Arslan Arinc Kayacelebi

Homoarginine and 3-nitrotyrosine in patients with takotsubo cardiomyopathy

In recent years, homoargininewas shown to be a cardiovascular risk factor [1], and to herald a poor prognosis in heart failure patients [2]. Yet, the underlying mechanism is elusive. Human and animal studies suggest that the enzyme responsible for the biosynthesis of homoarginine is arginine:glycine amidinotransferase (AGAT) [3–5]. Previously, excessive myocardial AGATgene expressionwas observed in heart failure; the authors implicated AGAT in cardiac creatine synthesis [6]. This finding suggests that homoarginine synthesis in the myocardiummay be elevated in heart failure. Thus far, there is no information about the homoarginine homeostasis in takotsubo cardiomyopathy (TTC) and which potential role this quite neglected non-proteinogenic amino acid may play in the development and recovery of TTC. In TTC patients we recently observed that the plasma concentration of asymmetric dimethylarginine (ADMA), another arginine homologue, is lower than the control, whereas the responsiveness to nitric oxide (NO) is substantially greater compared to healthy females [7]. This is of particular interest, because both L-arginine and L-homoarginine serve as substrates for NO synthases (NOS), while ADMA inhibits NOScatalyzed production of NO from these substrates [8]. The aim of the present study was to measure plasma homoarginine concentration inTTC patients and healthy controls of a previous study [7] and to determine its relationship to 3-nitrotyrosine, a biomarker of NO-related oxidative stress. Plasma homoarginine and 3nitrotyrosine were measured by validated, previously reported gas chromatography-tandem mass spectrometry (GC-MS/MS) methodologies [9,10]. Written informed consent was provided by all subjects included in the study, and the study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki as reflected in a priori approval by the local Ethics Committee of the Central Northern Adelaide Health Service: the Queen Elizabeth Hospital and Lyell McEwin Hospital (protocol number, 009014). The plasma concentration of homoarginine was significantly reduced in TTC patients compared to healthy controls (mean ± SEM; 1298 ± 112 nmol/L vs. 2094 ± 321 nmol/L; median 1403 nmol/L vs. 1634 nmol/L) (Fig. 1A). 3-Nitrotyrosine plasma concentrations were similar in TTC patients and in healthy controls (mean ± SEM; 2355 ± 217 pmol/L vs. 2227 ± 146 pmol/L; median 1915 pmol/L vs. 2170 pmol/L) (Fig. 1B). Pearson correlation between homoarginine and 3-nitrotyrosine concentrations revealed a significant negative relationship in TTC patients (Fig. 2A). In contrast, a positive relationship was observed in the control group (Fig. 2B). No relationship was obtained when all homoarginine and 3-nitrotyrosine data from TTC patients and controls were correlated (not shown). In the TTC patients, plasma homoarginine concentration correlated inversely with systolic blood pressure (SBP) (Fig. 2C). It is worth mentioning that plasma homoarginine concentrationwas found to correlate positively with SBP in an elderly population (50–87 years) non-suffering from takotsubo cardiomyopathy [11]. In contrast, plasma 3-nitrotyrosine concentration did not correlate with SBP (Fig. 2C). Our study indicates that plasma homoarginine concentrations are reduced in TTC patients. They are considerably lower than those measured by us and others in healthy subjects [1–5]. With the exception of four TTC patients, the plasma concentrations of 3nitrotyrosine measured in the other TTC patients and in the control subjects are comparable with those reported in the literature for healthy and ill subjects. The limited number of TTC patients and healthy controls investigated in our study limits the power of our findings. Nevertheless, the results of the present study in TTC supports recent studies indicating homoarginine as a novel marker of cardiovascular disease [1–5]. In contrast to elderly males and females with normal or impaired glucose metabolism or with type 2 diabetes mellitus but without TTC [11], in our TTC patients there was a negative correlation

Simultaneous GC-ECNICI-MS measurement of nitrite, nitrate and creatinine in human urine and plasma in clinical settings.

