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The pathogenesis of migraine, as well as cluster headache (CH), is yet a debated question. In this review, we discuss the possible role of tyrosine and tryptophan metabolism in the pathogenesis of primary headaches, including the abnormalities in the synthesis of neurotransmitters. High level of dopamine, low level of norepinephrine, and very elevated levels of octopamine and synephrine were found in the plasma of episodic migraine without aura. We hypothesize that the imbalance between the levels of neurotransmitters and elusive amines synthesis is due to a metabolic shift directing tyrosine toward increased decarboxylase and reduced hydroxylase enzyme activities, favored by a state of neuronal hyperexcitability and a reduced mitochondrial activity. In addition, we present biochemical studies performed in chronic migraine (CM) and chronic tension-type headache patients (CTTH) to verify if the same anomalies are present in these primary headaches and, if so, their possible role in the chronicity process of CM and CTTH. The results show that important abnormalities of tyrosine-related metabolites are present only in CM patients while tryptamine plasma levels were found significantly lower in both CM and CTTH patients. Because of this, we propose that migraine and, possibly, CH attacks derive from neurotransmitter and neuromodulator metabolic abnormalities in a hyperexcitable and hypoenergetic brain that spread from the frontal lobe, downstream, resulting in abnormally activated nuclei of the pain matrix. The low tryptamine plasma levels found in CM and CTTH patients suggest that these two primary chronic headaches are characterized by a common insufficient serotoninergic control of the pain threshold. [ABSTRACT FROM AUTHOR]

Background and aim: The pathogenesis of the pain that occurs in episodic migraine attack is due to the activation of the trigeminal system's first neuron receptors located on vessel wall. The release from the endothelium of nitric oxide, a product of arginine metabolism, causes vasodilation and stretching of the vascular trigeminal system and promotes pain. It is unknown whether this same metabolic event is involved in the pain accompanying chronic migraine. To understand the possible role of arginine in the pathogenesis of chronic migraine patients, we evaluated the metabolism of arginine in plasma of chronic migraine and control subjects. Methods: We evaluated the metabolism of arginine in a group of patients affected by chronic migraine. Quantification of arginine, ornithine, citrulline, monomethyl arginine (NMMA), dimethylarginines (ADMA, SDMA), and tyramine was performed by ultra-performance liquid chromatography coupled with a triple quadrupole mass spectrometer. Results: Chronic migraine patients showed low plasma levels of arginine, significantly elevated levels of ornithine, ADMA, and NMMA whereas the levels of citrulline and SDMA were in the range of controls. Conclusions: The elevated levels of ADMA and NMMA, inhibitors of nitric oxide synthase, suggest that the metabolism of arginine may be inhibited with a possible reduction of NO release in the circulation of chronic patients. This suggests that the origin of pain may not be related to the vasodilation of trigeminal vascular system that occurs in episodic migraine patients. [ABSTRACT FROM AUTHOR]

Abstract Background In 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. Methods We 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. Results In 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). Conclusion In ARDS the alveolar pool of surfactant is reduced and disaturated-phosphatidylcholine turnover is altered.

Objective: To describe the role of biochemical anomalies of tyrosine (TYR), tryptophan (TRP), and arginine (ARG) metabolism in patients suffering from episodic and chronic cluster headache (CCH). Background: The pathogenesis of cluster headache (CH) and the process that transforms the episodic into the chronic form are unknown. However, the accompanying symptoms suggest a dysfunction of the sympathetic system and hypothalamus along with anomalies of metabolism of catecholamines, elusive amines, and nitric oxide (NO) metabolism. Methods: We describe the results obtained from the last papers published on this issue. The level of metabolites were analyzed by different high‐performance liquid chromatography methods. Results: In both episodic and CH patients, the levels of dopamine and elusive amines are very elevated. The only biochemical difference found in studies between episodic and chronic cluster was that norepinephrine levels were significantly lower in episodic cluster in comparison to control and chronic subjects. In addition, the levels of ARG, homoarginine, and citrulline, precursors of synthesis of NO, were significantly lower in chronic cluster. Conclusions: All these results suggest that TYR, TRP, and ARG metabolism is abnormal and may constitute a biochemical fingerprint of CH patients. The increased levels of norepinephrine in chronic cluster constitute a possible cause of chronicity of this primary headache. The high levels of tryptamine and its activity on the central serotoninergic system may explain why the length of CH is brief in comparison to migraine and tension‐type headache. The low levels of ARG, homoarginine, and citrulline may be the consequence of high circulating levels of α1‐agonists, such as epinephrine and norepinephrine, and their biochemical interaction with endothelial trace amine‐associated receptor 1 that induces activation of NO synthase, resulting in NO synthesis in the circulation, NO release, intense vasodilation, and as a result, the cluster attack. [ABSTRACT FROM AUTHOR]

