Development of a radioimmunoassay for 15-hete and its application to 15-hete production by reticulocytes

Robert W. Bryant, Daniel H. Hwang

Research output: Contribution to journalArticle

8 Citations (Scopus)

Abstract

Mono-hydroxy-eicosatetraenoic acids (HETE's) are frequently the principal lipoxygenase-derived products in a number of cell types. This paper describes the development of a selective and sensitive radioimmunoassay procedure for 15-HETE, a metabolite which has previously been shown to be both an activator and inhibitor of leukotriene formation in various cells. Initially, rabbits were immunized with 15-HETE conjugated to bovine serum albumin. After seven months, the anti-plasma showed significant binding of tritiated 15-HETE (40- 45% binding with a 1:600 dilution of the anti-plasma) which was displaceable by cold 15-HETE. The sensitivity of the assay was approximately 20 pg. of 15-HETE. The anti-plasma exhibited very little (<1%) cross-reactivity with arachidonic acid, 5-, 8-, 9-, 11- and 12-HETE's, HHT, TXB2, PGE2 and 6-Keto-PGF. Significant cross-reactivity was observed with 5,15-diHETE (53%), 8, 15-diHETE (6.6%), and several other 15-hydroxy-eicosanoids. Rabbit reticulocytes have a very active 15S-lipoxygenase and converted arachidonic acid (final concentration 7 μM) principally to 15-HETE. Unstimulated reticulocytes were found to release negligible amounts of 15-HETE as determined by radio-immunoassay Treatment of these cells with the calcium ionophore A23187 (0.16 to 4.0 μg/ml) elicited a level of 15-HETE release (8 - 14 ng/ml) that was twenty to forty times less than obtained with exogenous arachidonic acid (2.5 μg/ml). The radioimmunoassay reported here may be useful for identifying factors which stimulate cellular release of 15-HETE and other 15-hydroxy-eicosanoids from endogenous arachidonic acid.

Original languageEnglish (US)
Pages (from-to)375-386
Number of pages12
JournalProstaglandins
Volume26
Issue number3
DOIs
StatePublished - 1983
Externally publishedYes

Fingerprint

Reticulocytes
Radioimmunoassay
Arachidonic Acid
Eicosanoids
Plasmas
12-Hydroxy-5,8,10,14-eicosatetraenoic Acid
Arachidonate Lipoxygenases
Arachidonic Acids
Rabbits
Hydroxyeicosatetraenoic Acids
15-hydroxy-5,8,11,13-eicosatetraenoic acid
Hydroxy Acids
Lipoxygenase
Calcium Ionophores
Leukotrienes
Calcimycin
Bovine Serum Albumin
Metabolites
Radio
Immunoassay

ASJC Scopus subject areas

  • Biochemistry
  • Endocrinology

Cite this

Development of a radioimmunoassay for 15-hete and its application to 15-hete production by reticulocytes. / Bryant, Robert W.; Hwang, Daniel H.

In: Prostaglandins, Vol. 26, No. 3, 1983, p. 375-386.

Research output: Contribution to journalArticle

Bryant, Robert W. ; Hwang, Daniel H. / Development of a radioimmunoassay for 15-hete and its application to 15-hete production by reticulocytes. In: Prostaglandins. 1983 ; Vol. 26, No. 3. pp. 375-386.
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abstract = "Mono-hydroxy-eicosatetraenoic acids (HETE's) are frequently the principal lipoxygenase-derived products in a number of cell types. This paper describes the development of a selective and sensitive radioimmunoassay procedure for 15-HETE, a metabolite which has previously been shown to be both an activator and inhibitor of leukotriene formation in various cells. Initially, rabbits were immunized with 15-HETE conjugated to bovine serum albumin. After seven months, the anti-plasma showed significant binding of tritiated 15-HETE (40- 45{\%} binding with a 1:600 dilution of the anti-plasma) which was displaceable by cold 15-HETE. The sensitivity of the assay was approximately 20 pg. of 15-HETE. The anti-plasma exhibited very little (<1{\%}) cross-reactivity with arachidonic acid, 5-, 8-, 9-, 11- and 12-HETE's, HHT, TXB2, PGE2 and 6-Keto-PGF1α. Significant cross-reactivity was observed with 5,15-diHETE (53{\%}), 8, 15-diHETE (6.6{\%}), and several other 15-hydroxy-eicosanoids. Rabbit reticulocytes have a very active 15S-lipoxygenase and converted arachidonic acid (final concentration 7 μM) principally to 15-HETE. Unstimulated reticulocytes were found to release negligible amounts of 15-HETE as determined by radio-immunoassay Treatment of these cells with the calcium ionophore A23187 (0.16 to 4.0 μg/ml) elicited a level of 15-HETE release (8 - 14 ng/ml) that was twenty to forty times less than obtained with exogenous arachidonic acid (2.5 μg/ml). The radioimmunoassay reported here may be useful for identifying factors which stimulate cellular release of 15-HETE and other 15-hydroxy-eicosanoids from endogenous arachidonic acid.",
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AB - Mono-hydroxy-eicosatetraenoic acids (HETE's) are frequently the principal lipoxygenase-derived products in a number of cell types. This paper describes the development of a selective and sensitive radioimmunoassay procedure for 15-HETE, a metabolite which has previously been shown to be both an activator and inhibitor of leukotriene formation in various cells. Initially, rabbits were immunized with 15-HETE conjugated to bovine serum albumin. After seven months, the anti-plasma showed significant binding of tritiated 15-HETE (40- 45% binding with a 1:600 dilution of the anti-plasma) which was displaceable by cold 15-HETE. The sensitivity of the assay was approximately 20 pg. of 15-HETE. The anti-plasma exhibited very little (<1%) cross-reactivity with arachidonic acid, 5-, 8-, 9-, 11- and 12-HETE's, HHT, TXB2, PGE2 and 6-Keto-PGF1α. Significant cross-reactivity was observed with 5,15-diHETE (53%), 8, 15-diHETE (6.6%), and several other 15-hydroxy-eicosanoids. Rabbit reticulocytes have a very active 15S-lipoxygenase and converted arachidonic acid (final concentration 7 μM) principally to 15-HETE. Unstimulated reticulocytes were found to release negligible amounts of 15-HETE as determined by radio-immunoassay Treatment of these cells with the calcium ionophore A23187 (0.16 to 4.0 μg/ml) elicited a level of 15-HETE release (8 - 14 ng/ml) that was twenty to forty times less than obtained with exogenous arachidonic acid (2.5 μg/ml). The radioimmunoassay reported here may be useful for identifying factors which stimulate cellular release of 15-HETE and other 15-hydroxy-eicosanoids from endogenous arachidonic acid.

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