Elusive amines: migraine depends on biochemical abnormalities.

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.

Study of arginine metabolism in medication overuse chronic migraine: possible defect in NO synthesis.

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.

Grape Seeds Proanthocyanidins: Advanced Technological Preparation and Analytical Characterization.

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.

Breathomics in Asthmatic Children Treated with Inhaled Corticosteroids.

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.

Pathogenesis of Cluster Headache: From Episodic to Chronic Form, the Role of Neurotransmitters and Neuromodulators.

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.

Quantification of Underivatized Amino Acids on Dry Blood Spot, Plasma, and Urine by HPLC-ESI-MS/MS.

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.

Different Circulating Trace Amine Profiles in De Novo and Treated Parkinson's Disease Patients.

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), ß-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.

A prospective pilot study of the effect on catecholamines of mindfulness training vs pharmacological prophylaxis in patients with chronic migraine and medication overuse headache.

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.

Lipopolysaccharide-induced chorioamnionitis and postnatal lung injury: The beneficial effects of L-citrulline in newborn rats.

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-1ß 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.

Metabolomics reveals new metabolic perturbations in children with type 1 diabetes.

OBJECTIVE: Using an untargeted metabolomics approach we investigated the metabolome of children with type 1 diabetes (T1D) in comparison with healthy peers and explored the contribution of HbA1c and clinical features to the observed difference. RESEARCH DESIGN AND METHODS: We enrolled children with T1D aged 6-15 years, attending the pediatric diabetes clinic of University of Padova (Italy). Healthy controls were enrolled on voluntary basis and matched for age, sex, pubertal status, body mass index (BMI). We performed a liquid chromatography and mass spectrometry analysis (LC-MS) on fasting urinary samples of the 2 groups. RESULTS: A total of 56 patients with T1D aged (11.4 ± 2.2) years, and 30 healthy controls (10.7 ± 2.8) years were enrolled. We identified 59 urinary metabolites having a higher level in children with T1D, mainly represented by gluco- and mineralcorticoids, phenylalanine and tryptophan catabolites (kynurenine), small peptides, glycerophospholipids, fatty acids, and gut bacterial products. We did not find any association between HbA1c, pubertal status, disease duration, and metabolome profile within the case group. CONCLUSIONS: T1D profoundly disrupts the metabolome of pediatric patients. The excess of cortisol and aldosterone may contribute to the development of macrovascular complications in adulthood, while the increase of tryptophan derivates may have a role in neuronal damage associated to hyperglycemia. Determinants of such findings, other than HbA1c, should be explored.

Asymmetric dimethylarginine and related metabolites in exhaled breath condensate of children with cystic fibrosis.

INTRODUCTION: Asymmetric dimethylarginine (ADMA) competitively inhibits nitric oxide synthase (NOS). Its levels in specimens from murine models and asthmatic patients are related to inflammation and oxidative stress. Patients with cystic fibrosis(CF) reportedly have higher arginase activity, lower NO production and NOS expression than healthy controls. OBJECTIVE: The objective was to assess the role of ADMA and related metabolites as disease biomarkers in exhaled breath condensate (EBC) of pediatric CF patients, compared with age-matched healthy controls (HC). METHODS: A longitudinal design was conceived and 34 CF patients (21 stable, 13 at the onset of exacerbation) and 16 HC were enrolled. All CF patients underwent clinical examination, spirometry and EBC collection at enrolment; the same tests were performed also after an antibiotic course in those patients with exacerbation. Metabolites levels in EBC were measured with an ultra-performance liquid chromatography and tandem mass spectrometry technique. RESULTS: All CF patients had ADMA levels (expressed as ratio to tyrosine) similar to those in HC (median 0.0112, IQR 0.0103-0.0120 and median 0.0114, IQR 0.0090-0.0128, respectively; P = 0.983), while a significant increase in the citrulline/tyrosine ratio was found in CF patients (median 0.6419, IQR 0.5738-0.6899 in CF vs median 0.4176, IQR 0.2986-0.5082 in HC; P = 0.00003). No differences in ADMA levels emerged between stable patients and those with exacerbation. CONCLUSION: ADMA and related aminoacids were measured simultaneously for the first time in EBC from CF patients. Higher citrulline/tyrosine ratios were found in CF children with normal ADMA levels, suggesting a dysregulated ADMA metabolism in these patients.

Improved synthesis of glycine, taurine and sulfate conjugated bile acids as reference compounds and internal standards for ESI-MS/MS urinary profiling of inborn errors of bile acid synthesis.