Aim of the study: The lung architecture of newborns appears to be affected by an inflammatory reaction to maternal choriodecidual layer infection. L-citrulline (L-Cit) was administered to pregnant rats exposed to intra-amniotic lipopolysaccharide (LPS)-induced chorioamnionitis to investigate its effect on neonatal lung injury. Materials and Methods: The pups were assigned to four experimental groups: 1- pups exposed to intra-amniotic NaCl but not to postnatal L-Cit (Controls); 2 - pups exposed to intra-amniotic NaCl as well as to postnatal L-Cit treatment (L-Cit group); 3 - pups exposed to prenatal LPS but not to postnatal (LPS); 4- pups exposed to prenatal LPS as well as to postnatal L-Cit treatment (LPS + L-Cit). Some pups in each group were sacrificed on postnatal (P) day 3 and others on day 7. The pups' lungs were harvested for morphometric analysis; cytokine, arginase 1, and VEGF values were quantified. Serum arginine, citrulline, asymmetric dimethylarginine (ADMA), symmetric dimethylarginine, NG-monomethyl arginine, and homoarginine levels were determined using UPLC-MS/MS. Results: L-Cit attenuated the disruption of alveolar growth in the LPS + L-Cit group. Arginine, homo-arginine, and ADMA levels fell in the LPS treated groups. Arginine and ADMA rose at P7 in the L-Cit group whose members also showed higher VEGF levels with respect to the Controls. The Controls, instead, showed higher IL-10 and IL-1β values with respect to the L-Cit group at P7. Arginase 1 was higher in the LPS groups with respect to the Controls at P7. Conclusions: L-Cit improved alveolar and vascular growth diminishing the lung inflammatory response in the newborn rats exposed to intra-amniotic LPS. The ADMA/DDAH/NO pathway appeared to counteract proinflammatory cytokine production and to sustain macrophage migration. [ABSTRACT FROM AUTHOR]

Bile acid synthesis defects are rare genetic disorders characterized by a failure to produce normal bile acids (BAs), and by an accumulation of unusual and intermediary cholanoids. Measurements of cholanoids in urine samples by mass spectrometry are a gold standard for the diagnosis of these diseases. In this work improved methods for the chemical synthesis of 30 BAs conjugated with glycine, taurine and sulfate were developed. Diethyl phosphorocyanidate (DEPC) and diphenyl phosphoryl azide (DPPA) were used as coupling reagents for glycine and taurine conjugation. Sulfated BAs were obtained by sulfur trioxide-triethylamine complex (SO 3 -TEA) as sulfating agent and thereafter conjugated with glycine and taurine. All products were characterized by NMR, IR spectroscopy and high resolution mass spectrometry (HRMS). The use of these compounds as internal standards allows an improved accuracy of both identification and quantification of urinary bile acids. [ABSTRACT FROM AUTHOR]

ABSTRACT 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. Copyright © 2014 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

