Germain Bessard

Determination of isoprostaglandin F2α type III in human urine by gas chromatography-electronic impact mass spectrometry. Comparison with enzyme immunoassay

F2-Isoprostanes are stable lipid peroxidation products of arachidonic acid, the quantification of which provides an index of oxidative stress in vivo. We describe a method for analysing isoprostaglandin F2alpha type III (15-F2t-IsoP) in biological fluids. The method involves solid-phase extraction on octadecyl endcapped and aminopropyl cartridges. After conversion to trimethylsilyl ester trimethylsilyl ether derivatives, isoprostaglandin F2alpha type III is analysed by mass spectrometry, operated in electronic impact selected ion monitoring mode. We have compared enzyme immunoassay (EIA; Cayman, Ann Arbor, MI, USA) to this method with 30 human urine aliquots following the same extraction procedure in order to determine the agreement between both methods. Isoprostaglandin F2alpha type III concentrations determined with gas chromatography-mass spectrometry (GC-MS) did not agree with those determined with EIA. Our results suggest that GC-MS and EIA do not measure the same compounds. As a consequence, comparison of clinical results using GC-MS and EIA should be avoided.

Lipid Peroxidation Is Not Increased in Patients With Untreated Mild-to-Moderate Hypertension

Abstract—In contrast with the huge amount of experimental data available, only few and somewhat unconvincing clinical studies support the hypothesis that oxidative stress is involved in the early stages of essential hypertension in humans. Isoprostanes are chemically stable lipid peroxidation products of arachidonic acid, the quantification of which provides a novel approach to the assessment of oxidative stress in vivo. The main objective of this study was to quantify the urinary levels of 15-F2t-IsoP in the early stages of essential hypertension, using gas chromatography/mass spectrometry, by comparing 30 patients with never-treated mild-to-moderate hypertension with 30 gender- and age-paired healthy controls. Urinary 15-F2t-IsoP levels were not significantly different in hypertensive patients (69±36 pmol/mmol creatinine) compared with controls (75±34 pmol/mmol creatinine, 95% confidence intervals on differences: −23 to 13). No significant correlation was found between basal urinary 15-F2t-IsoP levels and age, low-density lipoprotein cholesterol, glucose, clinical pulse pressure, carotid intima-media thickness, left ventricular mass index, or aortic pulse wave velocity. In conclusion, this study shows that lipid peroxidation is not increased in never-treated mild-to-moderate hypertension. This suggests that oxidative stress is not implicated in the pathogenesis of human essential hypertension, at least in the early stages.

Increased urinary F2-isoprostanes in patients with Crohn’s disease

OBJECTIVE:Reactive oxygen metabolites have been suggested to participate in the pathogenesis of Crohns disease, but the evidence supporting this contention in vivo is incomplete. Isoprostaglandin F2α type III (iPF2α-III, or 15-F2t-IsoP) is a prostaglandin F2α isomer produced in vivo by free radical-catalyzed peroxidation of arachidonic acid. We aimed to investigate urinary iPF2α-III concentrations as an index of lipid peroxidation in 23 patients with Crohns disease compared with 23 healthy controls, and to test whether lipid peroxidation correlates to clinical relapse and inflammation.METHODS:Urinary iPF2α-III was measured by gas chromatography/electronic impact mass spectrometry.RESULTS:Urinary iPF2α-III concentrations were significantly higher in patients with Crohns disease than in healthy controls (median [range] = 130 [38–622] vs 91 [35–152] pmol/mmol of creatinine, respectively; p < 0.01). There was a trend toward significance for patients with clinical relapse versus patients with clinical remission (median [range] = 155 [38–622] vs 96 [64–253] pmol/mmol of creatinine, respectively; p = 0.09). A significant correlation was found between urinary iPF2α-III and plasma C-reactive protein concentrations, suggesting a link between lipid peroxidation and inflammation.CONCLUSION:This study provides evidence of increased lipid peroxidation in patients suffering from Crohns disease, especially in patients with clinical relapse. iPF2α-III quantification has to be investigated as a prognosis biomarker in patients suffering from Crohns disease.

Urinary leukotriene E4 excretion: a biomarker of inflammatory bowel disease activity.

Background: Crohns disease (CD) and ulcerative colitis (UC) are chronic inflammatory disorders collectively referred to as inflammatory bowel diseases (IBD). Cysteinyl leukotrienes are proinflammatory 5‐lipoxygenase‐derived products that play a major role in the immune and inflammatory response. Consequently, they may be involved in the pathogenesis of IBD. The aim of this study was therefore to evaluate 1) the urinary excretion of leukotriene E4 (LTE4) in IBD patients and healthy volunteers, and 2) the association between LTE4 production and the activity (relapse/remission) of the disease. Methods: IBD patients and healthy volunteers were prospectively recruited. CD and UC activity was determined on inclusion with the Crohns Disease Activity Index and Clinical Activity Index, respectively. Urine was collected and the urinary excretion of LTE4 was measured by liquid chromatography tandem mass spectrometry. Results: 32 CD patients, 28 UC patients, and 30 controls were enrolled in the study. LTE4 urinary excretion was significantly increased (P < 0.01) in CD [52.0 pg/mg creatinine (10th–90th percentiles: 26.2–148.0)] and UC [64.1 pg/mg creatinine (10th–90th percentiles: 26.7–178.0)] patients compared to controls [32.3 pg/mg creatinine (10th–90th percentiles: 21.8–58.8)]. LTE4 levels were higher (P < 0.001) in patients with active disease than in patients in remission, for whom the levels of LTE4 were similar to the levels of controls. Conclusions: Cysteinyl leukotriene pathway activation could contribute to the inflammation associated with IBD. The quantification of urinary LTE4 could be an interesting noninvasive biomarker for the assessment of IBD activity.

Protease inhibitors and diltiazem increase tacrolimus blood concentration in a patient with renal transplantation: a case report

Tacrolimus is a potent calcineurin inhibitor currently used for prophylaxis and treatment of allograft rejection. As tacrolimus metabolism occurs for the most part via the cytochrome P450-3A4 (CYP3A4) enzyme system, co-administration of drugs activating or inhibiting this metabolic pathway may affect tacrolimus pharmacokinetics. We report a case of dramatic increase in tacrolimus blood concentration that could be attributed, at least in part, to enzymatic inhibition by protease inhibitors and diltiazem.

Physiological Variations of Isoprostanes: A Step Forward?

To the Editor: Ide et al1 recently reported that 15-F2t-IsoP urinary levels were higher in healthy young men compared with healthy premenopausal women, showing that lipid peroxidation is increased in the former group. Isoprostanes appear to be specific and sensitive biomarkers of lipid peroxidation2 and are widely used in clinical trials. Unlike the 5-series and the 15-F2t-IsoP metabolites, our knowledge of the physiological variations of 15-F2t-IsoP is increasing, essential criteria before assessing this biomarker in pathological states. The study by Ide et al provides important but incomplete information concerning the physiological variations of 15-F2t-IsoP in humans. This study was meticulously performed, using a validated assay.3 The numerous exclusion criteria (smoking, hypertension, dyslipidemia, diabetes, alcoholism, medication including vitamins and oral contraceptives) give weight to the validity of the results, because few biases are likely to have been introduced. Furthermore, four potential limitations of the study that the authors outline in the discussion can reasonably be ruled out: the confounding effects of exercise or of …