RESUMEN
The expansion of national newborn screening (NBS) programmes has provided significant benefits in the diagnosis and early treatment of several rare, heritable conditions, preventing adverse health outcomes for most affected infants. New technological developments have enabled the implementation of testing panel covering over 50 disorders. Consequently, the increment of false positive rate has led to a high number of healthy infants recalled for expensive and often invasive additional testing, opening a debate about the harm-benefit ratio of the expanded newborn screening. The false-positive rate represents a challenge for healthcare providers working in NBS systems. Here, we give an overview on the most commonly used strategies for decreasing the adverse effects due to inconclusive screening results. The focus is on NBS performance improvement through the implementation of analytical methods, the application of new and more informative biomarkers, and by using post-analytical interpretive tools. These strategies, used as part of the NBS process, can to enhance the positive predictive value of the test and reduce the parental anxiety and healthcare costs related to the unnecessary tests and procedures.
RESUMEN
Tandem mass spectrometry (MS/MS) has become a leading technology used in clinical chemistry and has shown to be particularly sensitive and specific when used in newborn screening (NBS) tests. The success of tandem mass spectrometry is due to important advances in hardware, software and clinical applications during the last 25 years. MS/MS permits a very rapid measurement of many metabolites in different biological specimens by using filter paper spots or directly on biological fluids. Its use in NBS give us the chance to identify possible treatable metabolic disorders even when asymptomatic and the benefits gained by this type of screening is now recognized worldwide. Today the use of MS/MS for second-tier tests and confirmatory testing is promising especially in the early detection of new disorders such as some lysosomal storage disorders, ADA and PNP SCIDs, X-adrenoleucodistrophy (X-ALD), Wilson disease, guanidinoacetate methyltransferase deficiency (GAMT), and Duchenne muscular dystrophy. The new challenge for the future will be reducing the false positive rate by using second-tier tests, avoiding false negative results by using new specific biomarkers and introducing new treatable disorders in NBS programs.
Asunto(s)
Espectrometría de Masas/métodos , Tamizaje Neonatal/métodos , Espectrometría de Masas en Tándem/métodos , Adrenoleucodistrofia/diagnóstico , Adrenoleucodistrofia/prevención & control , Anemia de Células Falciformes/diagnóstico , Anemia de Células Falciformes/prevención & control , Guanidinoacetato N-Metiltransferasa/deficiencia , Degeneración Hepatolenticular/diagnóstico , Degeneración Hepatolenticular/prevención & control , Humanos , Recién Nacido , Trastornos del Desarrollo del Lenguaje/diagnóstico , Trastornos del Desarrollo del Lenguaje/prevención & control , Enfermedades por Almacenamiento Lisosomal/diagnóstico , Enfermedades por Almacenamiento Lisosomal/prevención & control , Trastornos del Movimiento/congénito , Trastornos del Movimiento/diagnóstico , Trastornos del Movimiento/prevención & control , Inmunodeficiencia Combinada Grave/diagnóstico , Inmunodeficiencia Combinada Grave/prevención & controlRESUMEN
BACKGROUND: Purine nucleoside phosphorylase (PNP) deficiency has been recently introduced in the newborn screening program in Tuscany. In order to improve the PNP screening efficiency, we developed a 2nd tier test to quantify PNP primary markers deoxyguanosine (dGuo) and deoxyinosine (dIno). METHODS: Dried blood spots (DBS) samples were extracted with 200 µL of methanol and 100 µL of water (by two steps). Internal standards were added at a final concentration of 10 µmol/L. After extraction, samples were analysed by LC-MS/MS. The chromatographic run was performed in gradient mode by using a Synergi Fusion column. RESULTS: The assay was linear over a concentration range of 0.05-50 µmol/L (R2>0.999) for dGuo and 0.5-50 µmol/L (R2>0.998) for dIno. Intra- and interassay imprecision (mean CVs) for dIno and dGuo ranged from 2.9% to 12%. Limit of quantitaion (LOQ) were found to be 0.05 µmol/L and 0.5 µmol/L for dGuo and dIno, respectively. The reference ranges, obtained by measuring dGuo and dIno concentrations on DBS, were close to zero for both biomarkers. Moreover, DBS samples from seven patients with confirmed PNP were retrospectively evaluated and correctly identified. CONCLUSIONS: The LC-MS/MS method can reliably measure dIno and dGuo in DBS for the diagnosis of PNP. Validation data confirm the present method is characterised by good reproducibility, accuracy and imprecision for the quantitation of dIno and dGuo. The assay also appears suitable for use in monitoring treatment of PNP patients.