Creatinine in urine is a useful biochemical parameter to correct the urinary excretion rate of endogenous and exogenous substances. Nitrite (ONO-) and nitrate (ONO2-) are metabolites of nitric oxide (NO), a signalling molecule with multiple biological functions. Under certain and standardized conditions, the concentration of nitrate in the urine is a suitable measure of whole body NO synthesis. The urinary nitrate-to-nitrite molar ratio (UNOxR) may indicate nitrite-dependent renal carbonic anhydrase (CA) activity. In clinical studies, urine is commonly collected by spontaneous micturition. In those cases the nitrate and nitrite excretion must be corrected for creatinine excretion. Pentafluorobenzyl (PFB) bromide (PFB-Br) is a useful derivatization reagent of numerous inorganic and organic compounds, including urinary nitrite, nitrate and creatinine, for highly sensitive and specific quantitation by GC-MS. Here, we report on the simultaneous PFB-Br derivatization (60min, 50°C) of ONO-, O15NO-, ONO2-, O15NO2-, creatinine (do-Crea) and [methylo-2H3]creatinine (d3-Crea) in acetonic dilutions of native human urine and plasma samples (4:1, v/v) and their simultaneous quantification by GC-MS as PFBNO2, PFB15NO2, PFBONO2, PFBO15NO2, do-Crea-PFB and d3-Crea-PFB, respectively. Electron capture negative-ion chemical ionization (ECNICI) of these derivatives generates anions due to [M-PFB]-, i.e., the starting analytes. Quantification is performed by selected-ion monitoring (SIM) of m/z 46 (ONO-), m/z 47 (O15NO-), m/z 62 (ONO2-), m/z 63 (O15NO2-), m/z 112 (do-Crea), and m/z 115 (d3-Crea). Retention times were 2.97min for PFB-ONO2/PFB-O15NO2, 3.1min for PFB-NO2/PFB-15NO2, and 6.7min for do-Crea-PFB/d3-Crea-PFB. We used this method to investigate the effects of long-term oral NaNO3 or NaCl (serving as placebo) supplementation (each 0.1mmol/kg body weight per day for 3 weeks) on creatinine excretion and UNOxR in 17 healthy young men. Compared to NaCl (n=8), NaNO3 (n=9) supplementation increased UNOxR (1709±355 vs. 369±77, P<0.05). Creatinine excretion did not differ between the groups (6.67±1.34mM vs. 5.72±1.27mM, P=0.57). The method is also applicable to human plasma. In 78 adults patients newly diagnosed for cerebrovascular disease (CVD), there was a close correlation (r=0.9833) between the creatinine concentrations measured in plasma by GC-ECNICI-MS and those measured in serum by an enzymatic assay. Creatinine-corrected plasma nitrate and nitrite concentrations (P=0.035 and P=0.004, respectively) but not their concentrations (P=0.68 and P=0.40, respectively) differ between male (n=54) and female (n=24) CVD patients. No such differences were found between preterm newborn boys (n=25) and girls (n=22). Like in urine, circulating creatinine may be useful to correct for gender-specific differences in plasma nitrite and nitrate in adults. Chronic NaNO3 supplementation to healthy young men does not affect renal CA-dependent nitrite excretion or creatinine synthesis and excretion.

Plasma homoarginine (hArg) and asymmetric dimethylarginine (ADMA) in patients with rheumatoid arthritis: Is homoarginine a cardiovascular corrective in rheumatoid arthritis, an anti-ADMA?

