Antonina Gucciardi

Simultaneous quantitative determination of NG,NG-dimethyl-L-arginine or asymmetric dimethylarginine and related pathway's metabolites in biological fluids by ultrahigh-performance liquid chromatography/electrospray ionization-tandem mass spectrometry

BACKGROUND Asymmetric dimethylarginine (ADMA), an endogenous nitric oxide (NO) formation inhibitor, has emerged as a promising biomarker of NO-associated endothelial dysfunction in cardiovascular diseases as well in chronic renal failure. The interest in potentially fundamental role of this metabolite, in basic and clinical research, led to the development of numerous analytical methods for the quantitative determination of ADMA and dimethylarginines in biological systems, notably plasma, serum and urine. OBJECTIVES The aim of this work was to present a simple, fast and accurate UPLC-tandem-MS-based method for the simultaneous determination and quantification of arginine, ADMA, SDMA, NMMA, homo-arginine and citrulline. This method is designed for high sample throughput of only 10 μL of human plasma, serum or urine. METHODS The analysis time is reduced to 1.9 min by an ultrahigh-performance liquid chromatography run coupled with electrospray ionization (ESI) in the positive mode tandem mass spectrometry detection. RESULTS The method was validated in plasma, serum and urine. Correlation coefficients (r(2)) of the calibration curves in all matrices considered ranged from 0.9810 to 0.9993. Inter- and intra-assay precision, accuracy, recovery and carry-over were evaluated for validation. The LOD was 0.01 μM for all compounds in water, plasma and serum and 0.1 μM in urine. The LOQ was 0.05 μM for ADMA, SDMA, NMMA and H-Arg and 0.5 μM for Arg and Cit in water, plasma and serum; while in urine was 0.1 μM for ADMA, SDMA, NMMA and H-Arg and 0.5 μM for Arg and Cit. The precision was ranged from 1% to 15% expressed as CV% and the accuracy (bias %) was <±7% for all added concentrations with the exception of NMMA (-10%). ADMA mean plasma levels, measured in healthy adults and newborns, were in accord with literature data published: (M±SD) 0.56±0.10 μM and 0.84±0.21 μM, respectively, showing that ADMA levels in plasma decreased with age. In serum we have similar data (0.54±0.18 μM and 1.14±0.36 μM), while in neonatal urine ADMA was 11.98±7.13 μmol mmol(-1) creatinine. CONCLUSIONS Data from calibration curves and method validation reveal that the method is accurate and precise. The fast run time, the feasibility of high sample throughput and the small amount of sample required make this method very suitable for routine analysis in the clinical setting.

Surfactant-associated protein B kinetics in vivo in newborn infants by stable isotopes.

Surfactant-associated protein B (SP-B) is critical to the biophysical function of pulmonary surfactant. No information is available on SP-B synthesis and kinetics in humans. We administered a 24-h i.v. infusion of 13C-valine as metabolic precursor of SP-B to six newborn infants (weight 3.5 ± 0.5 kg; age 12 d, range 1–43 d). Three of the study infants also received i.v. 2H-palmitate to label surfactant disaturated phosphatidylcholine (DSPC). SP-B and DSPC were isolated from tracheal aspirates, and their respective 13C and 2H enrichments were measured by gas chromatography-mass spectrometry. SP-B kinetics was measured successfully in all six infants. SP-B median (range) fractional synthesis rate was 30% per day (20–78% per day), secretion time was 4.5 h (1–9 h), time to peak was 24 h (12–36 h), and half-life was 21 h (8–35 h). The ascending part of the SP-B kinetic curve was similar to the DSPC curve, suggesting similar secretion pathways. SP-B half-life seemed to be shorter than DSPC half-life. These results agree with existing animal data. We conclude that the measurement of SP-B kinetics is feasible in vivo in humans by stable isotope technology.

Surfactant status in preterm neonates recovering from respiratory distress syndrome.