Asunto(s)
Pruebas con Sangre Seca , Tamizaje Neonatal , Purina-Nucleósido Fosforilasa/deficiencia , Errores Innatos del Metabolismo de la Purina-Pirimidina/sangre , Adulto , Cromatografía Liquida , Humanos , Recién Nacido , Enfermedades de Inmunodeficiencia Primaria , Purina-Nucleósido Fosforilasa/sangre , Purina-Nucleósido Fosforilasa/metabolismo , Errores Innatos del Metabolismo de la Purina-Pirimidina/diagnóstico , Errores Innatos del Metabolismo de la Purina-Pirimidina/metabolismo , Espectrometría de Masas en TándemRESUMEN
Abstract The isodecyl neopentanoate is an ingredient used in the cosmetic industry to prepare a nipple fissure balm. We report on 12 newborns that showed elevated C5-acylcarnitine levels upon newborn screening following treatment with balm. The first 3 neonates were immediately recalled for confirmatory tests and resulted negative for both isovaleric acidemia and short/branched chain acyl-CoA dehydrogenase deficiency. In the other 9 cases, the immediate recall was avoided by applying a new second-tier test able to confirm the presence of pivaloylcarnitine. The concentration of C5-acylcarnitine was measured in the days following the suspension of balm application. Abnormal concentrations of C5-acylcarnitine did not seem to be associated with free carnitine deficiency, probably due to the short time of exposure. A direct correlation between balm ingestion and the elevation in pivaloylcarnitine has been demonstrated in 10 adult volunteers. The commercial balm containing a pivalic acid derivative is causal of false-positive results during newborn screening, and it could have the potential to cause secondary carnitine deficiency when used chronically.
RESUMEN
BACKGROUND: 3-Hydroxypalmitoleoyl-carnitine (C16:1-OH) has recently been reported to be elevated in acylcarnitine profiles of patients with propionic acidemia (PA) or methylmalonic acidemia (MMA) during expanded newborn screening (NBS). High levels of C16:1-OH, combined with other hydroxylated long chain acylcarnitines are related to long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHADD) and trifunctional protein (TFP) deficiency. METHODS: The acylcarnitine profile of two LCHADD patients was evaluated using liquid chromatography-tandem mass spectrometric method. A specific retention time was determined for each hydroxylated long chain acylcarnitine. The same method was applied to some neonatal dried blood spots (DBSs) from PA and MMA patients presenting abnormal C16:1-OH concentrations. RESULTS: The retention time of the peak corresponding to C16:1-OH in LCHADD patients differed from those in MMA and PA patients. Heptadecanoylcarnitine (C17) has been identified as the novel biomarker specific for PA and MMA patients through high resolution mass spectrometry (Orbitrap) experiments. We found that 21 out of 23 neonates (22 MMA, and 1PA) diagnosed through the Tuscany region NBS program exhibited significantly higher levels of C17 compared to controls. Twenty-three maternal deficiency (21 vitamin B12 deficiency, 1 homocystinuria and 1 gastrin deficiency) samples and 82 false positive for elevated propionylcarnitine (C3) were also analyzed. CONCLUSIONS: We have characterized a novel biomarker able to detect propionate disorders during expanded newborn screening (NBS). The use of this new biomarker may improve the analytical performances of NBS programs especially in laboratories where second tier tests are not performed.
