A targeted metabolomic procedure for amino acid analysis in different biological specimens by ultra-high-performance liquid chromatography-tandem mass spectrometry.

INTRODUCTION: Amino acid analysis in biological fluids is essential for the study of inborn errors of metabolism (IEM) and other diseases. OBJECTIVES: Our aim was to develop a UPLC-MS/MS procedure for the analysis of 25 amino acids and identification of 17 related compounds. METHODS: Sample treatment conditions were optimized for plasma, urine, cerebrospinal fluid (CSF) and dried blood spots. Amino acids and related compounds were analyzed on a Waters ACQUITY UPLC H-class instrument with a reversed-phase C-18 column using water and acetonitrile with 0.1% formic acid as the mobile phases (run time = 9 min). The detection was performed with a Waters Xevo TQD triple-quadrupole mass spectrometer using positive electrospray ionization in the multiple reaction monitoring mode. RESULTS: The method linearity, intra-assay and inter-assay precision, detection limit, quantification limit and trueness analysis displayed adequate results in both physiological and pathological conditions. Method comparison was performed between UPLC-MS/MS and ion exchange chromatography (IEC) with ninhydrin derivatization, and the methods showed good agreement, except for 4-hydroxyproline, aspartate and citrulline. Paediatrics age-related reference values in plasma, urine and CSF were established and patients with different IEM were easily identified. CONCLUSION: We report a modified UPLC-MS/MS procedure for the analysis of 42 amino acids and related compounds in different specimens. The method is fast, sensitive and robust, and it has been validated to be an alternative to the traditional IEC procedure as the routine method used in metabolic laboratories. The method greatly decreases the run time of the analysis while displaying good metrological results.

Cerebrospinal fluid monoamines, pterins, and folate in patients with mitochondrial diseases: systematic review and hospital experience.

Mitochondrial diseases are a group of genetic disorders leading to the dysfunction of mitochondrial energy metabolism pathways. We aimed to assess the clinical phenotype and the biochemical cerebrospinal fluid (CSF) biogenic amine profiles of patients with different diagnoses of genetic mitochondrial diseases. We recruited 29 patients with genetically confirmed mitochondrial diseases harboring mutations in either nuclear or mitochondrial DNA (mtDNA) genes. Signs and symptoms of impaired neurotransmission and neuroradiological data were recorded. CSF monoamines, pterins, and 5-methyltetrahydrofolate (5MTHF) concentrations were analyzed using high-performance liquid chromatography with electrochemical and fluorescence detection procedures. The mtDNA mutations were studied by Sanger sequencing, Southern blot, and real-time PCR, and nuclear DNA was assessed either by Sanger or next-generation sequencing. Five out of 29 cases showed predominant dopaminergic signs not attributable to basal ganglia involvement, harboring mutations in different nuclear genes. A chi-square test showed a statistically significant association between high homovanillic acid (HVA) values and low CSF 5-MTHF values (chi-square = 10.916; p = 0.001). Seven out of the eight patients with high CSF HVA values showed cerebral folate deficiency. Five of them harbored mtDNA deletions associated with Kearns-Sayre syndrome (KSS), one had a mitochondrial point mutation at the mtDNA ATPase6 gene, and one had a POLG mutation. In conclusion, dopamine deficiency clinical signs were present in some patients with mitochondrial diseases with different genetic backgrounds. High CSF HVA values, together with a severe cerebral folate deficiency, were observed in KSS patients and in other mtDNA mutation syndromes.

Importance of the determination of enzymatic activity in the diagnosis of deficiency of adenosine deaminase 2 (DADA2). / Importancia de la determinación de actividad enzimática en el diagnóstico del déficit de adenosina desaminasa 2 (DADA2).

OBJECTIVE: To describe the usefulness of determining the enzymatic activity of adenosine deaminase 2 (ADA2) in patients with suspected ADA2 deficiency (DADA2). METHOD: Retrospective multicenter study. Review with analysis of the clinical, biochemical and genetic data of the patients in whom the enzymatic activity of ADA2 has been determined by spectrophotometric method. RESULT: In 3 of the 20 patients, the diagnosis of DADA2 was confirmed by the combination of reduced enzyme activity and biallelic pathogenic variants in the CECR1 gene. In 2 patients with variants of uncertain significance in CECR1, the study of enzymatic activity allowed to rule out the disease. CONCLUSIONS: The reduced enzymatic detection of ADA2 confirms the diagnosis of DADA2, particularly important in carriers of variants of uncertain significance in CECR1.