We previously found a shorter surfactant disaturated phosphatidylcholine palmitate (DSPCPA) half-life in infants with bronchopulmonary dysplasia (BPD) by using a single stable isotope tracer and simple formulas based on a one-exponential fit of the final portion of the enrichment decay curve. The aim of this study was to apply noncompartmental and compartmental analysis on the entire enrichment decay curve of DSPC-PA and to compare the kinetic data with our previous results. We analyzed 10 preterm newborns with BPD (gestational age 26 ± 0.6 wk, weight 777 ± 199 g) and 6 controls (gestational age 26 ± 1.4 wk, weight 787 ± 259 g). All took part in our previous study. Endotracheal 13C-labeled dipalmitoyl phosphatidylcholine was administered, and the 13C-enrichment of surfactant DSPC-PA was measured from serial tracheal aspirates by gas chromatography-mass spectrometry. Noncompartmental and compartmental models were numerically identified from the tracer-to-tracee ratio and kinetic parameters related to the accessible (pool accessible to sampling, likely to be the lung alveolar pool) and to the nonaccessible pools (pools not accessible to samplings, likely to be the intracellular storage pool) were estimated in the two study groups. Comparison was performed by Mann-Whitney test. A two-compartment model provided the most reliable assessment of DSPC-PA kinetics. In BPD vs. controls, mean ± SE residence time of DSPC-PA in the accessible was 17.5 ± 2.6 vs. 32.2 ± 6.4 h (P < 0.05), whereas it was 49.7 ± 3.5 vs. 54.4 ± 3.9 h (NS, not significant) in the nonaccessible pool; DSPC-PA recycling was 0.26 ± 0.05 vs. 0.43 ± 0.04% (NS), respectively. A two-compartment model of surfactant DSPC-PA kinetics allowed a thorough assessment of DSPC-PA kinetics, including masses, synthesis, and fluxes between pools. The most important findings of this study are that in BPD infants DSPC-PA loss from the alveolar pool was higher and recycling through the intracellular pool lower than in controls. [ABSTRACT FROM AUTHOR]

A "green" solvent-free industrial process (patent pending) is here described for a grape seed extract (GSE) preparation (Ecovitis™) obtained from selected seeds of Veneto region wineries, in the northeast of Italy, by water and selective tangential flow filtration at different porosity. Since a comprehensive, non-ambiguous characterization of GSE is still a difficult task, we resorted to using an integrated combination of gel permeation chromatography (GPC) and electrospray ionization high resolution mass spectrometry (ESI-HRMS). By calibration of retention time and spectroscopic quantification of catechin as chromophore, we succeeded in quantifying GPC polymers up to traces at n = 30. The MS analysis carried out by the ESI-HRMS method by direct-infusion allows the detection of more than 70 species, at different polymerization and galloylation, up to n = 13. This sensitivity took advantage of the nanoscale shotgun approach, although paying the limit of missed separation of stereoisomers. GPC and MS approaches were remarkably well cross-validated by overlapping results. This simple integrated analytical approach has been used for quality control of the production of Ecovitis™. The emerging feature of Ecovitis™ vs. a popular benchmark in the market, produced by a different technology, is the much lower content of species at low n and the corresponding increase of species at high n. [ABSTRACT FROM AUTHOR]

Background: "breathomics" enables indirect analysis of metabolic patterns underlying a respiratory disease. In this study, we analyze exhaled breath condensate (EBC) in asthmatic children before (T0) and after (T1) a three-week course of inhaled beclomethasone dipropionate (BDP). Methods: we recruited steroid-naive asthmatic children for whom inhaled steroids were indicated and healthy children, evaluating asthma control, spirometry and EBC (in asthmatics at T0 and T1). A liquid-chromatography–mass-spectrometry untargeted analysis was applied to EBC and a mass spectrometry-based target analysis to urine samples. Results: metabolomic analysis discriminated asthmatic (n = 26) from healthy children (n = 16) at T0 and T1, discovering 108 and 65 features relevant for the discrimination, respectively. Searching metabolomics databases, seven putative biomarkers with a plausible role in asthma biochemical–metabolic processes were found. After BDP treatment, asthmatic children, in the face of an improved asthma control (p < 0.001) and lung function (p = 0.01), showed neither changes in EBC metabolomic profile nor in urinary endogenous steroid profile. Conclusions: "breathomics" can discriminate asthmatic from healthy children, with prostaglandin, fatty acid and glycerophospholipid as putative markers. The three-week course of BDP—in spite of a significant clinical improvement—was not associated with changes in EBC metabolic arrangement and urinary steroid profile. [ABSTRACT FROM AUTHOR]

