglobulin (porcine)

TMB:

3,3′,5,5′,-tetramethylbenzidine

VSM:

vascular smooth muscle

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Authors and Affiliations

  1. Oxford Biomedical Research, P.O. Box 522, 48371, Oxford, MI, USA

    Harry E. Grates, Richard M. McGowen, Smiti V. Gupta, Thomas R. Brown, Denis M. Callewaert & Diane M. Sasaki

  2. Department of Biochemistry, University of Texas Southwestern Medical Center, 75390, Dallas, TX, USA

    John R. Falck

  3. Department of Chemistry and Center for Biomedical Research, Oakland University, 48309, Rochester, MI, USA

    Denis M. Callewaert

Authors
  1. Harry E. Grates
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  2. Richard M. McGowen
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  3. Smiti V. Gupta
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  4. John R. Falck
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  5. Thomas R. Brown
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  6. Denis M. Callewaert
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  7. Diane M. Sasaki
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Correspondence to Diane M. Sasaki.

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Grates, H.E., McGowen, R.M., Gupta, S.V. et al. Quantification of 20-hydroxyeicosatetraenoic acid by colorimetric competitive enzyme linked immunosorbent assay. J. Biosci. 28, 109–113 (2003). https://doi.org/10.1007/BF02970140

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