In patients with rheumatoid arthritis (RA), cardiovascular disease(CVD)-related mortality is profoundly increased [1]. The risk of heartfailure in RA is almost twice that of the general population. Theincreasedriskcannotbeentirelyexplainedbytraditionalcardiovascularrisk factors. Instead, blood vessels may be damaged through inflamma-tion and immune-mediated processes [2–4].Asymmetricdimethylarginine(ADMA)isacardiovascularriskfactorandplaysaparticular,notyetfullyunderstoodroleinRA[5–7].ADMAisan endogenous inhibitor of nitric oxide (NO) synthesis, and this mech-anism may be one possible explanation for ADMAs involvement inCVD.However,inacohortof201RApatients,ADMAwasnotassociatedwith subendocardial viability ratio [8], a surrogate marker of coronarymicrovascular perfusion.Recently,homoarginine(hArg),anotherarginineanalogue,emergedas a novel cardiovascular risk factor [9–12], but the underlying mecha-nisms are still unresolved and much less understood than those ofADMA. As circulating hArg and ADMA may have opposing influenceson CVD, we proposed to use the plasma hArg-to-ADMA (hArg/ADMA)molar ratio to improve CVD risk prediction [13].Thus far, hArg has not been investigated in rheumatic diseasesincluding RA. In the present study, we newly determined hArg andADMA concentrations in plasma samples of 100 RA patients (88females, 12 males) collected in a previously reported study involvingcombined add-on supplementation for 12 weeks of omega-3 fattyacids, vitamins E and A, copper, and selenium or placebo (soy oil) [14].Written informed consent was provided by all subjects included in thestudy. The study protocol conformed to the ethical guidelines of the1975 Declaration of Helsinki as reflected in a priori approval by theFreiburg Ethics Committee International (Freiburg, Germany) and theEthics Committee of Charite-University of Medicine (Berlin, Germany).The results of the present study are shown in Figs. 1 and 2. Concentra-tions are reported as mean ± SEM.Plasma hArg concentrations in RA patients were comparable tothose measured by us and others in healthy subjects [13], and did notchange after 12 weeks in the placebo (2858 ± 152 vs. 2950 ±169 nmol/L) and in the verum (2952 ± 158 vs. 2854 ± 185 nmol/L)group. Plasma ADMA concentrations in RA patients were comparableto those in elderly subjects with CVD as measured by us and others[13], and did not change after 12 weeks in the placebo (561 ± 13 vs.566 ± 15 nmol/L) and in the verum (574 ± 21 vs. 576 ± 15 nmol/L)group. The plasma hArg/ADMA ratio was comparable to that measuredinhealthysubjects[13]anddidnotchangeafter12 weeksintheplace-bo (5.17 ± 0.29 vs. 5.32 ± 0.32) and in the verum (5.32 ± 0.33 vs.5.05 ± 0.34) group. At baseline, the plasma hArg/ADMA ratio did notdiffer betweenfemales and males(P = 0.56). There wasnocorrelationbetween the hArg and ADMA plasma concentrations in both groups atbaseline and after 12 weeks. Also, there was no correlation betweenthe hArg or ADMA plasma concentrations and C-reactive protein(CRP) in both groups at the beginning and after 12 weeks (data notshown). In subgroups of patients of both groups, add-on supplementa-tion with omega-3fatty acidsand antioxidants orplacebofor 12 weeksdidnotsignificantlychangethecreatinine-correctedexcretionofnitriteand nitrate, indicators of NO synthesis [20],orof15(S)-8-iso-PGF

A validated, rapid UPLC–MS/MS method for simultaneous ivabradine, reboxetine, and metoprolol analysis in human plasma and its application to clinical trial samples ☆

A recent clinical trial assessing human autonomic cardiovascular regulation applied pacemaker channel inhibition with ivabradine, norepinephrine transporter blockade with reboxetine, and beta-adrenoreceptor blockade with metoprolol. To verify patient adherence, we developed and validated a fast UPLC-MS/MS assay measuring all three compounds simultaneously. Deuterium-labeled drugs, d3-ivabradine, d5-reboxetine and d7-metoprolol, served as internal standards. Sample preparation of 200μL human plasma consisted of a single liquid-liquid extraction step by means of ethyl acetate. Chromatographic separation was performed on a 50-mm long BEH C18 column with gradient elution using a mixture of water and methanol each containing 2mM ammonium acetate over 4.5min. The mass spectrometer was operated in the positive electrospray ionization (ESI+) mode. Characteristic product ions resulting from collision-induced dissociation of unlabeled and deuterium-labeled drugs with argon were used for quantification in the selected-reaction monitoring mode. We validated the method according to the European Medicines Agency (EMA) guideline on bioanalytical method validation over the range from 1ng/mL to 500ng/mL for all three analytes. Linear responses with correlation coefficients>0.99 over that range were acquired. The LOQ value was 1ng/mL for each drug. Regulatory criteria for accuracy (80-120%) and precision (RSD<15%) were met for all drugs. The internal standard-normalized matrix factor was close to 1 for low and high analyte concentrations. We successfully measured ivabradine, reboxetine, and metoprolol concentrations in 107 human plasma samples from a clinical trial. Quality control samples processed in parallel confirmed the methods reliability in a clinical setting.