Asunto(s)
Errores Innatos del Metabolismo de los Aminoácidos/sangre , Errores Innatos del Metabolismo de los Aminoácidos/diagnóstico , Carnitina/análogos & derivados , Carnitina/sangre , Tamizaje Neonatal , Acidemia Propiónica/sangre , Acidemia Propiónica/diagnóstico , Biomarcadores/sangre , Humanos , Recién Nacido , Estudios RetrospectivosRESUMEN
Carbamazepine (CBZ) is a first-line drug for the treatment of different forms of epilepsy and the first choice drug for trigeminal neuralgia. CBZ is metabolized in the liver by oxidation into carbamazepine-10,11-epoxide (CBZE), its major metabolite which is equipotent and known to contribute to the pharmacological activity of CBZ. The aim of the present study was to develop and validate a reliable, selective and sensitive liquid chromatography-tandem mass spectrometry method for the simultaneous quantification of CBZ and its active metabolite in dried blood spots (DBS). The extraction process was carried out from DBS using methanol-water-formic acid (80:20:0.1, v/v/v). Chromatographic elution was achieved by using a linear gradient with a mobile phase consisting of acetonitrile-water-0.1% formic acid at a flow rate of 0.50mL/min. The method was linear over the range 1-40mg/L and 0.25-20mg/L for CBZ and CBZE, respectively. The limit of quantification was 0.75mg/L and 0.25mg/L for CBZ and CBZE. Intra-day and inter-day assay precisions were found to be lower than 5.13%, 6.46% and 11.76%, 4.72% with mean percentage accuracies of 102.1%, 97.5% and 99.2%, 97.8% for CBZ and CBZE. We successfully applied the method for determining DBS finger-prick samples in paediatric patients and confirmed the results with concentrations measured in matched plasma samples. This novel approach allows quantification of CBZ and its metabolite from only one 3.2mm DBS disc by LC-MS/MS thus combining advantages of DBS technique and LC-MS/MS in clinical practice.
Asunto(s)
Anticonvulsivantes/sangre , Carbamazepina/sangre , Monitoreo de Drogas/métodos , Adolescente , Biotransformación , Calibración , Niño , Preescolar , Cromatografía Líquida de Alta Presión , Femenino , Hematócrito , Humanos , Lactante , Límite de Detección , Masculino , Estándares de Referencia , Reproducibilidad de los Resultados , Manejo de Especímenes , Espectrometría de Masas en TándemRESUMEN
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.
Asunto(s)
Biomarcadores/sangre , Biomarcadores/orina , Errores Innatos del Metabolismo/diagnóstico , Metabolómica/métodos , Tamizaje Neonatal/métodos , Femenino , Cromatografía de Gases y Espectrometría de Masas , Humanos , Recién Nacido , MasculinoRESUMEN
Phenytoin (PHT) is one of the most commonly used anticonvulsant drugs for the treatment of epilepsy and bipolar disorders. The large amount of plasma required by conventional methods for drug quantification makes mass spectrometry combined with dried blood spot (DBS) sampling crucial for pediatric patients where therapeutic drug monitoring or pharmacokinetic studies may be difficult to realize. DBS represents a new convenient sampling support requiring minimally invasive blood drawing and providing long-term stability of samples and less expensive shipment and storage. The aim of this study was to develop a LC-MS/MS method for the quantification of PHT on DBS. This analytical method was validated and gave good linearity (r(2)=0.999) in the range of 0-100mg/l. LOQ and LOD were 1.0mg/l and 0.3mg/l, respectively. The drug extraction from paper was performed in a few minutes using a mixture composed of organic solvent for 80%. The recovery ranged from 85 to 90%; PHT in DBS showed to be stable at different storage temperatures for one month. A good correlation was also obtained between PHT plasma and DBS concentrations. This method is both precise and accurate and appears to be particularly suitable to monitor treatment with a simple and convenient sample collection procedure.
