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3-Methylcrotonylglycinuria is an organic aciduria resulting from deficiency of 3-methylcrotonyl-CoA carboxylase (3-MCC), a biotin-dependent mitochondrial enzym carboxylating 3-methylcrotonyl-CoA to 3-methylglutaconyl-CoA during leucine catabolism. Its deficiency, due to mutations on MCCC1 and MCCC2 genes, leads to accumulation of 3-methylcrotonyl-CoA metabolites in blood and/or urine, primarily 3-hydroxyisovaleryl-carnitine (C5-OH) in plasma and 3-methylcrotonyl-glycine (3-MCG) and 3-hydroxyisovaleric acid (3-HIVA) in the urine. The phenotype of 3-MCC deficiency is highly variable, ranging from severe neurological abnormalities and death in infancy to asymptomatic adults. Here we report the biochemical and molecular characterization of an Italian asymptomatic girl, positive for the newborn screening test. Molecular analysis showed two mutations in the MCCC2 gene, an already described missense mutation, c.691A > T (p.I231F), and a novel splicing mutation, c.1150-1G > A. We characterized the expression profile of the splice mutation by functional studies.
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Methylmalonic acidemia with homocystinuria, cobalamin deficiency type C (cblC) (MMACHC) is the most common inborn error of cobalamin metabolism. Despite a multidrug treatment, the long-term follow-up of early-onset patients is often unsatisfactory, with progression of neurological and ocular impairment. Here, the in-vivo proteome of control and MMACHC lymphocytes (obtained from patients under standard treatment with OHCbl, betaine, folate and L-carnitine) was quantitatively examined by two dimensional differential in-gel electrophoresis (2D-DIGE) and mass spectrometry. Twenty three proteins were found up-regulated and 38 proteins were down-regulated. Consistent with in vivo studies showing disturbance of glutathione metabolism, a deregulation in proteins involved in cellular detoxification, especially in glutathione metabolism was found. In addition, relevant changes were observed in the expression levels of proteins involved in intracellular trafficking and protein folding, energy metabolism, cytoskeleton organization and assembly. This study demonstrates relevant changes in the proteome profile of circulating lymphocytes isolated from treated cblC patients. Some results confirm previous observations in vivo on fibroblast, thus concluding that some dysregulation is ubiquitous. On the other hand, new findings could be tissue-specific. These observations expand our current understanding of the cblC disease and may ignite new research and therapeutic strategies to treat this disorder.
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Homocistinúria/metabolismo , Proteoma/análise , Deficiência de Vitamina B 12/congênito , Adolescente , Estudos de Casos e Controles , Criança , Pré-Escolar , Eletroforese em Gel Bidimensional , Feminino , Homocistinúria/sangue , Humanos , Lactente , Linfócitos/metabolismo , Linfócitos/patologia , Masculino , Proteômica , Transdução de Sinais , Deficiência de Vitamina B 12/sangue , Deficiência de Vitamina B 12/metabolismoRESUMO
A pilot expanded newborn screening programme to detect inherited metabolic disorders by means of liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) began in the Campania region, southern Italy, in 2007. By October 2009, >8,800 dried blood samples on filter paper from 11 hospitals had been screened. Within this screening programme, we identified a case of mitochondrial acetoacetyl-coenzyme A (CoA) thiolase deficiency [ß-ketothiolase (ß-KT) deficiency] by analysing the acylcarnitine profile from a dried blood spot with LC-MS/MS. Gas chromatography coupled with mass spectrometry analysis of urinary organic acids and LC-MS/MS analysis of urinary acylcarnitines were in line with this disorder. In fact, concentrations were well beyond the cut-off values of tiglyl carnitine, 3-hydroxybutyrylcarnitine and 2-methyl-3-hydroxybutyrylcarnitine, 2-methyl-3-hydroxybutyric acid and tiglyl glycine. The absence of 2-methylacetoacetic acid in urine may be attributed to: (i) the instability of this ß-ketoacid because it undergoes spontaneous decarboxylation to 2-butanone, which is highly volatile and thus difficult to detect, and (ii) the good health of the patient in the first days of life. ß-KT deficiency was subsequently diagnosed in the patient's older sister, who showed increased levels of the same metabolites but also small amounts of 2-methylacetoacetic acid, which is considered a key marker for ß-KT diagnosis. Genomic analysis revealed mutation c.1189C >G in exon 12 of the ACAT1 gene, which results in a severe defect because of the p.H397D amino acid change in both alleles of both patients.