GC–MS and GC–MS/MS measurement of ibuprofen in 10-μL aliquots of human plasma and mice serum using [α-methylo-2H3]ibuprofen after ethyl acetate extraction and pentafluorobenzyl bromide derivatization: Discovery of a collision energy-dependent H/D isotope effect and pharmacokinetic application to inhaled ibuprofen-arginine in mice

GC-MS and GC-MS/MS methods were developed and validated for the quantitative determination of ibuprofen (d0-ibuprofen), a non-steroidal anti-inflammatory drug (NSAID), in human plasma using α-methyl-2H3-4-(isobutyl)phenylacetic acid (d3-ibuprofen) as internal standard. Plasma (10μL) was diluted with acetate buffer (80μL, 1M, pH 4.9) and d0- and d3-ibuprofen were extracted with ethyl acetate (2×500μL). After solvent evaporation d0- and d3-ibuprofen were derivatized in anhydrous acetonitrile by using pentafluorobenzyl (PFB) bromide and N,N-diisopropylethylamine as the base catalyst. Under electron-capture negative-ion chemical ionization (ECNICI), the PFB esters of d0- and d3-ibuprofen readily ionize to form their carboxylate anions [M-PFB]- at m/z 205 and m/z 208, respectively. Collision-induced dissociation (CID) of m/z 205 and m/z 208 resulted in the formation of the anions at m/z 161 and m/z 164, respectively, due to neutral loss of CO2 (44 Da). A collision energy-dependent H/D isotope effect was observed, which involves abstraction/elimination of H- from d0-ibuprofen and D- from d3-ibuprofen and is minimum at a CE value of 5eV. Quantitative GC-MS determination was performed by selected-ion monitoring of m/z 205 and m/z 208. Quantitative GC-MS/MS determination was performed by selected-reaction monitoring of the mass transitions m/z 205 to m/z 161 for d0-ibuprofen and m/z 208 to m/z 164 for d3-ibuprofen. In a therapeutically relevant concentration range (0-1000μM) d0-ibuprofen added to human plasma was determined with accuracy (recovery, %) and imprecision (relative standard deviation, %) ranging between 93.7 and 110%, and between 0.8 and 4.9%, respectively. GC-MS (y) and GC-MS/MS (x) yielded almost identical results (y=4.00+0.988x, r2=0.9991). In incubation mixtures of arachidonic acid (10μM), d3-ibuprofen (10μM) or d0-ibuprofen (10μM) with ovine cyclooxygenase (COX) isoforms 1 and 2, the concentration of d3-ibuprofen and d0-ibuprofen did not change upon incubation at 37°C up to 60min. The trough pharmacokinetics of an inhaled arginine-containing ibuprofen preparation in mice was studied after once-daily treatment (0.0, 0.07, 0.4 and 2.5mg/kg body weight) for three days. A linear relationship between ibuprofen concentration in serum (10μL) and administered dose 24h after the last drug administration was observed.

Gas chromatographic–mass spectrometric analysis of the tripeptide glutathione in the electron-capture negative-ion chemical ionization mode

The dicarboxylic tripeptide glutathione (GSH) is the most abundant intracellular thiol. GSH analysis by liquid chromatography is routine. Yet, GSH analysis by gas chromatography is challenged due to thermal instability and lacking volatility. We report a high-yield laboratory method for the preparation of 2H-labeled GSH dimethyl ester ((d3Me)2-GSH) for use as internal standard (IS) which was characterized by LC–MS/MS. For GC–MS analysis, the dimethyl esters of GSH and the IS were derivatized with pentafluoropropionic (PFP) anhydride. Electron-capture negative-ion chemical ionization of the (Me)2-(PFP)3-GSH provided high sensitivity. We encourage increasing use of GC–MS in the analysis of amino acids as their Me-PFP derivatives in the ECNICI mode.