Asunto(s)
Cromatografía Liquida/métodos , Pruebas con Sangre Seca/métodos , Monitoreo de Drogas/métodos , Fenitoína/sangre , Espectrometría de Masas en Tándem/métodos , Calibración , Estabilidad de Medicamentos , Humanos , Límite de Detección , Modelos Lineales , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Relación Señal-RuidoRESUMEN
Enzyme replacement therapy is currently the only approved treatment for Pompe disease, due to acid α-glucosidase deficiency. Clinical efficacy of this approach is variable, and more effective therapies are needed. We showed in preclinical studies that chaperones stabilize the recombinant enzyme used for enzyme replacement therapy. Here, we evaluated the effects of a combination of enzyme therapy and a chaperone on α-glucosidase activity in Pompe disease patients. α-Glucosidase activity was analyzed by tandem-mass spectrometry in dried blood spots from patients treated with enzyme replacement therapy, either alone or in combination with the chaperone N-butyldeoxynojirimycin given at the time of the enzyme infusion. Thirteen patients with different presentations (3 infantile-onset, 10 late-onset) were enrolled. In 11 patients, the combination treatment resulted in α-glucosidase activities greater than 1.85-fold the activities with enzyme replacement therapy alone. In the whole patient population, α-glucosidase activity was significantly increased at 12 hours (2.19-fold, P = 0.002), 24 hours (6.07-fold, P = 0.001), and 36 hours (3.95-fold, P = 0.003). The areas under the curve were also significantly increased (6.78-fold, P = 0.002). These results suggest improved stability of recombinant α-glucosidase in blood in the presence of the chaperone.
Asunto(s)
1-Desoxinojirimicina/análogos & derivados , Enfermedad del Almacenamiento de Glucógeno Tipo II/sangre , Enfermedad del Almacenamiento de Glucógeno Tipo II/tratamiento farmacológico , Inhibidores de Glicósido Hidrolasas/farmacología , alfa-Glucosidasas/farmacología , 1-Desoxinojirimicina/farmacología , Adolescente , Adulto , Animales , Niño , Preescolar , Modelos Animales de Enfermedad , Pruebas con Sangre Seca , Sinergismo Farmacológico , Terapia de Reemplazo Enzimático/métodos , Estabilidad de Enzimas , Femenino , Humanos , Masculino , Ratones , Persona de Mediana Edad , Adulto Joven , alfa-Glucosidasas/sangre , alfa-Glucosidasas/uso terapéuticoRESUMEN
BACKGROUND: Purine nucleoside phosphorylase (PNP) deficiency is a rare form of autosomal recessive combined primary immunodeficiency caused by a enzyme defect leading to the accumulation of inosine, 2'-deoxy-inosine (dIno), guanosine, and 2'-deoxy-guanosine (dGuo) in all cells, especially lymphocytes. Treatments are available and curative for PNP deficiency, but their efficacy depends on the early approach. PNP-combined immunodeficiency complies with the criteria for inclusion in a newborn screening program. OBJECTIVE: This study evaluate whether mass spectrometry can identify metabolite abnormalities in dried blood spots (DBSs) from affected patients, with the final goal of individuating the disease at birth during routine newborn screening. METHODS: DBS samples from 9 patients with genetically confirmed PNP-combined immunodeficiency, 10,000 DBS samples from healthy newborns, and 240 DBSs from healthy donors of different age ranges were examined. Inosine, dIno, guanosine, and dGuo were tested by using tandem mass spectrometry (TMS). T-cell receptor excision circle (TREC) and kappa-deleting recombination excision circle (KREC) levels were evaluated by using quantitative RT-PCR only for the 2 patients (patients 8 and 9) whose neonatal DBSs were available. RESULTS: Mean levels of guanosine, inosine, dGuo, and dIno were 4.4, 133.3, 3.6, and 3.8 µmol/L, respectively, in affected patients. No indeterminate or false-positive results were found. In patient 8 TREC levels were borderline and KREC levels were abnormal; in patient 9 TRECs were undetectable, whereas KREC levels were normal. CONCLUSION: TMS is a valid method for diagnosis of PNP deficiency on DBSs of affected patients at a negligible cost. TMS identifies newborns with PNP deficiency, whereas TREC or KREC measurement alone can fail.