Assuntos
Acetil-CoA C-Aciltransferase/deficiência , Erros Inatos do Metabolismo dos Aminoácidos/diagnóstico , Carnitina/análogos & derivados , Cromatografia Líquida , Teste em Amostras de Sangue Seco , Triagem Neonatal/métodos , Espectrometria de Massas em Tandem , Acetil-CoA C-Acetiltransferase/deficiência , Acetil-CoA C-Acetiltransferase/genética , Acetil-CoA C-Aciltransferase/sangue , Acetil-CoA C-Aciltransferase/genética , Acetil-CoA C-Aciltransferase/urina , Erros Inatos do Metabolismo dos Aminoácidos/sangue , Erros Inatos do Metabolismo dos Aminoácidos/enzimologia , Erros Inatos do Metabolismo dos Aminoácidos/genética , Erros Inatos do Metabolismo dos Aminoácidos/urina , Biomarcadores/sangue , Biomarcadores/urina , Carnitina/sangue , Análise Mutacional de DNA , Predisposição Genética para Doença , Testes Genéticos , Hereditariedade , Humanos , Recém-Nascido , Itália , Mutação , Linhagem , Fenótipo , Valor Preditivo dos TestesRESUMO
Mucopolysaccharidoses are a group of lysosomal storage disorders (LSDs) characterized by the accumulation of glycosaminoglycans (GAGs). Recently, LC-MS/MS has been widely applied in GAGs analysis combined with different sample preparations for cleaving GAGs to disaccharide units. The aim of the present is paper is to present a new method for the simultaneous quantification of urinary dermatan sulfate (DS) and heparan sulfate (HS) by LC-MS/MS, after butanolysis reaction. Chromatographic separation was achieved with a gradient of acetonitrile and water in 0.1% formic acid on a Kinetex Biphenyl analytical column in 21â¯min. Calibration curves ranging from 0.78 to 50⯵g/mL for HS and from 1.56 to 100⯵g/mL for DS were prepared and the coefficient of determination (r2) was higher than 0.99 for both analytes. Intra-day and inter-day imprecisions and the bias for both compounds were <10.0%. Up to now, most analytical procedures for quantifying GAGs have not had a high level of reproducibility among laboratories, despite the availability of various techniques. The adoption of a new protocol incorporating the methods outlined in this paper could significantly improve the quality and reproducibility of MS results. A procedure using simple steps for preparing samples and reagents that are easily available on the market could promote the standardization of analytical procedures and increase the use of these measurements in clinical practice.
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Butanóis/química , Dermatan Sulfato/urina , Heparitina Sulfato/urina , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Cromatografia Líquida , Humanos , Lactente , Recém-Nascido , Pessoa de Meia-Idade , Espectrometria de Massas em Tandem , Adulto JovemRESUMO
New treatment options and improved strategies for Lysosomal Storage Disorders (LSDs) diagnosis on dried blood spot (DBS) have led to the development of several pilot newborn screening programs. Building on a previously published protocol, we devised a new 6-plex assay based on a single DBS punch incubated into a buffer containing a combination of substrates (GAA, GLA, ASM, GALC, ABG and IDUA). This new protocol incorporates a new trapping and clean-up procedure using perfusion chromatography connected on-line with an analytical column for analyte separation, after enzymatic reaction. Results are available after 4.5â¯min. Several incubation times were tested in order to reduce sample preparation times and to improve accuracy and reproducibility, also regarding the quenching of the reaction within the time window of linear product accumulation. The collected data demonstrate that an incubation time of 4â¯h is enough to achieve good reaction efficiency without any impact on sensitivity. The method proved versatile and robust for various instrument configurations. The fast sample preparation and running times allow a high sample throughput; an advantage in newborn screening procedures. This method can also be used for diagnostic purposes, allowing a rapid diagnosis in a few hours.
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Cromatografia Líquida/métodos , Doenças por Armazenamento dos Lisossomos/diagnóstico , Triagem Neonatal/métodos , Espectrometria de Massas em Tandem/métodos , Teste em Amostras de Sangue Seco/métodos , Humanos , Recém-Nascido , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Fatores de TempoRESUMO
This article provides supplementary data for the paper "LC-MS/MS method for simultaneous quantification of heparan sulfate and dermatan sulfate in urine by butanolysis derivatization" (Forni et al., 2018). Several parameters were tested to optimize sample preparation by butanolysis in order to carry out simultaneous quantifications of HS and DS by tandem mass spectrometry. Here we describe step-by-step instructions to perform HS and DS analysis in urine samples using external calibration curves of standards of known concentration. Sample are quantified by interpolation from the calibration curve and reported in µg/mL. Then, HS and DS are normalized to creatinine concentration and reported as mg/g uCr.