OBJECTIVE. The goal was to establish whether reduced amounts of pulmonary surfactant contribute to postextubation respiratory failure in preterm infants recovering from respiratory distress syndrome. METHODS. We prospectively recruited preterm infants who needed mechanical ventilation and exogenous surfactant for treatment of moderate/severe respiratory distress syndrome and could not be extubated before day 3 of life. 13C-labeled dipalmitoyl-phosphatidylcholine was administered endotracheally as tracer before extubation, for estimation of surfactant disaturated phosphatidylcholine pool size and half-life. Patients were retrospectively divided into 3 groups, that is, extubation failure if, after extubation, they needed reintubation or continuous positive airway pressure treatment of ≥6 cmH2O and fraction of inspired oxygen of >0.4, extubation success if they did not meet the failure criteria, and not extubated if they needed ongoing ventilation. Clinical and respiratory parameters were recorded hourly. RESULTS. Reliable kinetic data could be obtained for 63 of the 88 enrolled neonates. Sixteen, 23, and 24 neonates were categorized in the extubation failure, extubation success, and not extubated groups, respectively. Clinical and demographic characteristics did not differ between the extubation failure and extubation success groups. Disaturated phosphatidylcholine pool size was smaller in the extubation failure group than in the extubation success group (25 ± 12 vs 43 ± 24 mg/kg) and was 37 ± 32 mg/kg in the not extubated group. Disaturated phosphatidylcholine half-life was 19 ± 7, 24 ± 12, and 28 ± 18 hours in the extubation failure, extubation success, and not extubated groups, respectively. CONCLUSIONS. In a selected population of preterm infants with moderate/severe respiratory distress syndrome who could not be extubated in the first 3 days of life, infants who were reintubated or needed high continuous positive airway pressure settings after extubation had a smaller disaturated phosphatidylcholine pool size than did those who were successfully extubated or needed low continuous positive airway pressure settings.

Surfactant disaturated-phosphatidylcholine kinetics in acute respiratory distress syndrome by stable isotopes and a two compartment model.

BackgroundIn patients with acute respiratory distress syndrome (ARDS), it is well known that only part of the lungs is aerated and surfactant function is impaired, but the extent of lung damage and changes in surfactant turnover remain unclear. The objective of the study was to evaluate surfactant disaturated-phosphatidylcholine turnover in patients with ARDS using stable isotopes.MethodsWe studied 12 patients with ARDS and 7 subjects with normal lungs. After the tracheal instillation of a trace dose of 13C-dipalmitoyl-phosphatidylcholine, we measured the 13C enrichment over time of palmitate residues of disaturated-phosphatidylcholine isolated from tracheal aspirates. Data were interpreted using a model with two compartments, alveoli and lung tissue, and kinetic parameters were derived assuming that, in controls, alveolar macrophages may degrade between 5 and 50% of disaturated-phosphatidylcholine, the rest being lost from tissue. In ARDS we assumed that 5–100% of disaturated-phosphatidylcholine is degraded in the alveolar space, due to release of hydrolytic enzymes. Some of the kinetic parameters were uniquely determined, while others were identified as lower and upper bounds.ResultsIn ARDS, the alveolar pool of disaturated-phosphatidylcholine was significantly lower than in controls (0.16 ± 0.04 vs. 1.31 ± 0.40 mg/kg, p < 0.05). Fluxes between tissue and alveoli and de novo synthesis of disaturated-phosphatidylcholine were also significantly lower, while mean resident time in lung tissue was significantly higher in ARDS than in controls. Recycling was 16.2 ± 3.5 in ARDS and 31.9 ± 7.3 in controls (p = 0.08).ConclusionIn ARDS the alveolar pool of surfactant is reduced and disaturated-phosphatidylcholine turnover is altered.

