Urinary and plasma metabolite differences detected by HPLC-ESI-QTOF-MS in systemic sclerosis patients.

Systemic Sclerosis (SSc) is a chronic autoimmune disease whose origin and pathogenesis are not yet well known. Recent studies are allowing a better definition of the disease. However, few studies have been performed based on metabolomics. In this way, this study aims to find altered metabolites in SSc patients in order to improve their diagnosis, prognosis and treatment. For that, 59 SSc patients and 28 healthy volunteers participated in this study. Urine and plasma samples were analysed by a fingerprinting metabolomic approach based on HPLC-ESI-QTOF-MS. We observed larger differences in urine than plasma metabolites. The main deregulated metabolic families in urine were acylcarnitines, acylglycines and metabolites derived from amino acids, specifically from proline, histidine and glutamine. These results indicate perturbations in fatty acid beta oxidation and amino acid pathways in scleroderma patients. On the other hand, the main plasma biomarker candidate was 2-arachidonoylglycerol, which is involved in the endocannabinoid system with potential implications in the induction and propagation of systemic sclerosis and autoimmunity.

Simultaneous ultra-high performance liquid chromathograpy-electrospray ionization-quadrupole-time of flight mass spectrometry quantification of endogenous anandamide and related N-acylethanolamides in bio-matrices.

We describe and validate a sensitive UHPLC-ESI-QTOF-MS method for the simultaneous quantification of seven endocannabinoids and non-endocannabinoids related N-acylethanolamides: N-arachidonoylethanolamide, N-palmitoylethanolamide, N-stearoylethanolamide, N-oleoylethanolamide, N-linoleoylethanolamide, N-α-linolenoylethanolamide and N-eicosapentaenoylethanolamide in several bio-matrices for the purpose of research and clinical application. We examined effects of different liquid-liquid and solid phase extraction on the recovery of endocannabinoids and N-acylethanolamides. Protein precipitation with cooled acetone and extraction with acetonitrile (1% v/v formic acid) using OASIS HLB cartridge gave better results. Separation was performed on a Waters Acquity UPLC HSST3 column using a 9min elution gradient coupled with high resolution mass spectrometry (QTOF/MS). The high sensitivity of the developed method allow its application on sample with low volumes or low levels of endocannabinoids and N-acylethanolamides and make the method suitable for routine measurement in human bio-matrices, such as plasma, serum (500µL), urine (1mL) and tissues (10-30mg). Its application in clinical research could contribute to unravel pathophysiological roles of these family of lipid mediators and disclose novel diagnostic and prognostic markers.

Analytical approaches for the determination of phytocannabinoids and endocannabinoids in human matrices.

Over the last two decades, the role played by phytocannabinoids and endocannabinoids in medicine has gained increasing interest in the scientific community. Upon identification of the plant compound Δ(9)-tetrahydrocannabinol (THC) and of the endogenous substance anandamide (AEA), different methodological approaches and innovative techniques have been developed, in order to evaluate the content of these molecules in various human matrices. In this review, we discuss the analytical methods that are currently used for the identification of phytocannabinoids and endocannabinoids, and we summarize the benefits and limitations of these procedures. Moreover, we provide an overview of the main biological matrices that have been analyzed to date for qualitative detection and quantitative determination of these compounds.

Enhanced lipid peroxidation and platelet activation as potential contributors to increased cardiovascular risk in the low-HDL phenotype.

