Determination of 9α, 11β-prostaglandin F2 in human urine. Combination of solid-phase extraction and radioimmunoassay

I. Muchá, A. Riutta

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4 Citations (Scopus)

Abstract

This paper describes a new iodine-125 radioimmunoassay of 9α,11β-PGF2, and its use for the determination of urinary 9α,11β-prostaglandin F2 after a selective one-step solid-phase extraction. The newly reported immunoassay is based on the use of 125l-tyrosyl methyl ester derivative of 9α,11β-PGF2 and specific polyclonal antibody raised in rabbits. The assay detected as low as 0.85 pg/tube 9α,11β-PGF2, and the antibody showed less than 0.01 cross-reaction with PGF-ring metabolites (e.g., 8-iso-PGF2α, PGF2α, 2,3-dinor-6-keto-PGF1α, and 5 more PGF-ring compounds). Both the intra-assay, and inter-assay CVs were less than 20% for internal controls containing low, medium and high concentrations of 9α, 11β-PGF2. Immuno-HPLC analysis showed a very low ratio of specific immunoreactivity in both non-extracted urine (6.5%), and in urine extracted on C18-silica cartridge (14.8%). By contrast, approximately 80% specific immunoreactivity could be achieved by using C2-silica as the sorbent, acetonitrile: water (15: 85, v/v) as wash solvent, and ethyl acetate as eluent of 9α,11β-PGF2. This extraction procedure enabled a reasonably high extraction efficiency of 80.4±0.855 (mean ± SEM, n=82), as determined by 3H-9α, 11 β-PGF2. The new SPE/RIA method was applied for the determination of urinary 9α, 11β -PGF2 values in 50 healthy human volunteers. For the concentration and for the excretion rate 37.52±4.61 pg/ml (mean ± SEM), and 3.50±0.35 ng/mmol creatinine (mean ± SEM), respectively, was measured. The specificity of the SPE/RIA method was supported by the observed 69% decrease in 9α, 11β-PGF2 excretion rate after acetylsalicylic acid treatment. The effect of nicotinic acid, a PGD2-stimulatory agent, was monitored by the urinary excretion of 9α11β-PGF2 in 6 patients, by using the new SPE/RIA method. In, patients responding with flushing symptoms nicotinic acid induced an increase of the urinary excretion of 9α,11β-PGF2 in the range between 11% and 187%. In summary, the combination of the newly developed specific [125l] radioimmunoassay with solid-phase extraction on C2-silica cartridges enables the specific, sensitive, and reliable determination of 9α,11β-PGF2 in human urine without the need for further laborious chromatographic purification before radioimmunoassay.

Original languageEnglish
Pages (from-to)271-280
Number of pages10
JournalProstaglandins Leukotrienes and Essential Fatty Acids
Volume65
Issue number5-6
DOIs
Publication statusPublished - 2001

Fingerprint

Dinoprost
Solid Phase Extraction
Radioimmunoassay
Urine
Silicon Dioxide
Assays
Niacin
Prostaglandins F
Scanning electron microscopy
Prostaglandin D2
Antibodies
Cross Reactions
Metabolites
Sorbents
Immunoassay
Iodine
Aspirin
Purification
Creatinine

ASJC Scopus subject areas

  • Clinical Biochemistry
  • Endocrinology, Diabetes and Metabolism

Cite this

@article{4aedc0ed617f49568ed57bcd3f066315,
title = "Determination of 9α, 11β-prostaglandin F2 in human urine. Combination of solid-phase extraction and radioimmunoassay",
abstract = "This paper describes a new iodine-125 radioimmunoassay of 9α,11β-PGF2, and its use for the determination of urinary 9α,11β-prostaglandin F2 after a selective one-step solid-phase extraction. The newly reported immunoassay is based on the use of 125l-tyrosyl methyl ester derivative of 9α,11β-PGF2 and specific polyclonal antibody raised in rabbits. The assay detected as low as 0.85 pg/tube 9α,11β-PGF2, and the antibody showed less than 0.01 cross-reaction with PGF-ring metabolites (e.g., 8-iso-PGF2α, PGF2α, 2,3-dinor-6-keto-PGF1α, and 5 more PGF-ring compounds). Both the intra-assay, and inter-assay CVs were less than 20{\%} for internal controls containing low, medium and high concentrations of 9α, 11β-PGF2. Immuno-HPLC analysis showed a very low ratio of specific immunoreactivity in both non-extracted urine (6.5{\%}), and in urine extracted on C18-silica cartridge (14.8{\%}). By contrast, approximately 80{\%} specific immunoreactivity could be achieved by using C2-silica as the sorbent, acetonitrile: water (15: 85, v/v) as wash solvent, and ethyl acetate as eluent of 9α,11β-PGF2. This extraction procedure enabled a reasonably high extraction efficiency of 80.4±0.855 (mean ± SEM, n=82), as determined by 3H-9α, 11 β-PGF2. The new SPE/RIA method was applied for the determination of urinary 9α, 11β -PGF2 values in 50 healthy human volunteers. For the concentration and for the excretion rate 37.52±4.61 pg/ml (mean ± SEM), and 3.50±0.35 ng/mmol creatinine (mean ± SEM), respectively, was measured. The specificity of the SPE/RIA method was supported by the observed 69{\%} decrease in 9α, 11β-PGF2 excretion rate after acetylsalicylic acid treatment. The effect of nicotinic acid, a PGD2-stimulatory agent, was monitored by the urinary excretion of 9α11β-PGF2 in 6 patients, by using the new SPE/RIA method. In, patients responding with flushing symptoms nicotinic acid induced an increase of the urinary excretion of 9α,11β-PGF2 in the range between 11{\%} and 187{\%}. In summary, the combination of the newly developed specific [125l] radioimmunoassay with solid-phase extraction on C2-silica cartridges enables the specific, sensitive, and reliable determination of 9α,11β-PGF2 in human urine without the need for further laborious chromatographic purification before radioimmunoassay.",
author = "I. Much{\'a} and A. Riutta",
year = "2001",
doi = "10.1054/plef.2001.0325",
language = "English",
volume = "65",
pages = "271--280",
journal = "Prostaglandins Leukotrienes and Essential Fatty Acids",
issn = "0952-3278",
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number = "5-6",

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TY - JOUR

T1 - Determination of 9α, 11β-prostaglandin F2 in human urine. Combination of solid-phase extraction and radioimmunoassay

AU - Muchá, I.