Simplified method for microlitre deuterium measurements in water and urine by gas chromatography–high‐temperature conversion–isotope ratio mass spectrometry

Deuterium (2H) in water and urine can be measured by off-line and, more recently, on-line techniques using isotope ratio mass spectrometry (IRMS). We describe a new simple on-line pyrolysis method for the analysis of 2H/1H in water and urine samples by continuous flow IRMS, normally used for 2H/1H measurements in organic compounds. A deactivated column connected the split injector to a high-temperature conversion reactor (TC HD), and 0.5 microL of sample was injected. Accuracy and precision were determined with Vienna Standard Mean Ocean Water (VSMOW), Standard Light Antarctic Precipitation (SLAP), and Greenland Ice Sheet Precipitation (GISP). The range of linearity was measured with a calibration curve of enriched water from 0 up to 0.1 atom percent excess (APE) (i.e. -72 up to 6323 delta per mil (deltaD per thousand)) with a precision of <5 per thousand and accuracy ranging between 1 and 55 per thousand. Blinded reanalysis of urine samples by an equilibration device (Gas Bench) and by a dedicated pyrolysis system (TC/EA) was performed and results compared by the Bland-Altman test. Enrichments ranged between 600 and 2400 per thousand deltaD(VSMOW) with a precision of +/-5 per thousand. Urine enrichments described by our method were strongly correlated with values obtained by Gas Bench and TC/EA (p < 0.0001). There was a significant memory effect that was reduced by injecting the sample 15 times and discarding the first 10 injections, together with accurate furnace conditioning and appropriate cleaning of the syringe. Data indicate that the method is accurate, and that it can be used for water and urine deuterium determination when a Gas Bench or TC/EA instrument is not available and the amount of sample is limited.

Abnormal tyrosine metabolism in chronic cluster headache

Objective Episodic cluster headache is characterized by abnormalities in tyrosine metabolism (i.e. elevated levels of dopamine, tyramine, octopamine and synephrine and low levels of noradrenalin in plasma and platelets.) It is unknown, however, if such biochemical anomalies are present and/or constitute a predisposing factor in chronic cluster headache. To test this hypothesis, we measured the levels of dopamine and noradrenaline together with those of elusive amines, such as tyramine, octopamine and synephrine, in plasma of chronic cluster patients and control individuals. Methods Plasma levels of dopamine, noradrenaline and trace amines, including tyramine, octopamine and synephrine, were measured in a group of 23 chronic cluster headache patients (10 chronic cluster ab initio and 13 transformed from episodic cluster), and 16 control participants. Results The plasma levels of dopamine, noradrenaline and tyramine were several times higher in chronic cluster headache patients compared with controls. The levels of octopamine and synephrine were significantly lower in plasma of these patients with respect to control individuals. Conclusions These results suggest that anomalies in tyrosine metabolism play a role in the pathogenesis of chronic cluster headache and constitute a predisposing factor for the transformation of the episodic into a chronic form of this primary headache.

Quantification of Underivatised Amino Acids on Dry Blood Spot, Plasma, and Urine by HPLC–ESI–MS/MS

Enzyme deficiencies in amino acid (AA) metabolism affecting the levels of amino acids and their derivatives in physiological fluids may serve as diagnostically significant biomarkers for one or a group of metabolic disorders. Therefore, it is important to monitor a wide range of free amino acids simultaneously and to quantify them. This is time consuming if we use the classical methods and more than ever now that many laboratories have introduced Newborn Screening Programs for the semiquantitative analysis, detection, and quantification of some amino acids needed to be performed in a short time to reduce the rate of false positives.We have modified the stable isotope dilution HPLC-electrospray ionization (ESI)-MS/MS method previously described by Qu et al. (Anal Chem 74: 2034-2040, 2002) for a more rapid, robust, sensitive, and specific detection and quantification of underivatised amino acids. The modified method reduces the time of analysis to 10 min with very good reproducibility of retention times and a better separation of the metabolites and their isomers.The omission of the derivatization step allowed us to achieve some important advantages: fast and simple sample preparation and exclusion of artefacts and interferences. The use of this technique is highly sensitive, specific, and allows monitoring of 40 underivatized amino acids, including the key isomers and quantification of some of them, in order to cover many diagnostically important intermediates of metabolic pathways.We propose this HPLC-ESI-MS/MS method for underivatized amino acids as a support for the Newborn Screening as secondary test using the same dried blood spots for a more accurate and specific examination in case of suspected metabolic diseases. In this way, we avoid plasma collection from the patient as it normally occurs, reducing anxiety for the parents and further costs for analysis.The same method was validated and applied also to plasma and urine samples with good reproducibility, accuracy, and precision. The fast run time, feasibility of high sample throughput, and small amount of sample required make this method very suitable for routine analysis in the clinical setting.