Host, technical, and environmental factors affecting QuantiFERON-TB Gold In-Tube performance in children below 5 years of age.

Interferon-gamma release assays performance can be impaired by host-related, technical and environmental factors, but data in young children are limited. We performed a cross-sectional study of children < 5 years-of-age at risk of tuberculosis (TB), using QuantiFERON-TB Gold In-Tube (QFT-GIT) assays. The impact of the following was evaluated: (i) host-related [age; hematological parameters; erythrocyte sedimentation rate (ESR); C-reactive protein (CRP); and tobacco smoke exposure (TSE) based on serum cotinine concentrations], (ii) technical (pre-analytical delay) and (iii) environmental factors (annual season; monthly temperatures). Of 204 children, 35 (17.2%) were diagnosed with latent TB infection or TB disease. QFT-GIT results were indeterminate in 14 (6.9%) patients. In multivariate analysis, younger age and higher ESR were associated with lower positive control responses (beta: 0.247, p = 0.002 and - 0.204, p = 0.007, respectively), and increasing age was associated with lower rates of indeterminate QFT-GIT results [OR (95% CI) 0.948 (0.903-0.996) per month, p = 0.035]. In children with positive QFT-GIT results, average monthly temperatures correlated with antigen responses (r = 0.453, p = 0.020); also, antigen responses were lower in winter than in other seasons (p = 0.027). Serum cotinine concentrations determined in a subgroup of patients (n = 41) indicated TSE in 36 (88%), positive control responses being lower in children with TSE (p = 0.034). In children < 5 years-of-age, young age, elevated ESR, temperature, annual season and TSE can affect the performance of QFT-GIT assays.

Effect of blood contamination of cerebrospinal fluid on amino acids, biogenic amines, pterins and vitamins.

BACKGROUND: Cerebrospinal fluid (CSF) metabolomic investigations are a powerful tool for studying neurometabolic diseases. We aimed to assess the effect of CSF contamination with blood on the concentrations of selected biomarkers. METHODS: CSF samples were spiked in duplicate with increasing volumes of whole blood under two conditions: (A) pooled CSF spiked with fresh whole blood and frozen to cause red blood cell (RBC) lysis; (B) pooled CSF spiked with fresh blood and centrifuged (the supernatant with no RBCs was frozen until the moment of analysis). CSF concentrations of amino acids, biogenic amines, pterins, and vitamins were analysed by HPLC coupled with tandem mass spectrometry, electrochemical and fluorescence detection. RESULTS: Aspartate, glutamate, taurine, ornithine, glycine, citrulline, pyridoxal 5´-phosphate, 5-methyltetrahydrofolate, and thiamine showed higher values when RBCs were lysed when compared with those of CSF with no RBC, while arginine, 5-hydroxyindoleacetic and homovanillic acids showed lower values. When RBCs were removed from CSF, only some amino acids, thiamine and pyridoxal 5´-phosphate showed moderately higher values when compared with the non-spiked CSF sample. CONCLUSIONS: CSF-targeted metabolomic analysis is feasible even when substantial RBC contamination of CSF has occurred since CSF centrifugation to remove RBC prior to freezing eliminated most of the interferences observed.

Discovery of Biomarker Panels for Neural Dysfunction in Inborn Errors of Amino Acid Metabolism.

Patients with inborn errors of amino acid metabolism frequently show neuropsychiatric symptoms despite accurate metabolic control. This study aimed to gain insight into the underlying mechanisms of neural dysfunction. Here we analyzed the expression of brain-derived neurotrophic factor (BDNF) and 10 genes required for correct brain functioning in plasma and blood of patients with Urea Cycle Disorders (UCD), Maple Syrup Urine Disease (MSUD) and controls. Receiver-operating characteristic (ROC) analysis was used to evaluate sensitivity and specificity of potential biomarkers. CACNA2D2 (α2δ2 subunit of voltage-gated calcium channels) and MECP2 (methyl-CpG binding protein 2) mRNA and protein showed an excellent neural function biomarker signature (AUC ≥ 0,925) for recognition of MSUD. THBS3 (thrombospondin 3) mRNA and AABA gave a very good biomarker signature (AUC 0,911) for executive-attention deficits. THBS3, LIN28A mRNA, and alanine showed a perfect biomarker signature (AUC 1) for behavioral and mood disorders. Finally, a panel of BDNF protein and at least two large neural AAs showed a perfect biomarker signature (AUC 1) for recognition of psychomotor delay, pointing to excessive protein restriction as central causative of psychomotor delay. To conclude, our study has identified promising biomarker panels for neural function evaluation, providing a base for future studies with larger samples.