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Síndromes de Inmunodeficiencia/diagnóstico , Mutación , Purina-Nucleósido Fosforilasa/deficiencia , Purina-Nucleósido Fosforilasa/genética , Errores Innatos del Metabolismo de la Purina-Pirimidina/diagnóstico , Adolescente , Preescolar , Reparación del ADN , Desoxiguanosina/análisis , Desoxiguanosina/metabolismo , Pruebas con Sangre Seca , Femenino , Guanosina/análisis , Guanosina/metabolismo , Humanos , Síndromes de Inmunodeficiencia/genética , Síndromes de Inmunodeficiencia/patología , Lactante , Recién Nacido , Inosina/análogos & derivados , Inosina/análisis , Inosina/metabolismo , Linfocitos/patología , Masculino , Tamizaje Neonatal , Enfermedades de Inmunodeficiencia Primaria , Errores Innatos del Metabolismo de la Purina-Pirimidina/genética , Errores Innatos del Metabolismo de la Purina-Pirimidina/patología , Espectrometría de Masas en TándemRESUMEN
Severe combined immunodeficiency due to adenosine-deaminase defect (ADA-SCID) is usually deadly in childhood because of severe recurrent infections. When clinical diagnosis is done, permanent damages due to infections or metabolite accumulation are often present. Gene therapy, bone marrow transplantation or enzyme replacement therapy may be effective if started early. The aim of this study was to set-up a robust method suitable for screening with a minimized preparation process and with inexpensive running costs, for diagnosing ADA-SCID by tandem mass spectrometry. ADA-SCID satisfies all the criteria for inclusion in a newborn screening program. We describe a protocol revised to incorporate adenosine and 2-deoxyadenosine testing into an expanded newborn screening program. We assessed the effectiveness of this approach testing dried blood spots from 4 genetically confirmed early-onset and 5 delayed-onset ADA-SCID patients. Reference values were established on 50,000 healthy newborns (deoxyadenosine <0.09µmol/L, adenosine <1.61µmol/L). We also developed a second tier test to distinguish true positives from false positives and improve the positive predictive value of an initial abnormal result. In the first 18 months, the pilot project has identified a newborn with a genetically confirmed defect in adenosine deaminase (ADA) gene. The results show that the method having great simplicity, low cost and low process preparations can be fully applicable to a mass screening program.
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Adenosina Desaminasa/sangre , Pruebas con Sangre Seca , Tamizaje Neonatal/métodos , Inmunodeficiencia Combinada Grave/sangre , Espectrometría de Masas en Tándem , Calibración , Humanos , Recién Nacido , Proyectos Piloto , Valor Predictivo de las Pruebas , Valores de Referencia , Reproducibilidad de los Resultados , Estudios Retrospectivos , Inmunodeficiencia Combinada Grave/diagnósticoRESUMEN
Propranolol, a non-selective beta blocker drug, is used in young infants and newborns for treating several heart diseases; its pharmacokinetics has been extensively evaluated in adult patients using extrapolation to treat pediatric population. The purpose of the present study was to develop and validate a method to measure propranolol levels in dried blood spots. The analysis was performed by using liquid chromatography/tandem mass spectrometry operating in multiple reaction monitoring mode. The calibration curve in matrix was linear in the concentration range of 2.5-200 µg/L with correlation coefficient r=0.9996. Intra-day and inter-day precisions and biases were less than 8.0% (n=10) and 11.5% (n=10) respectively. The recoveries ranged from 94 to 100% and the matrix effect did not result in a severe signal suppression. Propranolol on dried blood spot showed a good stability at three different temperatures for one month. This paper describes a micromethod for measuring propranolol levels on dried blood spot, which determines a great advantage in neonates or young infants during pharmacokinetic studies because of less invasive sampling and small blood volume required.