A column-switching HPLC-MS/MS method for mucopolysaccharidosis type I analysis in a multiplex assay for the simultaneous newborn screening of six lysosomal storage disorders

Lysosomal storage disorders comprise a group of rare genetic diseases in which a deficit of specific hydrolases leads to the storage of undegraded substrates in lysosomes. Impaired enzyme activities can be assessed by MS/MS quantification of the reaction products obtained after incubation with specific substrates. In this study, a column-switching HPLC-MS/MS method for multiplex screening in dried blood spot of the lysosomal enzymes activities was developed. Mucopolysaccharidosis type I, Fabry, Gaucher, Krabbe, Niemann-Pick A/B and Pompe diseases were simultaneously assayed. Dried blood spots were incubated with substrates and internal standards; thereafter, supernatants were collected with minor manipulations. Samples were injected, trapped into an online perfusion column and, by a six-port valve, switched online through the C18 analytical column to perform separation of metabolites followed by MS/MS analysis. A total of 1136 de-identified newborn screening samples were analyzed to determine references for enzymes activity values. As positive controls, we analyzed dried blood spots from three patients with Pompe, one with Fabry, one with Krabbe disease and two with MPS I, and in all cases the enzyme activities were below the cutoff values measured for newborns, except for an MPS I patient after successful hematopoietic stem cell transplantation.

Analysis and interpretation of acylcarnitine profiles in dried blood spot and plasma of preterm and full-term newborns

Background:Acylcarnitines are biomarkers of fatty acid metabolism, and examining their patterns in preterm newborn may reveal metabolic changes associated with particular conditions related to prematurity. Isomeric acylcarnitines in dried blood spots (DBS) and plasma have never been assessed in preterm infants.Methods:We studied 157 newborn divided into four groups by weeks of gestational age (GA), as follows: 22–27 wk in group 1; 28–31 wk in group 2; 32–36 wk in group 3; and 37–42 wk in group 4. Samples were collected on the third day of life. Acylcarnitines were separated and quantified using ultra-performance liquid chromatography tandem mass spectrometry.Results:Acylcarnitine concentrations correlated significantly with GA and birth weight in both DBS and plasma samples. Concentrations were lower in preterm newborn, except for acylcarnitines derived from branched-chain amino acids, which were higher and correlated with enteral feeding. On day 3 of life, no correlations emerged with gender, respiratory distress syndrome, bronchopulmonary dysplasia, surfactant administration, or mechanical ventilation.Conclusion:We established GA-based reference ranges for isomeric acylcarnitine concentrations in preterm newborn, which could be used to assess nutritional status and the putative neuroprotective role of acylcarnitines.

Kinetics of surfactant in respiratory diseases of the newborn infant

Deficiency or dysfunction of pulmonary surfactant plays a critical role in the pathogenesis of respiratory diseases in the newborn. We describe the studies made by applying two recently developed methods to measure surfactant kinetics. The first allows the measurement of endogenous surfactant phosphatidylcholine (PC) synthesis and kinetics by a constant intravenous infusion of glucose or fatty acids labeled with stable isotope 13C. The second method consists of endotracheal administration of a tracer dose of 13C-labeled dipalmitoyl-phosphatidylcholine (DPPC) to measure disaturated-phosphatidylcholine (DSPC) half-life and apparent pool size. We present the results of surfactant kinetics in some of the respiratory diseases of the newborn infant.