Analysis of human cerebrospinal fluid monoamines and their cofactors by HPLC.

The presence of monoamines and their cofactors (the pterins and vitamin B6 (pyridoxal phosphate (PLP))) in human cerebrospinal fluid (CSF) can be used as indicators of the biosynthesis and turnover of dopamine and serotonin in the brain. In addition, abnormalities in the CSF levels of these molecules are associated with various neurological diseases, including genetic diseases leading to dopamine and serotonin deficiency. Here, we provide a set of quantitative high-performance liquid-chromatography (HPLC) approaches to determine CSF levels of monoamines and their cofactors. This protocol describes step-by-step procedures for CSF sample preparation for the analysis of different molecules, HPLC calibration and analysis, and data quantification and interpretation. Unlike plasma/tissue/blood samples, CSF requires minimal sample preparation: in this protocol, only the analysis of PLP requires mixing with trichloroacetic acid to release the protein-bound vitamin, centrifugation, and mixing of the supernatant with phosphate buffer and sodium cyanide for derivatization in alkaline conditions. Monoamines are analyzed by HPLC with coulometric electrochemical detection (ED), pterins are analyzed by HPLC with coupled coulometric electrochemical and fluorescence detection, and PLP is analyzed by HPLC with fluorescence detection. The quantification of all compounds is achieved by external calibration procedures, and internal quality control and standards are analyzed in each run. We anticipate that investigation of dopamine and serotonin disturbances will be facilitated by measurements of these compounds in human CSF and other biological samples. The estimated time for the different procedures primarily depends on the electrochemical detector stabilization. Overnight stabilization of this detector is advised, and, after that step, preanalytical equilibration rarely exceeds 3 h.

Urinary sulphatoxymelatonin as a biomarker of serotonin status in biogenic amine-deficient patients.

Melatonin is synthesized from serotonin and it is excreted as sulphatoxymelatonin in urine. We aim to evaluate urinary sulphatoxymelatonin as a biomarker of brain serotonin status in a cohort of patients with mutations in genes related to serotonin biosynthesis. We analized urinary sulphatoxymelatonin from 65 healthy subjects and from 28 patients with genetic defects. A total of 18 patients were studied: 14 with autosomal dominant and recessive guanosine triphosphate cyclohydrolase-I deficiency; 3 with sepiapterin reductase deficiency; and 1 with aromatic L-amino acid decarboxylase deficiency. Further 11 patients were studied after receiving serotoninergic treatment (serotonin precursors, monoamine oxidase inhibitors, selective serotonin re-uptake inhibitors): 5 with aromatic L-amino acid decarboxylase deficiency; 1 with sepiapterin reductase deficiency; 3 with dihydropteridine reductase deficiency; and 2 with 6-pyruvoyltetrahydropterin synthase deficiency. Among the patients without therapy, 6 presented low urinary sulphatoxymelatonin values, while most of the patients with guanosine triphosphate cyclohydrolase-I deficiency showed normal values. 5 of 11 patients under treatment presented low urine sulphatoxymelatonin values. Thus, decreased excretion of sulphatoxymelatonin is frequently observed in cases with severe genetic disorders affecting serotonin biosynthesis. In conclusion, sulphatoxymelatonin can be a good biomarker to estimate serotonin status in the brain, especially for treatment monitoring purposes.

Biochemical Analyses of Cerebrospinal Fluid for the Diagnosis of Neurometabolic Conditions. What Can We Expect?

In this article, we review the state-of-the-art analysis of different biomarkers in the cerebrospinal fluid for the diagnosis of genetically conditioned, rare, neurometabolic diseases, including glucose transport defects, neurotransmitter (dopamine, serotonin, and gamma-aminobutyric acid) and pterin deficiencies, and vitamin defects (folate, vitamin B6, and thiamine) that affect the brain. The analysis of several key metabolites are detailed, which thus highlights the preanalytical and analytical factors that should be cautiously controlled to avoid misdiagnosis; moreover, these factors may facilitate an adequate interpretation of the biochemical profiles in the context of severe neuropediatric disorders. Secondary disturbances in these biomarkers, which are associated with other genetic or environmental conditions, are also detailed. Importantly, the early biochemical identification of biochemical disturbances in the cerebrospinal fluid may improve the clinical outcomes of a remarkable number of patients, who may exhibit good neurologic outcomes using the available therapies for these disorders.