Analytical challenges in the assessment of NO synthesis from L-arginine in the MELAS syndrome.

Article history: Received 29 November 2016 Accepted 16 December 2016 Available online xxxx and of de novo synthesized N2-ADMA. Args compartmentalization [5] and concomitant conversion of infused N2-Arg to L-N2homoarginine and N2-guanidinoacetate alter Args metabolism and leads to variable plasma steady states, e.g., for N2-Arg and [N]nitrite/[N]nitrate. Secondly, GC-MSmeasurement of N-enrichmenent in [N]nitrite/

Cross-validated stable-isotope dilution GC-MS and LC-MS/MS assays for monoacylglycerol lipase (MAGL) activity by measuring arachidonic acid released from the endocannabinoid 2-arachidonoyl glycerol.

2-Arachidonoyl glycerol (2AG) is an endocannabinoid that activates cannabinoid (CB) receptors CB1 and CB2. Monoacylglycerol lipase (MAGL) inactivates 2AG through hydrolysis to arachidonic acid (AA) and glycerol, thus modulating the activity at CB receptors. In the brain, AA released from 2AG by the action of MAGL serves as a substrate for cyclooxygenases which produce pro-inflammatory prostaglandins. Here we report stable-isotope GC-MS and LC-MS/MS assays for the reliable measurement of MAGL activity. The assays utilize deuterium-labeled 2AG (d8-2AG; 10μM) as the MAGL substrate and measure deuterium-labeled AA (d8-AA; range 0-1μM) as the MAGL product. Unlabelled AA (d0-AA, 1μM) serves as the internal standard. d8-AA and d0-AA are extracted from the aqueous buffered incubation mixtures by ethyl acetate. Upon solvent evaporation the residue is reconstituted in the mobile phase prior to LC-MS/MS analysis or in anhydrous acetonitrile for GC-MS analysis. LC-MS/MS analysis is performed in the negative electrospray ionization mode by selected-reaction monitoring the mass transitions [M-H]-→[M-H - CO2]-, i.e., m/z 311→m/z 267 for d8-AA and m/z 303→m/z 259 for d0-AA. Prior to GC-MS analysis d8-AA and d0-AA were converted to their pentafluorobenzyl (PFB) esters by means of PFB-Br. GC-MS analysis is performed in the electron-capture negative-ion chemical ionization mode by selected-ion monitoring the ions [M-PFB]-, i.e., m/z 311 for d8-AA and m/z 303 for d0-AA. The GC-MS and LC-MS/MS assays were cross-validated. Linear regression analysis between the concentration (range, 0-1μM) of d8-AA measured by LC-MS/MS (y) and that by GC-MS (x) revealed a straight line (r2=0.9848) with the regression equation y=0.003+0.898x, indicating a good agreement. In dog liver, we detected MAGL activity that was inhibitable by the MAGL inhibitor JZL-184. Exogenous eicosatetraynoic acid is suitable as internal standard for the quantitative determination of d8-AA produced from d8-2AG by hepatic MAGL activity. The formation of d8-prostaglandin E2 by the consecutive catalytic action of recombinant MAGL on d8-2AG and recombinant cyclooxygenase-2 (COX) on d8-AA was demonstrated by GC-MS/MS.

The l-arginine/NO pathway in the MELAS syndrome: An insufficiently explored and controversial research area

Article history: Received 21 October 2016 Accepted 2 November 2016 Available online xxxx the L-arginine/NO pathway in the MELAS syndrome is insufficiently explored and controversial. Further systematic investigations in larger patient cohorts are necessary in order to address remaining issues properly [1]. Unfortunately, Finsterer and Zarrouk-Mahjoub [1] neither clarified any of the reported issues in our case work up [2] nor added to the understanding of the L-arginine/NO pathway in the MELAS syn-