AU - Riutta, A.

PY - 2001

Y1 - 2001

N2 - This paper describes a new iodine-125 radioimmunoassay of 9α,11β-PGF2, and its use for the determination of urinary 9α,11β-prostaglandin F2 after a selective one-step solid-phase extraction. The newly reported immunoassay is based on the use of 125l-tyrosyl methyl ester derivative of 9α,11β-PGF2 and specific polyclonal antibody raised in rabbits. The assay detected as low as 0.85 pg/tube 9α,11β-PGF2, and the antibody showed less than 0.01 cross-reaction with PGF-ring metabolites (e.g., 8-iso-PGF2α, PGF2α, 2,3-dinor-6-keto-PGF1α, and 5 more PGF-ring compounds). Both the intra-assay, and inter-assay CVs were less than 20% for internal controls containing low, medium and high concentrations of 9α, 11β-PGF2. Immuno-HPLC analysis showed a very low ratio of specific immunoreactivity in both non-extracted urine (6.5%), and in urine extracted on C18-silica cartridge (14.8%). By contrast, approximately 80% specific immunoreactivity could be achieved by using C2-silica as the sorbent, acetonitrile: water (15: 85, v/v) as wash solvent, and ethyl acetate as eluent of 9α,11β-PGF2. This extraction procedure enabled a reasonably high extraction efficiency of 80.4±0.855 (mean ± SEM, n=82), as determined by 3H-9α, 11 β-PGF2. The new SPE/RIA method was applied for the determination of urinary 9α, 11β -PGF2 values in 50 healthy human volunteers. For the concentration and for the excretion rate 37.52±4.61 pg/ml (mean ± SEM), and 3.50±0.35 ng/mmol creatinine (mean ± SEM), respectively, was measured. The specificity of the SPE/RIA method was supported by the observed 69% decrease in 9α, 11β-PGF2 excretion rate after acetylsalicylic acid treatment. The effect of nicotinic acid, a PGD2-stimulatory agent, was monitored by the urinary excretion of 9α11β-PGF2 in 6 patients, by using the new SPE/RIA method. In, patients responding with flushing symptoms nicotinic acid induced an increase of the urinary excretion of 9α,11β-PGF2 in the range between 11% and 187%. In summary, the combination of the newly developed specific [125l] radioimmunoassay with solid-phase extraction on C2-silica cartridges enables the specific, sensitive, and reliable determination of 9α,11β-PGF2 in human urine without the need for further laborious chromatographic purification before radioimmunoassay.

AB - This paper describes a new iodine-125 radioimmunoassay of 9α,11β-PGF2, and its use for the determination of urinary 9α,11β-prostaglandin F2 after a selective one-step solid-phase extraction. The newly reported immunoassay is based on the use of 125l-tyrosyl methyl ester derivative of 9α,11β-PGF2 and specific polyclonal antibody raised in rabbits. The assay detected as low as 0.85 pg/tube 9α,11β-PGF2, and the antibody showed less than 0.01 cross-reaction with PGF-ring metabolites (e.g., 8-iso-PGF2α, PGF2α, 2,3-dinor-6-keto-PGF1α, and 5 more PGF-ring compounds). Both the intra-assay, and inter-assay CVs were less than 20% for internal controls containing low, medium and high concentrations of 9α, 11β-PGF2. Immuno-HPLC analysis showed a very low ratio of specific immunoreactivity in both non-extracted urine (6.5%), and in urine extracted on C18-silica cartridge (14.8%). By contrast, approximately 80% specific immunoreactivity could be achieved by using C2-silica as the sorbent, acetonitrile: water (15: 85, v/v) as wash solvent, and ethyl acetate as eluent of 9α,11β-PGF2. This extraction procedure enabled a reasonably high extraction efficiency of 80.4±0.855 (mean ± SEM, n=82), as determined by 3H-9α, 11 β-PGF2. The new SPE/RIA method was applied for the determination of urinary 9α, 11β -PGF2 values in 50 healthy human volunteers. For the concentration and for the excretion rate 37.52±4.61 pg/ml (mean ± SEM), and 3.50±0.35 ng/mmol creatinine (mean ± SEM), respectively, was measured. The specificity of the SPE/RIA method was supported by the observed 69% decrease in 9α, 11β-PGF2 excretion rate after acetylsalicylic acid treatment. The effect of nicotinic acid, a PGD2-stimulatory agent, was monitored by the urinary excretion of 9α11β-PGF2 in 6 patients, by using the new SPE/RIA method. In, patients responding with flushing symptoms nicotinic acid induced an increase of the urinary excretion of 9α,11β-PGF2 in the range between 11% and 187%. In summary, the combination of the newly developed specific [125l] radioimmunoassay with solid-phase extraction on C2-silica cartridges enables the specific, sensitive, and reliable determination of 9α,11β-PGF2 in human urine without the need for further laborious chromatographic purification before radioimmunoassay.

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