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Quantification of arginine and its metabolites in human erythrocytes using liquid chromatography-tandem mass spectrometry
No full textErythrocytes may affect several physiological processes because they are scavengers, vehicles, and (as recently highlighted) a producer of nitric oxide (NO). NO bioavailability is linked to arginine, its metabolic products ornithine and citrulline, and methylarginines. Here we describe a liquid chromatography–tandem
mass spectrometry method for the simultaneous quantification of analytes involved in the Arg/NO metabolic pathway in erythrocytes. Calibration functions were linear, and the interday coefficients of variation were less than 10%. Limit of quantification values make this method suitable for low concentration samples. The method presented here allows easy sample preparation and provides a valuable tool for the evaluation of the Arg/NO metabolic pathway in erythrocytes
Direct measurement of reduced and oxidized glutathione in human blood by liquid chromatography tandem mass spectrometry – comparison with HPLC with electrochemical detection method
No full textGlutathione (GSH) is an important non-protein thiol compound that plays a key role in maintaining cellular homeostasis as well as protecting cells against oxidative stress. Glutathione depletion has been identified in various disease processes such as cardiovascular diseases, carcinogenesis and aging. Under pathological conditions GSH is converted into glutathione disulfide (GSSG) the oxidized form, resulting in a decreased GSH/GSSG ratio. As this ratio is considered to be an early indicator of oxidative stress and/or disease risk, it is important to measure both GSH and GSSG in whole blood samples which may reflect glutathione status in other less accessible tissues.
An analytical method based on liquid-chromatography with positive electrospray ionization (ESI) coupled to tandem mass spectrometric detection (LC-MS/MS) was developed for the determination of both GSH and GSSG in human whole blood.
Separation of analytes was conducted on a Luna PFP(2) analytical column (100x2.0 mm, 3 μm; Phenomenex, USA). A Thermo TSQ Quantum Access triple quadrupole mass spectrometer coupled with ESI was operated in multiple reaction monitoring (MRM) mode with the precursor-to-product ion transitions m/z 308.1→76.2, 84.2, 161.9 (GSH) and 613.2→230.5, 234.6, 354.8 (GSSG) used for quantification.
The assay was fully validated and found to perform well in terms of validation parameters. The method is linear over a concentration range of 0.01 to 50 μM with a lower limit of quantitation (LLOQ) of 0.01 μM for both compounds. The intra-day precision values for all quality control (QC) samples were 2.7% for GSH and 5.4% for GSSG. The inter-day assay precision values for all QC samples were 6.7% for GSH and 6.5% for GSSG. The method also showed adequate accuracy values for both analytes.
The new method was compared with the HPLC coupled with electrochemical detector (HPLC-ECD) assay which we routinely use, and the two assays were found to show a good agreement.
In conclusion although both methods are reliable, easy and fast to perform, the major benefits of the LC-MS/MS are related to the small whole blood volume required (only one-twentieth if that needed by HPLC-ECD assay) and to the higher sensitivity and selectivity, with a better precision and accuracy, thus allowing the quantification of both analytes also in low-concentration samples
Metabolomic evaluation of NO production in human erythrocytes: an LC-MS/MS method to asses arginine and its metabolites
No full textExperimental studies suggest that red blood cells are involved in nitric oxide (NO) synthesis and delivery. Erythrocytes acts as producers, scavengers and vehicles of NO affecting several physiological processes. NO bioavailability is linked not only to arginine and its metabolic products, ornithine and citrulline, but also to methylarginines which are inhibitors of NO sinthesis. Existing methodologies do not permit a systematic evaluation of the metabolic and biosynthetic pathway of NO in RBC. A metabolomic approach, considering a larger number of compounds involved in NO metabolic pathway, could be important to better understand the role of red blood cells in physiological and pathological conditions.