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Organic acidurias are inherited metabolic diseases due to the deficiency of an enzyme or a transport protein involved in one of the several cellular metabolic pathways devoted to the catabolism of amino acids, carbohydrates or lipids. These deficiencies result in abnormal accumulation of organic acids in the body and their abnormal excretion in urine. More than 65 organic acidurias have been described; the incidence varies, individually, from 1 out of 10,000 to >1 out of 1000,000 live births. Collectively, their incidence approximates 1 out of 3000 live births. Among these disorders, methyl malonic aciduria, propionic aciduria, maple syrup urine disease and isovaleric aciduria are sometimes referred to as classical organic acidurias. In this review, we focused on the basic GC-MS-based methodologies employed in the diagnosis of classical organic acidurias and provided updated reference values for the most common involved organic acids. We also attempted to provide the most recent updates on the pathogenetic bases of these diseases.
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Ácidos Carboxílicos/urina , Técnicas de Laboratório Clínico/métodos , Testes Diagnósticos de Rotina/métodos , Cromatografia Gasosa-Espectrometria de Massas/métodos , Erros Inatos do Metabolismo/diagnóstico , Erros Inatos do Metabolismo/patologia , Urina/química , Humanos , Incidência , Erros Inatos do Metabolismo/epidemiologiaRESUMO
Organic acidemias (OAs) are inherited metabolic disorders caused by deficiency of enzymatic activities in the catabolism of amino acids, carbohydrates, or lipids. These disorders result in the accumulation of mono-, di-, or tricarboxylic acids, generally referred to as organic acids. The OA outcomes can involve different organs and/or systems. Some OA disorders are easily managed if promptly diagnosed and treated, whereas, in others cases, such as propionate metabolism-related OAs (propionic acidemia, PA; methylmalonic acidemia, MMA), neither diet, vitamin therapy, nor liver transplantation appears to prevent multiorgan impairment. Here, we review the recent developments in dissecting molecular bases of OAs by using integration of mass spectrometry- (MS-) based metabolomic and proteomic strategies. MS-based techniques have facilitated the rapid and economical evaluation of a broad spectrum of metabolites in various body fluids, also collected in small samples, like dried blood spots. This approach has enabled the timely diagnosis of OAs, thereby facilitating early therapeutic intervention. Besides providing an overview of MS-based approaches most frequently used to study the molecular mechanisms underlying OA pathophysiology, we discuss the principal challenges of metabolomic and proteomic applications to OAs.
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Erros Inatos do Metabolismo dos Aminoácidos/metabolismo , Espectrometria de Massas/métodos , Metabolômica/métodos , Proteômica/métodos , Animais , Biomarcadores/metabolismo , HumanosRESUMO
Methylmalonic acidemia (MMA) is a heterogeneous and severe autosomal recessive inborn error of metabolism most commonly caused by the deficient activity of the vitamin B12 dependent enzyme, methylmalonyl-CoA mutase (MUT). The main treatment for MMA patients is the dietary restriction of propiogenic amino acids and carnitine supplementation. Despite treatment, the prognosis for vitamin B12 non-responsive patients remains poor and is associated with neonatal lethality, persistent morbidity and decreased life expectancy. While multi-organ pathology is a feature of MMA, the liver is severely impacted by mitochondrial dysfunction which likely underlies the metabolic instability experienced by the patients. Liver and/or combined liver/kidney transplantation is therefore sometimes performed in severely affected patients. Using liver specimens from donors and MMA patients undergoing elective liver transplantation collected under a dedicated natural history protocol (clinicaltrials.gov: NCT00078078), we employed proteomics to characterize the liver pathology and impaired hepatic metabolism observed in the patients. Pathway analysis revealed perturbations of enzymes involved in energy metabolism, gluconeogenesis and Krebs cycle anaplerosis. Our findings identify new pathophysiologic and therapeutic targets that could be valuable for designing alternative therapies to alleviate clinical manifestations seen in this disorder.