BACKGROUND: Low high-density lipoprotein (HDL) levels are major predictors of cardiovascular (CV) events, even in patients on statin treatment with low-density lipoprotein (LDL) at target. In animal models HDLs protect LDL from oxidation and blunt platelet activation. Our study aimed to examine whether HDL levels are related to in vivo oxidative stress and platelet activation, as determinants of atherothrombosis. METHODS AND RESULTS: Urinary 8-iso-PGF2α and 11-dehydro-TXB2, in vivo markers of oxidative stress and platelet activation, respectively, were measured in 65 coronary heart disease (CHD) normocholesterolemic patients with HDL ≤35 mg/dL, and in 47 CHD patients with HDL >35 mg/dL. The 2 eicosanoids were also measured before and after an intensive exercise program in sedentary people (n=18) and before and after fenofibrate treatment in otherwise healthy subjects with low HDL (n=10). Patients with HDL ≤35 mg/dL showed significantly higher urinary 8-iso-PGF2α (median [25th to 75th percentiles]: 289 [189 to 380] versus 216 [171 to 321] pg/mg creatinine, P=0.019) and 11-dehydro-TXB2 (563 [421 to 767] versus 372 [249 to 465] pg/mg creatinine, P=0.0001) than patients with higher HDL. A direct correlation was found between urinary 8-iso-PGF2α and 11-dehydro-TXB2 in the entire group of patients (ρ=0.77, P<0.0001). HDL levels were inversely related to both 8-iso-PGF2α (ρ=-0.32, P=0.001) and 11-dehydro-TXB2 (ρ=-0.52, P<0.0001). On multiple regression, only 8-iso-PGF2α (ß=0.68, P<0.0001) and HDL level (ß=-0.29, P<0.0001) were associated with urinary 11-dehydro-TXB2 excretion, independent of sex, age, smoking, hypertension, diabetes, previous myocardial infarction, total cholesterol, LDL, and triglycerides. Both intensive exercise and fenofibrate treatment significantly reduced the 2 eicosanoids in healthy subjects, in parallel with an HDL increase. CONCLUSIONS: A low HDL phenotype, both in CHD patients and in healthy subjects, is associated with increased lipid peroxidation and platelet activation. These data provide novel insight into the mechanisms linking low HDL with increased CV risk.

Simultaneous UPLC-MS/MS quantification of the endocannabinoids 2-arachidonoyl glycerol (2AG), 1-arachidonoyl glycerol (1AG), and anandamide in human plasma: minimization of matrix-effects, 2AG/1AG isomerization and degradation by toluene solvent extraction.

Analysis of the endocannabinoid (EC) system's key molecules 2-arachidonoyl glycerol (2AG) and arachidonoyl ethanolamide (anandamide, AEA) is challenging due to several peculiarities. 2AG isomerizes spontaneously to its biologically inactive analogue 1-arachidonoyl glycerol (1AG) by acyl migration and it is only chromatographically distinguishable from 1AG. Matrix-effects caused primarily by co-extracted phospholipids may further compromise analysis. In addition, 2AG and 1AG are unstable under certain conditions like solvent evaporation or reconstitution of dried extracts. We examined effects of different organic solvents and their mixtures, such as toluene, ethyl acetate, and chloroform-methanol, on 2AG/1AG isomerisation, 2AG/1AG stability, and matrix-effects in the UPLC-MS/MS analysis of 2AG and AEA in human plasma. Toluene prevented, both, 2AG isomerisation to 1AG and degradation of 2AG/1AG during evaporation. Toluene extracts contain only 2% of matrix-effect-causing plasma phospholipids compared to extracts from the traditionally used solvent mixture chloroform-methanol. Toluene and all other tested organic solvents provide comparable 2AG and AEA extraction yields (60-80%). Based on these favourable toluene properties, we developed and validated a UPLC-MS/MS method with positive electrospray ionization (ESI+) that allows for simultaneous accurate and precise measurement of 2AG and AEA in human plasma. The UPLC-MS/MS method was cross-validated with a previously described fully-validated GC-MS/MS method for AEA in human plasma. A close correlation (r(2)=0.821) was observed between the results obtained from UPLC-MS/MS (y) and GC-MS/MS (x) methods (y=0.01+0.85x). The UPLC-MS/MS method is suitable for routine measurement of 2AG and AEA in human plasma samples (1 mL) in clinical settings as shown by quality control plasma samples processed over a period of 100 days. The UPLC-MS/MS method was further extended to human urine. In urine, AEA was not detectable and 2AG was detected in only 3 out of 19 samples from healthy subjects at 160, 180 and 212 pM corresponding to 12.3, 14.5 and 9.9 pmol/mmol creatinine, respectively.

Simultaneous measurement of three N-acylethanolamides in human bio-matrices using ultra performance liquid chromatography-tandem mass spectrometry.