We set up an HPLC-electrospray ionization-tandem mass spectrometry method, to simultaneously detect and quantify arginine metabolome, i.e. arginine, symmetric and asymmetric dimethylarginine, monomethyarginine, ornithine, citrulline. Stable isotope-labelled internal standards have been used to minimize analytical variations before organic protein precipitation of the isolated and lysated erythrocytes. The ion source was operating in positive ion mode and the analytes were detected using multiple reaction monitoring after hydrophilic-interaction chromatography.
A 8 min run time allowed the simultaneous evaluation of all the analytes involved in the NO pathway. The chromatograms were free from interferences, the calibration functions were linear and the inter-day CV were<10% for all the analytes. LOQ values make this method suitable for low concentration samples.
Our validated LC-MS/MS method might be useful to clarify the role of RBCs in the synthesis of NO and will be of help for studies assessing the involvement of these cells in the regulation of blood flow under physiologic or pathological conditions. Moreover, a simple sample processing without the derivatization step, allows to consider this method a valuable tool for diagnostic evaluation of arginine metabolome in red blood cells and it could be applied in clinical chemistry
Cytoskeleton and cyclooxigenase -2 expression in human endothelial cells: autocrine modulation by prostacyclin
No full textEndothelium is a highly dynamic tissue that governs physiological and pathological functions to preserve permeability, vasodilation and the anti-thrombotic properties of the vessel wall. As a result of their unique location, endothelial cells are exposed to a highly dynamic environment that requires a constant rearrangement, either in terms of morphology or function, to maintain an intact monolayer at rest and re-establishing it after disruption. Adaptive physiologic/pathophysiologic responses that are either systematically or regionally compromised by hyperlipidemia, hypertension, diabetes and inflammatory disorders, depend upon the function of cytoskeleton. Obviously, cytoskeletal alterations impact signalling pathways that regulate the expression of genes involved in endothelial “reprogramming”. In this respect, the microtubule-targeted drugs are considered useful tools for the identification of cytoskeletal sensitive genetic responses and for the clarification of signalling pathways associated with alteration of cytoskeletal integrity (Samarakoon et al., 2002). Arachidonic acid metabolites are important in the modulation of vascular homeostasis. The major rate-limiting enzymes involved in their synthesis are the cyclooxygenases: cyclooxygenase-1 is constitutively expressed in most tissues and has general housekeeping functions, whereas cyclooxygenase-2 (Cox-2) is responsible for high-level production of prostanoids in response to pro-inflammatory agents, tumor promoters and growth factors. Cox-2 activity may serve as a compensatory mechanism to preserve vasodilation and the anti-thrombotic properties of the vessel wall under damaging conditions (Cheng et al., 2002). Indeed, Cox-2 metabolites, particularly prostacyclin (PGI2) and prostaglandin E2 (PGE2), have profound influences on vascular tone, platelet activation, permeability and remodeling, angiogenesis and wound repair (Turini et al., 2002). Here, we show that nocodazole, a microtubule disrupting agent, strongly up-regulates Cox-2 in human endothelial cells, in close association with gap formation. In parallel, the levels of acetylated -tubulin, marker of microtubule stability, were reduced. All these effects were prevented by the microtubule stabilizer paclitaxel, relating them to the disruption of the tubular network. In addition, nocodazole increased the extent of glutathionylated actin with dissolution of F-actin cortical ring and stress fiber formation. Concomitantly, intracellular GSH levels were reduced. Of note, the Cox-2 metabolite prostacyclin (PGI2) down-regulated Cox-2 levels through a receptor-mediated mechanism, and restored, at least in part, the monolayer integrity. In a quest for signaling molecules that connect microtubule disruption to Cox-2 induction, we identified serine/threonine phosphatase 2A inhibition and phosphorylation of mitogen activated protein kinase p38 (p38 MAPK) as essential. Taken together data link alterations in microtubule and actin cytoskeleton to Cox-2 expression in human endothelial cells. This finding may represent a mechanistic perspective that set Cox-2 among the endothelial genes whose up-regulation by atherogenic/inflammatory agents is mediated by perturbation of cytoskeletal integrity. In this respect, Cox-2 expression in dysfunctional endothelial cells can be viewed as compensatory in virtue of the atheroprotective properties of PGI2.