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 (SO3-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.

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.

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.

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.

Online trapping and enrichment ultra performance liquid chromatography-tandem mass spectrometry method for sensitive measurement of "arginine-asymmetric dimethylarginine cycle" biomarkers in human exhaled breath condensate.

BACKGROUND: Exhaled breath condensate (EBC) is a biofluid collected non invasively that, enabling the measurement of several biomarkers, has proven useful in the study of airway inflammatory diseases, including asthma, COPD and cystic fibrosis. To the best of our knowledge, there is no previous report of any analytical method to detect ADMA in EBC. OBJECTIVES: Aim of this work was to develop an online sample trapping and enrichment system, coupled with an UPLC-MS/MS method, for simultaneous quantification of seven metabolites related to "Arginine-ADMA cycle", using the isotopic dilution. METHODS: Butylated EBC samples were trapped in an online cartridge, washed before and after each injection with cleanup solution to remove matrix components and switched inline into the high pressure analytical column. Multiple reaction monitoring in positive mode was used for analyte quantification by tandem mass spectrometry. RESULTS: Validation studies were performed in EBC to examine accuracy, precision and robustness of the method. For each compound, the calibration curves showed a coefficient of correlation (r(2)) greater than 0.992. Accuracy (%Bias) was <3% except for NMMA and H-Arg (<20%), intra- and inter-assay precision (expressed as CV%) were within ±20% and recovery ranged from 97.1 to 102.8% for all analytes. Inter-day variability analysis on 20 EBC of adult subjects did not demonstrate any significant variation of quantitative data for each metabolite. ADMA and SDMA mean concentrations (µmolL(-1)), measured in EBC samples of asthmatic adolescents are significantly increased (p<0.0001) than in normal controls (0.0040±0.0021 vs. 0.0012±0.0005 and 0.0020±0.0015 vs. 0.0002±0.0001, respectively), as well the ADMA/Tyr (0.34±0.09 vs. 0.12±0.02, p<0.0001) and the SDMA/Tyr ratio (0.10±0.04 vs. 0.015±0.004, p<0.0001). CONCLUSIONS: The proposed method features simple specimen preparation, maintenance of an excellent peak shape of all metabolites and reduced matrix effects as well mass spectrometer noise. Moreover, the possibility to perform different cycles of enrichment, using large injection volumes, compensated for the low concentration of analytes contained in EBC, leading to a good analytical sensitivity. Preliminary data obtained from asthmatic and healthy adolescents, demonstrated that the analytical method applied to EBC seems suitable not only for research purposes, but also for clinical routinely analysis.

A rapid UPLC-MS/MS method for simultaneous separation of 48 acylcarnitines in dried blood spots and plasma useful as a second-tier test for expanded newborn screening.

Acylcarnitine profiling in dried blood spots (DBS) is a useful method for high-throughput newborn screening of metabolic disorders, but differentiation of isobaric and isomeric compounds is not achievable. Chromatographic methods for separation have already been reported but are specific for short-chain acylcarnitines or time-consuming. The aim of this work was to develop a fast ultraperformance liquid chromatography (UPLC)-tandem mass spectrometry (MS/MS) method for separation and quantification of a large number of acylcarnitines, including dicarboxylic acylcarnitines and hydroxyacylcarnitines, in DBS and plasma samples. Acylcarnitines from DBS and plasma were converted to their butyl esters and analyzed by electrospray ionization MS/MS. Chromatographic separation was achieved using a UPLC system equipped with an ethylene-bridged hybrid C(18) column. The correlation coefficients of the calibration curves (r(2)) ranged from 0.990 to 0.999. The limit of detection ranged from 0.002 and 0.063 µM for all compounds, and the limit of quantification ranged from 0.004 and 0.357 µM. Precision ranged from 0.8 to 8.8% and the mean recovery was 103%. Profiles of acylcarnitine isomers were investigated in specimens obtained from patients diagnosed with different inborn errors of metabolism. Acylcarnitine concentrations were also measured in 58 term newborns and compared with flow injection analysis measurements. With this newly developed UPLC-MS/MS method, the simultaneous detection of 61 (13 of these labeled) acylcarnitines in DBS and plasma can be achieved in 15 min including postrun equilibration. The method has been validated and can be used as an important component of newborn screening methods as a second-tier test for discrimination and to confirm diagnosis.

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.

Simultaneous quantitative determination of N(G),N(G)-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 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. (13)C-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 >or=6 cmH(2)O 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.