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Antagonistas Adrenérgicos beta/sangre , Cromatografía Liquida/métodos , Propranolol/sangre , Espectrometría de Masas en Tándem/métodos , Calibración , Humanos , Reproducibilidad de los ResultadosRESUMEN
BACKGROUND: Adenosine deaminase (ADA)-severe combined immunodeficiency (SCID) is caused by genetic variants that disrupt the function of ADA. In its early-onset form, it is rapidly fatal to infants. Delayed or late-onset ADA-SCID is characterized by insidious progressive immunodeficiency that leads to permanent organ damage or death. Quantification of T-cell receptor excision circles (TRECs) or tandem mass spectrometry (tandem-MS) analysis of dried blood spots (DBSs) collected at birth can identify newborns with early-onset ADA-SCID and are used in screening programs. However, it is not clear whether these analyses can identify newborns who will have delayed or late-onset ADA-SCID before symptoms appear. OBJECTIVE: We performed a retrospective study to evaluate whether tandem-MS and quantitative TREC analyses of DBSs could identify newborns who had delayed-onset ADA-SCID later in life. METHODS: We tested stored DBSs collected at birth from 3 patients with delayed-onset ADA-SCID using tandem-MS (PCT EP2010/070517) to evaluate levels of adenosine and 2'-deoxyadenosine and real-time PCR to quantify TREC levels. We also analyzed DBSs from 3 newborns with early-onset ADA-SCID and 2 healthy newborn carriers of ADA deficiency. RESULTS: The DBSs taken at birth from the 3 patients with delayed-onset ADA-SCID had adenosine levels of 10, 25, and 19 µmol/L (normal value, <1.5 µmol/L) and 2'-deoxyadenosine levels of 0.7, 2.7, and 2.4 µmol/L (normal value, <0.07 µmol/L); the mean levels of adenosine and 2'-deoxyadenosine were respectively 12.0- and 27.6-fold higher than normal values. DBSs taken at birth from all 3 patients with delayed-onset ADA deficiency had normal TREC levels, but TRECs were undetectable in blood samples taken from the same patients at the time of diagnosis. CONCLUSION: Tandem-MS but not TREC quantification identifies newborns with delayed- or late-onset ADA deficiency.
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Adenosina Desaminasa/sangre , Agammaglobulinemia/diagnóstico , Receptores de Antígenos de Linfocitos T/sangre , Inmunodeficiencia Combinada Grave/diagnóstico , Espectrometría de Masas en Tándem , Adenosina Desaminasa/deficiencia , Adenosina Desaminasa/genética , Desoxiadenosinas/metabolismo , Activación Enzimática , Eritrocitos/metabolismo , Humanos , Inmunoglobulinas/sangre , Inmunofenotipificación , Recién Nacido , Subgrupos Linfocitarios/metabolismo , Receptores de Antígenos de Linfocitos T/genética , Estudios RetrospectivosRESUMEN
In recent years, new treatments have become available to treat some lysosomal storage disorders (LSDs) and many studies suggest that there is a benefit with starting therapy early. Newborn screening should detect diseases early enough for prompt treatment. Some countries include additional conditions, such as some LSDs, into their newborn screening panels. Mucopolysaccharidosis Type I (MPS I) is an autosomal recessive disorder caused by the deficiency of α-L-iduronidase (IDUA) activity. Currently, enzyme replacement therapy (ERT) or bone marrow transplantation is available and this has raised a growing interest for the development of a newborn screening test. In 2009, we reported a new fast and simplified tandem mass spectrometry-based method for quantifying five enzyme activities on dried blood spots. Here, we describe the inclusion of IDUA activity determination for the simultaneous detection of six lysosomal storage diseases. We have defined reference normal ranges by testing 680 healthy newborns and 240 adults. The assay was checked through three confirmed MPS I patients whose IDUA activity was below the normal range. Reproducibility of the assays has been established by assessing the intra-day and inter-day assay imprecisions. This quick assay has been devised to be implemented in newborn screening by liquid chromatography tandem mass spectrometry.