Samarakoon et al., (2002) J Cell Sci 115:3093-103.
Cheng et al., (2002) Science 296:539-41.
Turini et al., (2002) Annu Rev Med 53:35-57
Homocysteine lowering and glutathione increasing effects of N-acetylcysteine in acute coronary syndrome
No full textUrinary excretion of iPF2α-III predicts the risk of future thrombotic events. A 10-year follow-up
No full textObjective: F2-isoprostanes derived by arachidonate peroxidation link platelet activation and atherothrombosis. We evaluated whether urinary excretion of 8-iso-PGF2α that follows a methionine loading test predicts future thrombotic events.
Methods: Among those referred for a methionine loading test, 96 unrelated subjects (54 males/42 females, 52/96 with a history of thrombosis) were chosen for a lifelong follow-up.
Results: During 10 years of such follow-up, 39 new events occurred in 29 subjects, 25 of whom having a history of thrombosis. Mean post-methionine loading (PML) excretion of 8-iso-PGF2α was higher (p<0.05) among patients that developed new thrombotic events as compared with those who did not. New events occurred in 14 subjects (58.3%) among those of the upper quartile and in 3 (12.5%) among those of the lowest quartile of PML-8-iso-PGF2α excretion (RR:9.80; 95%CI:2.28-42.05, p=0.002). PML-8-iso-PGF2α OR:4.78, p=0.007), a history of thrombosis at baseline (OR:5.52, p=0.012), smoking habits (OR:4.97, p=0.020), and visceral obesity (OR:3.19, p=0.045) predicted new thrombotic events. Only PML-8-iso-PGF2α predicted arterial events (OR:9.97, p=0.004) in this setting.
Conclusion: oxidative stress that follows a methionine loading test, as reflected by urinary excretion of 8-iso-PGF2α, predicts the risk of future thrombotic events
Plasma dimethylarginines and chronic kidney disease in long-term outcome of non-ST elevation acute coronary sindrome
No full textUrinary levels of 11-dehydro-TxB2 are not an accurate marker of inhibition of platelet thromboxane A2 production by aspirin
No full textHomocysteine lowering and glutathione increasing effects of N-acetylcysteine in acute coronary syndrome
No full textSeveral studies have suggested that increased homocysteine (Hcy) is a risk factor for cardiovascular (CVD) and atherothrombotic diseases because it affect both the vascular wall structure and the blood coagulation system. However, large randomised clinical trials have shown that, even though B-vitamin and folic acid supplementation reduced Hcy levels, there was no significant effect on CVD risk. Oxidative stress is another contributory factor to the etiology of many CVD and we have previously reported low concentrations of reduced glutathione (GSH) in CVD. GSH is an endogenous tripeptide with antioxidant properties and the ratio between reduced and oxidized (GSSG) forms is a major mechanism by which cells maintain redox balance.
N-acetylcysteine (NAC), a drug used to prevent contrast-nephropathy after angiography, is a GSH synthesis precursor. Recent studies have also evidenced a Hcy-lowering activity of NAC in healthy subjects and in renal insufficiency bearing patients. In the present study we investigated the effect of NAC on Hcy and GSH levels in acute coronary syndrome (ACS) patients.
Plasmatic Hcy and whole blood GSH and GSSG were measured in patients with ACS (n=20) before and 2, 6 and 24 hrs after an intravenous bolus (600 mg) of NAC.
Mean Hcy concentrations significantly decreased up to 6 hours after bolus (-40.1 %, p<.0001), returning to basal levels at 24 hrs, while the profile of NAC concentrations showed an opposite behaviour. Whole blood GSH significantly increased after 24 hrs (+18.4 %, p=0.015) with a concomitant GSSG decrease (-23.8 %, p=0.03).
These preliminary results suggest a beneficial effect of NAC administration in ACS patients. In conclusion, NAC may represent a therapeutic tool able to counteract both the oxidative stress status, by increasing GSH levels, and hyperhomocysteinemia condition, by reducing Hcy plasmatic levels
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