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Erros Inatos do Metabolismo dos Aminoácidos/metabolismo , Fígado/metabolismo , Proteoma/metabolismo , Erros Inatos do Metabolismo dos Aminoácidos/cirurgia , Estudos de Casos e Controles , Pré-Escolar , Feminino , Humanos , Lactente , Transplante de Fígado , Masculino , Redes e Vias Metabólicas , Eletroforese em Gel Diferencial BidimensionalRESUMO
In contrast to the widely accepted consensus of the existence of a single RNA polymerase in bacteria, several actinomycetes have been recently shown to possess two forms of RNA polymerases due the to co-existence of two rpoB paralogs in their genome. However, the biological significance of the rpoB duplication is obscure. In this study we have determined the genome sequence of the lipoglycopeptide antibiotic A40926 producer Nonomuraea gerenzanensis ATCC 39727, an actinomycete with a large genome and two rpoB genes, i.e. rpoB(S) (the wild-type gene) and rpoB(R) (the mutant-type gene). We next analyzed the transcriptional and metabolite profiles in the wild-type gene and in two derivative strains over-expressing either rpoB(R) or a mutated form of this gene to explore the physiological role and biotechnological potential of the "mutant-type" RNA polymerase. We show that rpoB(R) controls antibiotic production and a wide range of metabolic adaptive behaviors in response to environmental pH. This may give interesting perspectives also with regard to biotechnological applications.
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Actinomycetales/genética , Proteínas de Bactérias/genética , RNA Polimerases Dirigidas por DNA/genética , Genoma Bacteriano , Transcriptoma , Actinomycetales/metabolismo , Antibacterianos/biossíntese , Concentração de Íons de Hidrogênio , Mutação , Teicoplanina/análogos & derivados , Teicoplanina/biossínteseRESUMO
A sex-gender approach in laboratory medicine is scarce; furthermore, the influence of sex on acylcarnitines and amino acid levels at birth has not been thoroughly investigated, even if sex impacts on newborn screening. We aimed to establish the influence of sex on amino acids and acylcarnitines levels in male and female newborns. Amino acids and acylcarnitines were analysed in dried blood spots using tandem mass spectrometry in male and female newborns. Data were analysed before and after body weight correction also using principal components analysis. This retrospective analytical study showed that females had small but significantly higher levels of amino acids and the correction for body weight amplified these differences. Acylcarnitines were overall higher in males before body weight correction with the exception of isovalerylcarnitine + methylbutyrylcarnitine (C5), which was significantly higher in females. Body weight correction decreased the sex differences in C5. Principal component analysis showed that both amino acids and acylcarnitines were necessary to describe the model for females, whereas only acylcarnitines were required for males. These metabolomics data underline the importance of including sex as a variable in future investigations of circulating metabolites; the existence of sex differences highlights the need for setting distinct reference values for female and male neonates in metabolite concentration.
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Metaboloma , Metabolômica , Fatores Etários , Aminoácidos/sangue , Carnitina/análogos & derivados , Carnitina/sangue , Teste em Amostras de Sangue Seco , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Metabolômica/métodos , Fatores Sexuais , Espectrometria de Massas em TandemRESUMO
Inborn errors of metabolism are genetic disorders due to impaired activity of enzymes, transporters, or cofactors resulting in accumulation of abnormal metabolites proximal to the metabolic block, lack of essential products or accumulation of by-products. Many of these disorders have serious clinical consequences for affected neonates, and an early diagnosis allows presymptomatic treatment which can prevent severe permanent sequelae and in some cases death. Expanded newborn screening for these diseases is a promising field of targeted metabolomics. Here we report the application, between 2007 and 2014, of this approach to the identification of newborns in southern Italy at risk of developing a potentially fatal disease. The analysis of amino acids and acylcarnitines in dried blood spots by tandem mass spectrometry revealed 24 affected newborns among 45,466 infants evaluated between 48 and 72 hours of life (overall incidence: 1 : 1894). Diagnoses of newborns with elevated metabolites were confirmed by gas chromatography-mass spectrometry, biochemical studies, and genetic analysis. Five infants were diagnosed with medium-chain acyl CoA dehydrogenase deficiency, 1 with methylmalonic acidemia with homocystinuria type CblC, 2 with isolated methylmalonic acidemia, 1 with propionic acidemia, 1 with isovaleric academia, 1 with isobutyryl-CoA dehydrogenase deficiency, 1 with beta ketothiolase deficiency, 1 with short branched chain amino acid deficiency, 1 with 3-methlycrotonyl-CoA carboxylase deficiency, 1 with formimino-transferase cyclodeaminase deficiency, and 1 with cystathionine-beta-synthase deficiency. Seven cases of maternal vitamin B12 deficiency and 1 case of maternal carnitine uptake deficiency were detected. This study supports the widespread application of metabolomic-based newborn screening for these genetic diseases.