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Cromatografía Liquida/métodos , Pruebas con Sangre Seca/métodos , Espectrometría de Masas/métodos , Mucopolisacaridosis I/diagnóstico , Tamizaje Neonatal/métodos , Cromatografía Liquida/normas , Pruebas con Sangre Seca/normas , Humanos , Iduronidasa/análisis , Iduronidasa/sangre , Iduronidasa/química , Recién Nacido , Espectrometría de Masas/normas , Reproducibilidad de los ResultadosRESUMEN
Linezolid is a new drug from the oxazolidinone class of antibiotics used against mycobacteria and multi-drug resistant (MDR) Gram-positive bacterial infections, which may are also glycopeptide-resistant. The drug usage in pediatric age needs an accurate drug monitoring for effective patient management. The aim of this study was to evaluate the use of dried blood spot (DBS) specimens to determinate linezolid levels during treatment. Advantages of DBS include short collection time, low invasiveness, ease and low cost of sample collection, transport and storage. The analysis was performed in LC-MS/MS operating in positive ion mode and multiple reaction monitoring (MRM) mode. The calibration curve in matrix was linear in the concentration range of 1-100 mg/L with correlation coefficient value of 0.9987. Intraday and interday coefficients of variation were within 3.6% and 13.0%, respectively. We also tested the thermal and temporal drug stability in dried blood spots at four different temperatures to evaluate the risks of sample delivery in different conditions. The short term stability studies showed that linezolid concentration remained stable for at least one month under all the conditions tested. This new assay has favorable characteristics being highly precise and accurate and allows a fast linezolid analysis with a total run time 22 min long, in gradient analysis. Concentration data for plasma and DBS samples from patients after treatment were compared showing a good correlation. Correlation between DBS data and serum samples measured by HPLC-UV was satisfactory. The benefit for patients is the ability to monitor the treatment with a simple and convenient sample collection at home.
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Acetamidas/sangre , Antiinfecciosos/sangre , Cromatografía Liquida/métodos , Oxazolidinonas/sangre , Espectrometría de Masas en Tándem/métodos , Calibración , Límite de Detección , LinezolidRESUMEN
Phenylketonuria (PKU) is one of the most common inborn errors of metabolism and is due to a deficit of phenylalanine hydroxylase, the enzyme that converts phenylalanine (Phe) into tyrosine (Tyr). The resultant hyperphenylalaninemia (HPA) leads to severe neurological impairment, whose pathogenesis has not been entirely elucidated. Treatment of PKU consists essentially in lifelong protein restriction and, in mild cases, in tetrahydrobiopterin supplementation. However, compliance to both strategies, particularly to the long-term diet, is low and therefore other therapies are desirable. We explored a gene therapy approach aimed at long-term correction of the pathologic phenotype of BTBR-PahEnu2 mice, a mouse model of PKU. To this aim, we developed a helper-dependent adenoviral (HD-Ad) vector expressing phenylalanine hydroxylase and administered it to 3-week-old PKU mice. This resulted in complete normalization of Phe and Tyr levels and reversal of coat hypopigmentation that lasted throughout the observation period of six months. The spatial learning deficits observed in PKU mice were also reversed and hippocampus levels of the N-methyl-D-Aspartate and 2-amino-3-(5-methyl-3-oxo-1,2- oxazol-4-yl) propanoic acid receptor subunits returned to normal. Long-term potentiation, which is impaired in PKU mice, was also restored by treatment. Therefore, HD-Ad vector-mediated gene therapy is a promising approach to PKU treatment.