Endocannabinoids including N-acylethanolamides (NAEs) are a family of lipid-related signaling molecules implicated in many physiological and disease states which elicit their activities via the cannabinoid receptors. Anandamide (N-arachidonoylethanolamine, AEA) is the most characterized endocannabinoid and has been detected in many tissues and bio-fluids including human plasma and the central nervous system. The endocannabinoid-like NAEs, oleoylethanolamide (OEA) and palmitoylethanolamide (PEA) are described as entourage compounds because they illicit similar physiological effects to AEA but have little or no affinity for cannabinoid receptors. As entourage compounds, levels of these NAEs can greatly influence the efficacy of AEA yet there are few studies which measure these compounds in bio-fluids. Here we describe a rapid, highly sensitive, specific and highly reproducible ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the analysis of AEA, OEA, and PEA in human bio-fluids including plasma, serum, breast milk, and amniotic fluids. This validated method using deuterated (AEA-d(8), OEA-d(2), and PEA-d(4)) internal standards, represents an improvement over previous analyses in terms of run time (4 min), limit of detection (0.9 fmol on column for AEA and PEA and 4.4 fmol on column for OEA), precision (relative standard deviations of peak areas: 3.1% (AEA), 2.9% (OEA), and 5.4% (PEA) for 133 fmol on column) and accuracy (95.1-104.9%). The sensitivity and precision of the validated method described here suggests that this method is suitable for the analysis of AEA, OEA, and PEA in clinical samples and may be utilized for the investigation of bio-matrices containing limited amounts of NAEs.

Cyclooxygenase-1-deficient mice have high sleep-to-wake blood pressure ratios and renal vasoconstriction.

We used cyclooxygenase-1 (COX-1)-deficient mice to test the hypothesis that COX-1 regulates blood pressure (BP) and renal hemodynamics. The awake time (AT) mean arterial pressures (MAPs) measured by telemetry were not different between COX-1(+/+) and COX-1(-/-) (131+/-2 versus 126+/-3 mm Hg; NS). However, COX-1(-/-) had higher sleep time (ST) MAP (93+/-1 versus 97+/-2 mm Hg; P<0.05) and sleep-to-awake BP ratio (+8.6%; P<0.05). Under anesthesia with moderate sodium loading, COX-1(-/-) had higher MAP (109+/-5 versus 124+/-4 mm Hg; P<0.05), renal vascular resistance (23.5+/-1.6 versus 30.7+/-1.7 mm Hg . mL(-1) . min(-1) . g(-1); P<0.05) and filtration fraction (33.7+/-2.1 versus 40.2+/-2.0%; P<0.05). COX-1(-/-) had a 89% reduction (P<0.0001) in the excretion of TxB2, a 76% reduction (P<0.01) in PGE2, a 40% reduction (P<0.0002) in 6-ketoPGF1alpha (6keto), a 27% reduction (P<0.02) in 11-betaPGF2alpha (11beta), a 35% reduction (P<0.01) in nitrate plus nitrite (NOx), and a 52% increase in metanephrine (P<0.02). The excretion of normetanephrine, a marker for sympathetic nervous activity, was reduced during ST in COX-1(+/+) (6.9+/-0.9 versus 3.2+/-0.6 g . g(-1) creatinine . 10(-3); P<0.01). This was blunted in COX-1(-/-) (5.1+/-0.9 versus 4.9+/-0.7 g . g(-1) creatinine . 10(-3); NS). Urine collection during ST showed lower excretion of 6keto, 11beta, NOx, aldosterone, sodium, and potassium than during AT in both COX-1(+/+) and COX-1(-/-), and there were positive correlations among these parameters (6keto versus NOx; P<0.005; 11beta versus NOx; P<0.005; and NOx versus sodium; P<0.005). In conclusion, COX-1 mediates a suppressed sympathetic nervous activity and enhanced NO, which may contribute to renal vasodilatation and a reduced MAP while asleep or under anesthesia. COX-1 contributes to the normal nocturnal BP dipping phenomenon.

Cannabinoid mimics in chocolate utilized as an argument in court.

A case is presented involving chocolate cannabinoid mimics which have been utilized in court by the defendant's lawyer in order to clear the accused of smoking and dealing in marijuana after he was found positive for cannabis in a routine urine immunoassay screening test. The argumentation in this case was that the accused had supposedly eaten a massive amount of chocolate which contained anandamide-related lipids. These lipids inhibit anandamide hydrolysis in the brain, act as cannabinoid mimics and, according to the lawyer, were the cause of the positive cannabinoid test. To investigate this in detail, we synthesized N-oleoyl- and N-linoleoylethanolamide and spiked these compounds together with N-arachidonoylethanolamide in urine for immunological investigations. None of the samples were found positive, indicating that no cross-reactivity occurs with cannabinoids. As a result, the lawyer's claim could be refuted and the accused was convicted.

Antioxidant susceptibility of pathogenic pathways in subjects with antiphospholipid antibodies: a pilot study.

