ARS | Publication request: ISOLATION AND MEASUREMENT OF URINARY 8-ISO-PROSTAGLANDIN F2ALPHA BY HPLC/ GAS CHROMATOGRAPHY-MASS SPECTOMETRY

Submitted to: The Journal of Chromatography B: Biomedical Applications
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: February 21, 1997
Publication Date: N/A

Interpretive Summary: Oxygen-containing free radicals are chemical entities that are formed in humans and animals as by-products of normal metabolism and under the influence of certain environmental agents. Free radicals have been implicated in the pathogenesis of a wide variety of human disorders such as cancer, cardiovascular disease, immune dysfunction and aging. One of the well recognized targets of free radical-induced injury is the peroxidation of lipids. Scientists at Vanderbilt University have recently discovered a series of products of lipid peroxidation called isoprostanes. The ability to reliably measure the rate of formation of isoprostanes in vivo should enable investigators to test the free radical hypothesis of human disease and to develop dietary strategies to reduce their formation. This paper reports the development and validation of a method to measure a specific isoprostane in human urine. This analytical tool will help us evaluate the erole of free radicals in human disease and assess the pharmacology of antioxidant agents (e.g., vitamin C, vitamin E, carotenoids and flavanoids) by using the reduction of isoprostane formation as an endpoint. It will also enable us to determine the efficacy of antioxidants to suppress various disease processed, e.g., atherosclerosis, and the optimal doses and combination of antioxidants that effectively suppress oxidant injury in humans.

Technical Abstract: 8-iso-prostaglandin F2alpha (8-iso-PGF2alpha) is a product of free radical catalyzed peroxidation of arachidonic acid. Measurement of its urinary excretion has been proposed as an index of oxidative status in vivo. A fully validated stable isotope dilution method for its quantification by gas chromatography-electron capture chemical ionization mass spectrometry is described. The interassay R.S.D. in two separate determinations was 1. (n=4) and 2.3% (n=4). The validity of the assay was assessed through recovery experiments. The equation of the regression plot correlating the amounts added and recovery was y=0.91x-0.31, R=0.9916 (n=12). The pair of fragment ions ([M-181]) at m/z 569 and m/z 573 were monitored for quantification. Intake of 80 mg/d of lycopene by eleven volunteers for four weeks resulted in a non-significant reduction of 8-iso-PGF2alpha excretion.