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.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 375-386 |
| Number of pages | 12 |
| Journal | Prostaglandins |
| Volume | 26 |
| Issue number | 3 |
| DOIs | |
| State | Published - 1983 |
| Externally published | Yes |
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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 journal › Article
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TY - JOUR
T1 - Development of a radioimmunoassay for 15-hete and its application to 15-hete production by reticulocytes
AU - Bryant, Robert W.
AU - Hwang, Daniel H.
PY - 1983
Y1 - 1983
N2 - 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.
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.
UR - http://www.scopus.com/inward/record.url?scp=0021056835&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0021056835&partnerID=8YFLogxK
U2 - 10.1016/0090-6980(83)90173-9
DO - 10.1016/0090-6980(83)90173-9
M3 - Article
C2 - 6419285
AN - SCOPUS:0021056835
VL - 26
SP - 375
EP - 386
JO - Prostaglandins
JF - Prostaglandins
SN - 0090-6980
IS - 3
ER -