Improved assay for the quantification of the major urinary metabolite of the isoprostane 15-F(2t)-Isoprostane (8-iso-PGF(2alpha)) by a stable isotope dilution mass spectrometric assay.
BACKGROUND
The F(2)-isoprostanes (IsoPs) are a series of novel prostaglandin (PG)-like compounds generated from the free radical catalyzed peroxidation of arachidonic acid. One IsoP, 15-F(2t)-IsoP (8-iso-PGF(2alpha)), has been shown to be formed in abundance in vivo and to exert potent biological activity.


METHODS
As a means to assess the endogenous production of this compound, we previously developed a method to quantify the major urinary metabolite of 15-F(2t)-IsoP, 2,3-dinor-5,6-dihydro-15-F(2t)-IsoP (2,3-dinor-5,6-dihydro-8-iso-PGF(2alpha), 15-F(2t)-IsoP-M ), by gas chromotography (GC)/negative ion chemical ionization mass spectrometry (MS) employing stable isotope dilution methodology. While useful, we found that the assay occasionally suffered from the presence of impurities that co-elute on GC with 15-F(2t)-IsoP-M, making the measurement of this compound difficult. We now report a modified assay for the quantification of 15-F(2t)-IsoP-M employing GC/MS that alleviates this problem.


RESULTS
Precision of the assay is +/-7% and the accuracy is 96%. The lower limit of sensitivity is approximately 8 pg. Normal concentrations of this metabolite in urine were found to be 0.46+/-0.09 ng/mg creatinine (mean+/-1 S.D.) Urinary excretion of 15-F(2t)-IsoP-M is markedly altered in situations associated with increased or decreased oxidant stress in vivo.


CONCLUSIONS
This assay provided a sensitive and accurate method to assess endogenous IsoP generation and can be used to further explore the role of oxidant injury in human disease.
Morales
C R
CR
Department of Medicine, Pharmacology and Pathology, Vanderbilt University, 526 Medical Research Building 1, 23rd and Pierce Aves., Nashville, TN 37232-6602, USA.
Terry
E S
ES
Zackert
W E
WE
Montine
T J
TJ
Morrow
J D
JD
D002849:Chromatography, Gas; D002855:Chromatography, Thin Layer; D015237:Dinoprost / Q000031:analogs & derivatives*; D028441:F2-Isoprostanes / Q000652:urine*; D006801:Humans; D007202:Indicators and Reagents; D013058:Mass Spectrometry; D010103:Oxygen Isotopes; D011865:Radioisotope Dilution Technique; D015203:Reproducibility of Results
D028441:F2-Isoprostanes; D007202:Indicators and Reagents; D010103:Oxygen Isotopes; C075750:8-epi-prostaglandin F2alpha; D015237:Dinoprost