Quantification of the major urinary metabolite of 15-F2t-isoprostane (8-iso-PGF2alpha) by a stable isotope dilution mass spectrometric assay

The isoprostanes (IsoPs) are a series of novel prostaglandin (PG)-like compounds generated from the free radical-catalyzed peroxidation of arachidonic acid. The first series of IsoPs characterized contained F-type prostane rings analogous to PGF2alpha. One F-ring IsoP, 15-F2t-IsoP (8-iso-PGF2alpha) has been shown to be formed in abundance in vivo and to exert potent biological activity. As a means to assess the endogenous production of this compound, we developed a method to quantify the major urinary metabolite of 15-F2t-IsoP, 2,3-dinor-5,6-dihydro-15-F2t-IsoP (2,3-dinor-5, 6-dihydro-8-iso-PGF2alpha), by gas chromotography/negative ion chemical ionization mass spectrometry. This metabolite was chemically synthesized and converted to an 18O2-labeled derivative for use as an internal standard. After purification, the compound was analyzed as a pentafluorobenzyl ester trimethylsilyl ether. Precision of the assay is +/-4% and accuracy is 97%. The lower limit of sensitivity is approximately 20 pg. Levels of the urinary excretion of this metabolite in 10 normal adults were found to be 0. 39 +/- 0.18 ng/mg creatinine (mean +/- 2 SD). Substantial elevations in the urinary excretion of the metabolite were found in situations in which IsoP generation is increased and antioxidants effectively suppressed metabolite excretion. Levels of 2,3-dinor-5, 6-dihydro-15-F2t-IsoP were not affected by cyclooxygenase inhibitors. Thus, this assay provides a sensitive and accurate method to assess endogenous production of 15-F2t-IsoP as a means to explore the pathophysiological role of this compound in human disease.     

Determination of urinary 8-epi-prostaglandin F(2alpha) using liquid chromatography-tandem mass spectrometry: increased excretion in diabetics

Liquid chromatography-tandem mass spectrometry (LC/MS-MS) was applied to the quantitative analysis of urinary 8-epi-prostaglandin F(2alpha) (8-epi-PGF(2alpha)) level. 8-Epi-PGF(2alpha) and its internal standard, [(2)H(4)]-8-epi-PGF(2alpha), were extracted from urine by using a solid phase extraction cartridge and loaded to LC/MS-MS in selected reaction monitoring (SRM) mode. The standard curve showed good linearity in the range of 40 pg to 10 ng (r = 0. 997). The accuracy of the added 8-epi-PGF(2alpha) ranged from 96.8 to 104.9% with a mean +/- SD of 99.5+/-2.5%. The average level +/- SD of urinary 8-epi-PGF(2alpha) in 13 healthy volunteers (five women and eight men, 31+/-7.4 years old) was 429.4+/-149.6 pg/mg creatinine. The level of seven patients with noninsulin dependent diabetes mellitus (two women and five men, 40+/-13.6 years old), 630.9+/-275.6 pg/mg creatinine, was statistically higher than that of healthy volunteers (P<0.05). This finding suggested that diabetics are in a highly oxidative condition. This simple and rapid LC/MS-MS method can be used to elucidate the pathophysiological feature of diabetes or for monitoring the curative effect.     

Urinary excretion of 8-iso-PGF(2 alpha) in three patients during sepsis,recovery and state of health

Sepsis is known to be associated with oxidative stress. Novel markers of oxidative stress are now believed to be F2-isoprostanes which are produced in situ in phospholipids and subsequently released into circulation and excreted in the urine. This study, therefore, sought to investigate whether the excretion of the isoprostane, 8-iso-PGF(2 alpha), is elevated during sepsis. The excretion of 8-iso-PGF(2 alpha), in the 24 h urine of three patients was studied in the septic stage, during mobilisation and in the state of health by a radioimmunological method. Extrapolating the urinary excretion of 8-iso-PGF(2 alpha) over time showed an insignificant variation in the excretion values during 24 h. The amount of mean 24 h urinary 8-iso-PGF(2 alpha) was about similar in the septic stage and in the state of health but increased remarkably during mobilisation in two of the patients. We suggest that mobilisation of septic patients can be associated with an increase of oxidative stress which may stem from an increase in oxygen consumption and/or from a depletion of antioxidants leading to the enhanced formation of free radicals.     

Liquid chromatography-tandem mass spectrometric quantification of the dehydration product of tetranor PGE-M, the major urinary metabolite of prostaglandin E(2) in human urine

A LC-MS/MS method has been developed to analyze tetranor PGE-M, the major urinary metabolite of PGE(2), that involves the acid-catalyzed dehydration of tetranor PGE-M and its deuterated (d(6)) analog followed by LC-MS/MS measurement of the dehydrated tetranor PGE-M product (tetranor PGA-M). We also report a method for quantification of creatinine in urine by LC-MS/MS to normalize tetranor PGE-M concentrations with that of urinary creatinine. These methods were used to study the effect of aspirin on urinary tetranor PGE-M levels in healthy male volunteers. Aspirin did not affect urinary creatinine concentrations but decreased urinary tetranor PGE-M concentrations by approximately 44%.     

Kinetic studies of bioactive products nitric oxide and 8-iso-PGF(2alpha) in Burkholderia pseudomallei infected human macrophages, and their role in the intracellular survival of these organisms

The oxidative response of Burkholderia pseudomallei and Escherichia coli infected macrophages from normal and melioidosis subjects was determined by measuring the production of nitric oxide which is one of the reactive nitrogen intermediates, and the activation state of these macrophages was determined by measuring the generation of 8-iso-PGF(2alpha), a bioactive product of free radical induced lipid peroxidation. Macrophages obtained from the melioidosis patients generated significantly lower levels of nitric oxide and 8-iso-PGF(2alpha) compared to macrophages obtained from the normal subjects (P<0.001). The reduced efficiency of the oxygen dependent microbicidal mechanism in macrophages of melioidosis patients may be one of the survival strategies developed by B. pseudomallei to remain viable intracellularly.     

Impact of diabetic polyneuropathy and cardiovascular autonomic neuropathy on the excretion of urinary 8-epi-PGF2alpha and its metabolites (2, 3-dinor and 2, 3-dinor-5, 6-dihydro)

The objective of this study was to establish if diabetes in the presence of polyneuropathy (PN) and/or cardiovascular autonomic neuropathy (CAN) is associated with alterations in the amounts of 8-epi-PGF2alpha (IP) and its metabolites including 2, 3-dinor-8-epi-PGF2alpha (dinor-IP) and 2, 3-dinor-5, 6 dihydro-8-epi-PGF2alpha (dinor-dihydro-IP) in urine. Mass spectrometric separation showed that excretion of IP was similar in the PN + /CAN- and PN+/CAN+ groups but higher than in the PN-/CAN- group (n = 103, 22 and 60, respectively; P < 0.05). By contrast, excretion of dinor-IP or dinor-dihydro-IP were similar in the PN-/CAN- and PN+/CAN- groups but higher than in PN+/CAN+ group. Correlations were obtained between IP and dinor-IP or dinor-dihydro-IP (r = 0.30; P < 0.001 and r = 0.31; P < 0.001, respectively). A significant association was also observed between dinor-IP and dinor-dihydro-IP (r = 0.48; P < 0.001). In conclusion, these biomarkers should prove useful in studies evaluating the impact of therapeutic drugs or antioxidant interventions aimed at delaying the onset of diabetic complications.