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Terapia Genética , Fenilalanina Hidroxilasa/genética , Fenilalanina Hidroxilasa/metabolismo , Fenilcetonurias/genética , Fenilcetonurias/metabolismo , Fenilcetonurias/terapia , Adenoviridae/genética , Animales , Modelos Animales de Enfermedad , Electrofisiología/métodos , Vectores Genéticos , Humanos , Discapacidades para el Aprendizaje/genética , Discapacidades para el Aprendizaje/terapia , Ratones , N-Metilaspartato/genética , N-Metilaspartato/metabolismo , Fenilalanina/sangre , Pigmentación/genética , Tirosina/sangreRESUMEN
Ertapenem (Invanz) is a newly developed carbapenem ß-lactam antimicrobial agent. The drug usage in pediatric age needs an accurate drug monitoring for effective patient management. The aim of this study was to evaluate the use of dried blood spot (DBS) specimens to measure ertapenem concentration during treatment. The analysis was performed by UPLC-MS/MS operating in multiple reaction monitoring (MRM) mode. The calibration curve in matrix was linear in the concentration range of 0.5-100 mg/L with correlation coefficient value higher than 0.997. Performance parameters of this method like lower limit of detection (LLOD, 0.2 mg/L), lower limit of quantification (LLOQ, 0.5 mg/L), matrix effect (20%), intra- and inter-day imprecision (CV within than 15%) and accuracy (between 94 and 155%) of drug concentrations have been evaluated. The drug stability at different temperatures was tested for one month, to evaluate the risks of sample delivery at different climatic conditions. The reported method allows now ertapenem analysis and offers many advantages for patients including the possibility of collecting samples at home. This new assay is both precise and accurate and is especially suitable for therapeutic drug monitoring and pharmacokinetic studies in neonates in whom obtaining larger blood samples is not convenient or possible.
Asunto(s)
Antibacterianos/sangre , Pruebas con Sangre Seca/métodos , Espectrometría de Masas en Tándem/métodos , beta-Lactamas/sangre , Antibacterianos/química , Recolección de Muestras de Sangre/métodos , Pruebas con Sangre Seca/normas , Ertapenem , Humanos , Espectrometría de Masas/métodos , Espectrometría de Masas/normas , Espectrometría de Masa por Ionización de Electrospray/métodos , Espectrometría de Masa por Ionización de Electrospray/normas , Espectrometría de Masas en Tándem/normas , beta-Lactamas/químicaRESUMEN
The analysis of urinary acylglycines is an important biochemical tool for the diagnosis of many organic acidemias and mitochondrial fatty acid ß-oxidation defects. A new rapid analytical method has been developed for quantification of acylglycines in urine by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). The method requires a simple sample preparation avoiding derivatization. It has high sensitivity, specificity, and throughput capability, and it requires minimal instrument maintenance. The use of chromatographic separation allows us to identify and quantify isomeric compounds that cannot be solved by appropriate multiple reaction monitoring (MRM) transitions. Urinary concentrations of the different acylglycines were determined using deuterated internal standards. The reference interval for the various metabolites was established using 120 healthy controls. The diagnostic usefulness of the method was demonstrated in three patients with propionic acidemia (PA), one patient with isovaleric acidemia (IVA), two patients with beta ketothiolase deficiency (BKTD), one patient with short branched chain amino acid deficiency (SBCAD), four patients with medium chain acyl-coenzyme A dehydrogenase deficiency (MCADD), one patient with isobutyryl-coenzyme A dehydrogenase deficiency (IBDHD), and one patient with multiple acyl-coenzyme A dehydrogenase deficiency (MADD).
Asunto(s)
Cromatografía Liquida/métodos , Glicina/orina , Errores Innatos del Metabolismo/diagnóstico , Errores Innatos del Metabolismo/orina , Espectrometría de Masas en Tándem/métodos , Adolescente , Estudios de Casos y Controles , Niño , Preescolar , Humanos , Lactante , Recién Nacido , Termodinámica , Factores de TiempoRESUMEN
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.