Abstract: 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 10μL of human plasma, serum or urine. Methods: The analysis time is reduced to 1.9min 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 0.01μM for all compounds in water, plasma and serum and 0.1μM in urine. The LOQ was 0.05μM for ADMA, SDMA, NMMA and H-Arg and 0.5μM for Arg and Cit in water, plasma and serum; while in urine was 0.1μM for ADMA, SDMA, NMMA and H-Arg and 0.5μM 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±0.10μM and 0.84±0.21μM, respectively, showing that ADMA levels in plasma decreased with age. In serum we have similar data (0.54±0.18μM and 1.14±0.36μM), while in neonatal urine ADMA was 11.98±7.13μmolmmol−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. [Copyright &y& Elsevier]

Aim: To address whether, in patients with chronic migraine and medication overuse headache, mindfulness-based treatment is associated with changes in plasma levels of catecholamines and elusive amines that are similar to those observed in patients undergoing pharmacological prophylaxis.Methods: In this non-randomized, clinic-based effectiveness study, patients aged 18-65, with a history of chronic migraine ≥ 10 years and overuse of triptans or non-steroidal anti-inflammatory drugs ≥ 5 years, were enrolled. Upon completion of a structured withdrawal program, patients received either pharmacological prophylaxis or six weekly sessions of mindfulness-based treatment and were followed for 12 months. Daily headache diaries were used to record headache frequency and medication intake; catecholamines (noradrenaline, epinephrine and dopamine) and levels of elusive amines were assayed from poor platelet plasma.Results: Complete follow-up data were available for 15 patients in the pharmacological prophylaxis-group (14 females, average age 44.1) and 14 in the mindfulness treatment-group (all females, average age 46.4), and all variables were comparable between groups at baseline. At 12 months, significant improvement ( p < .001) was found in the pharmacological prophylaxis group for headache frequency and medication intake (by 51% and 48.7%, respectively), noradrenaline, epinephrine and dopamine (by 98.7%, 120.8% and 501.9%, respectively); patients in the mindfulness treatment-group performed similarly. For elusive amines, no longitudinal changes were found.Conclusions: The similar improvement trends observed in the two groups of patients further support the utility of mindfulness-based treatment in migraine care, and reinforce the hypothesis that alteration and normalization of tyrosine metabolism are implicated in migraine chronification and in remission of chronic migraine. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Early diagnosis of Parkinson's disease (PD) remains a challenge to date. New evidence highlights the potential clinical value of circulating trace amines (TAs) in early-stage PD and their involvement in disease progression. A new ultra performance chromatography mass spectrometry (UPLC-MS/MS) method was developed to quantify plasmatic TAs, and the catecholamines and indolamines pertaining to the same biochemical pathways. Three groups of subjects were recruited: 21 de novo, drug untreated, PD patients, 27 in treatment PD patients and 10 healthy subjects as controls. Multivariate and univariate data analyses were applied to reveal metabolic changes among the groups in attempt to discover new putative markers for early PD detection and disease progression. Different circulating levels of tyrosine (p = 0.002), tyramine (p < 0.001), synephrine (p = 0.015), norepinephrine (p = 0.012), metanephrine (p = 0.001), β-phenylethylamine (p = 0.001) and serotonin (p = 0.006) were found among the three groups. While tyramine behaves as a putative biomarker for early-stage PD (AUC = 0.90) tyramine, norepinephrine, and tyrosine appear to act as biomarkers of disease progression (AUC > 0.75). The findings of this pilot cross-sectional study suggest that biochemical anomalies of the aminergic and indolic neurotransmitters occur in PD patients. Compounds within the TAs family may constitute putative markers for early stage detection and progression of PD.

Enzyme deficiencies in amino acid metabolism may increase the levels of a single or several compounds in physiological fluids becoming 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, especially now that many laboratories have introduced Newborn Screening Programs for the semiquantitative analysis, the detection and quantification of some amino acids need to be performed in a short time to reduce the rate of false positives.We have modified the stable isotope dilution HPLC-ESI-MS/MS method previously described by Qu (Qu et al., 2002) for a more rapid, robust, sensitive, and specific detection and quantification of underivatized 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, enabled to achieve some important advantages: fast and simple sample preparation, exclusion of artifacts, and interferences. The use of this technique is highly sensitive and specific and allowed to monitor 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, 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.

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.

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.

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.

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.

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.