The pathogenesis of antiphospholipid antibody (aPL) related thrombosis is multifactorial and includes, amongst others, enhanced coagulation activation measured as prothrombin fragment 1 + 2 (F1 + 2), elevated plasma levels of von Willebrand factor (vWF), plasminogen activator inhibitor (PAI) and endothelin-1 (ET-1) as well as heightened thromboxane generation and lipid peroxidation. To evaluate the antioxidant susceptibility of some of the above pathways, probucol (500 mg/d orally, a cholesterol lowering agent bearing antioxidant properties) was administered for a three week period to 14 subjects with aPL and to seven healthy controls. At baseline aPL participants showed higher plasma levels of vWF (P = 0.006), ET-1 (P = 0.0002) and enhanced urinary excretion of 11-dehydro-thromboxane-B2 (TXB2) (P = 0.0004), F2-isoprostanes (marker of lipid peroxidation) (P = 0.02) and albumin (P = 0.04) than controls. In the aPL group baseline IgG anticardiolipin (aCL) titre positively related with urinary TXB2 (r2 = 0.43, P = 0.01) and inversely with urinary NOx (r2 = -0.6, P = 0.005) whereas urinary NOx and TXB2 were negatively correlated (r2 = -0.42, P = 0.01). After the treatment period significant decreases from baseline values were noted for PAI (P = 0.01), ET-1 (P = 0.006), TXB2 (P = 0.02), F2-isoprostanes (P = 0.01) and albuminuria (P = 0.01) in aPL participants but not in controls. These pilot data support oxidative sensitive mechanisms and a potential role for antioxidant treatment in the pathogenesis of aPL induced vasculopathy.

Increase in circulating products of lipid peroxidation (F2-isoprostanes) in smokers. Smoking as a cause of oxidative damage.

BACKGROUND: It has been hypothesized that the pathogenesis of diseases induced by cigarette smoking involves oxidative damage by free radicals. However, definitive evidence that smoking causes the oxidative modification of target molecules in vivo is lacking. We conducted a study to determine whether the production of F2-isoprostanes, which are novel products of lipid peroxidation, is enhanced in persons who smoke. METHODS: We measured the levels of free F2-isoprostanes in plasma, the levels of F2-isoprostanes esterified to plasma lipids, and the urinary excretion of metabolites of F2-isoprostanes in 10 smokers and 10 nonsmokers matched for age and sex. The short-term effects of smoking (three cigarettes smoked over 30 minutes) and the effects of two weeks of abstinence from smoking on levels of F2-isoprostanes in the circulation were also determined in the smokers. RESULTS: Plasma levels of free and esterified F2-isoprostanes were significantly higher in the smokers (242 +/- 147 and 574 +/- 217 pmol per liter, respectively) than in the nonsmokers (103 +/- 19 and 345 +/- 65 pmol per liter; P = 0.02 for free F2-isoprostanes and P = 0.03 for esterified F2-isoprostanes). Smoking had no short-term effects on the circulating levels of F2-isoprostanes. However, the levels of free and esterified F2-isoprostanes fell significantly after two weeks of abstinence from smoking (250 +/- 156 and 624 +/- 214 pmol per liter, respectively, before the cessation of smoking, as compared with 156 +/- 67 and 469 +/- 108 pmol per liter after two weeks' cessation; P = 0.03 for free F2-isoprostanes and P = 0.02 for esterified F2-isoprostanes). CONCLUSIONS: The increased levels of F2-isoprostanes in the circulation of persons who smoke support the hypothesis that smoking can cause the oxidative modification of important biologic molecules in vivo.

Endogenous nitric oxide influences acetylcholine-induced bronchovascular dilation in sheep.

To test whether endogenous endothelial nitric oxide (NO) influences baseline bronchial vascular tone and mediates acetylcholine (ACh)-induced bronchial vascular dilation and/or modulates bronchoconstriction in ovine airways, we studied anesthetized ventilated open-chest sheep and measured bronchial blood flow (Qbr) and pulmonary resistance (RL). In six sheep we measured the response of Qbr and RL to the dose of ACh required to produce 50% of the maximal increase in Qbr at baseline during infusion of the NO synthase inhibitor NG-nitro-L-arginine (L-NNA; 10(-2) M). Infusion of L-NNA decreased both the baseline Qbr (28 +/- 13 to 8 +/- 2 ml/min, P < 0.01) and the change in Qbr (delta Qbr) from the baseline value (84 +/- 42 to 33 +/- 18 ml/min, P < 0.05). There was no difference in baseline RL or in the response of RL to ACh at any time. In another six sheep, phenylephrine (5 x 10(-6) to 5 x 10(-7) M) decreased baseline Qbr (22 +/- 6 to 10 +/- 3 ml/min, P < 0.05) but not delta Qbr (62 +/- 13 to 66 +/- 21 ml/min, not significant). Infusion of L-NNA in these sheep decreased the baseline Qbr to a similar extent (11 +/- 5 ml/min) and also decreased delta Qbr (42 +/- 16 ml/min, P < 0.05). We conclude that endogenous endothelial NO influences baseline vascular tone and ACh-induced vasodilation of the ovine bronchial vasculature but has no effect on baseline RL or ACh-induced bronchoconstriction.