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Biomarcadores/sangue , Biomarcadores/urina , Erros Inatos do Metabolismo/diagnóstico , Metabolômica/métodos , Triagem Neonatal/métodos , Feminino , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Recém-Nascido , MasculinoRESUMO
AIM: Considering that the effects of sex and oral contraceptives (OCs) on blood metabolites have been scarcely studied and the fact that protocol designs for clinical trials emphasise the use of contraception for women of childbearing potential, we examined if OCs and sex affect the serum levels of the physiologically relevant amino acids, carnitine and acylcarnitines, using metabolomics approaches. METHODS: Healthy adult men and women were enrolled. They were drug free with the exception of women taking cyclic format OCs (ethinylestradiol + different progestins). OCs-free women were analysed during the follicular phase, and amino acids, free carnitine and acylcarnitines were measured using HPLC or LC-MS/MS, respectively. RESULTS: Men had significantly higher leucine, isoleucine, methionine, asparagine, proline, valine, tyrosine, glutamine+glutamate, glutamate, histidine and citrulline than OCs-free women, while tryptophan was significantly lower in men. OCs use significantly decreased the levels of glycine, proline, histidine, lysine, hydroxyproline and ornithine and increased isoleucine levels when compared with non-user women. OCs use reduced, although not significantly, carnitine levels in women. Total esterified carnitines were higher in untreated women than in OCs users. Globally, the effect of OCs and sex was specific for the individual esterified carnitine. The observed metabolic changes were not attributable to renal or hepatic functions or to differences in body weight. CONCLUSIONS: The assessed parameters were specifically influenced by sex, highlighting the need to have reference values for women and men. The major novelty of this study is the demonstration that OCs specifically change the profiles of serum amino acids and carnitine, which suggests that OCs users and non-users should be represented in clinical trials.
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OBJECTIVES: Besides the inherited form, vitamin B(12) deficiency may be due to diet restrictions or abnormal absorption. The spread of newborn screening programs worldwide has pointed out that non-inherited conditions are mainly secondary to a maternal deficiency. The aim of our work was to study seven cases of acquired vitamin B12 deficiency detected during our newborn screening project. Moreover, we aimed to evaluate vitamin B(12) and related biochemical parameters status on delivering female to verify the consequences on newborns of eventually altered parameters. DESIGN AND METHODS: 35,000 newborns were screened; those showing altered propionyl carnitine (C3) underwent second-tier test for methylmalonic acid (MMA) on dried blood spot (DBS). Subsequently, newborns positive to the presence of MMA on DBS and their respective mothers underwent further tests: serum vitamin B(12), holo-transcobalamin (Holo-TC), folate and homocysteine; newborns were also tested for urinary MMA content. A control study was conducted on 203 females that were tested for the same parameters when admitted to hospital for delivery. RESULTS: Approximately 10% of the examined newborns showed altered C3. Among these, seven cases of acquired vitamin B(12) deficiency were identified (70% of the MMA-positive cases). Moreover, our data show a high frequency of vitamin B(12) deficiency in delivering female (approximately 48% of examined pregnants). CONCLUSIONS: We suggest to monitor vitamin B(12) and Holo-TC until delivery and to reconsider the reference interval of vitamin B(12) for a better identification of cases at risk. Finally, newborns from mothers with low or borderline levels of vitamin B(12) should undergo second-tier test for MMA; in the presence of MMA they should be supplemented with vitamin B(12) to prevent adverse effects related to vitamin B(12) deficiency.
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Triagem Neonatal/métodos , Complicações na Gravidez/diagnóstico , Deficiência de Vitamina B 12/diagnóstico , Vitamina B 12/metabolismo , Carnitina/análogos & derivados , Carnitina/sangue , Feminino , Ácido Fólico/sangue , Homocisteína/sangue , Humanos , Recém-Nascido , Ácido Metilmalônico/sangue , Ácido Metilmalônico/urina , Gravidez , Complicações na Gravidez/sangue , Transcobalaminas/metabolismo , Vitamina B 12/sangue , Deficiência de Vitamina B 12/sangueRESUMO
OBJECTIVES: Patients affected by Phenylketonuria (PKU) require lifelong management based on phenylalanine (Phe) and tyrosine (Tyr) restricted intake or tetrahydrobiopterin (BH4) administration. Frequent monitoring of blood concentration of both amino acids during treatment is the key point for clinicians to achieve the best long-term neuropsychological outcome. RESULTS: The present study develops and validates a rapid and simple method for Phe and Tyr quantization in dried blood spot (DBS) since this specimen has the advantage of being low invasive, easily withdrawn even at home and stable if mail-delivered. The validation studies showed the robustness of the method. CONCLUSIONS: Serum and DBS samples from PKU patients were analyzed and compared, finding a good correlation of Phe and Tyr concentrations between the two different matrixes.