Solid-phase extraction of prostanoids using an automatic sample preparation system.

A commercial automated solid-phase extraction system for cyclooxygenase arachidonic acid metabolites in urine samples has been evaluated. Comparison of manual and automatic batch (36 samples) extraction procedures for tritium labelled prostanoids added as tracers to urine samples has shown equivalent results with recoveries greater than 90% for prostaglandins E2, F2alpha and 6-keto prostaglandin F1alpha as well as thromboxane B2. Analyte stability is not affected by the automated procedure, which uses less solvents and has a faster overall processing time than the manual method. The automated system has been applied to the extraction of prostanoids in urine samples from workers exposed to dichloroethane.

Effect of magnesium lithospermate B on urinary excretion of arachidonate metabolites in rats with renal failure.

The effect of magnesium lithospermate B isolated from Salviae miltiorrhizae Radix on excretion of urinary arachidonate metabolites was examined in both normal rats and those given adenine. Urinary excretion of prostaglandin E2 (PGE2) and 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) decreased while urinary thromboxane B2 (TXB2) excretion increased markedly with the progression of renal failure. Rats administered magnesium lithospermate B showed an increase of urinary PGE2 excretion at the 6th and 12th days. Excretion of 6-keto-PGF1 alpha also showed a significant increase on the 6th and 12th days in rats with renal failure induced by the administration of adenine. However, these effects were lower than the corresponding values in normal rats. In addition, urinary PGE2 and 6-keto-PGF1 alpha excretions showed no appreciable difference in rats that exhibited progressive renal failure with continuation of the adenine administration period, as shown on the 18th and 24th days. There were no significant changes in TXB2 excretion between the control and magnesium lithospermate B-treated groups throughout the experimental period.

Effects of droxicam on in vivo prostaglandin synthesis and ex vivo platelet aggregation.

Droxicam, similar to piroxicam, inhibits in vivo renal synthesis of PGF2 alpha. This study was performed in rats; droxicam and piroxicam were administered orally at doses of 0.5, 1.2 and 8 mg/kg. Inhibitory activity of the two compounds was similar (40-60%) but showed no dose-effect relationship. Maximum inhibition was obtained with the 1 mg/kg dosage. In dogs droxicam has shown a clear inhibitory effect on arachidonic acid induced ex vivo platelet aggregation. Droxicam was administered orally at a dose of 2 mg/kg. Maximum inhibition (-40%) was achieved 24 hr post-administration and the effect was sustained up to 72 hr (-23%).

Measurement of renal and non-renal eicosanoid synthesis.

Enzymatic metabolites of arachidonic acid (eicosanoids) have potent biologic actions in vitro that suggest their pathophysiologic importance in vivo. To address this possibility, analytic methodology has been developed to permit study of the formation of these compounds in vivo. Both radioimmunoassay and gas chromatography-mass spectrometry have been used to measure stable but biologically inactive metabolites of the eicosanoids. Although indirect, such measures are presently the most reliable, because superfusion-bioassay lacks the specificity and precision necessary for quantitative analysis of eicosanoid formation in vivo. Measurement of eicosanoids and their hydration products and metabolites in urine represents a non-invasive approach to the assessment of eicosanoid biosynthesis. Although a tissue of origin cannot be ascribed definitely to a compound measured in urine, corroborative evidence can be obtained to indicate the predominant tissue source under physiologic and pathologic conditions. This relates particularly to the distinction between renal and extrarenal biosynthesis of these compounds. Although similar limitations apply to the measurement of eicosanoids in plasma, these may also be confounded by sources of artifact related to blood withdrawal. In the case of thromboxane B2, these concerns have been addressed by the development of methods to measure its enzymatic metabolites in plasma. Finally, formation of eicosanoids may be studied in localized compartments such as lavage or synovial fluid. Such an approach has recently provided biochemical evidence for increased formation of prostacyclin and prostaglandin E2 at the platelet-vascular interface during selective inhibition of thromboxane synthase in humans.