Development and validation of a 2nd tier test for identification of purine nucleoside phosphorylase deficiency patients during expanded newborn screening by liquid chromatography-tandem mass spectrometry.

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.

Expanded newborn screening by mass spectrometry: New tests, future perspectives.

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.

Reticular dysgenesis-associated AK2 protects hematopoietic stem and progenitor cell development from oxidative stress.

Adenylate kinases (AKs) are phosphotransferases that regulate the cellular adenine nucleotide composition and play a critical role in the energy homeostasis of all tissues. The AK2 isoenzyme is expressed in the mitochondrial intermembrane space and is mutated in reticular dysgenesis (RD), a rare form of severe combined immunodeficiency (SCID) in humans. RD is characterized by a maturation arrest in the myeloid and lymphoid lineages, leading to early onset, recurrent, and overwhelming infections. To gain insight into the pathophysiology of RD, we studied the effects of AK2 deficiency using the zebrafish model and induced pluripotent stem cells (iPSCs) derived from fibroblasts of an RD patient. In zebrafish, Ak2 deficiency affected hematopoietic stem and progenitor cell (HSPC) development with increased oxidative stress and apoptosis. AK2-deficient iPSCs recapitulated the characteristic myeloid maturation arrest at the promyelocyte stage and demonstrated an increased AMP/ADP ratio, indicative of an energy-depleted adenine nucleotide profile. Antioxidant treatment rescued the hematopoietic phenotypes in vivo in ak2 mutant zebrafish and restored differentiation of AK2-deficient iPSCs into mature granulocytes. Our results link hematopoietic cell fate in AK2 deficiency to cellular energy depletion and increased oxidative stress. This points to the potential use of antioxidants as a supportive therapeutic modality for patients with RD.

Dried blood spot assay for the quantification of phenytoin using Liquid Chromatography-Mass Spectrometry.

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.

Diagnosis of immunodeficiency caused by a purine nucleoside phosphorylase defect by using tandem mass spectrometry on dried blood spots.

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.

The inclusion of ADA-SCID in expanded newborn screening by tandem mass spectrometry.

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.

Proteinuria impairs podocyte regeneration by sequestering retinoic acid.

In CKD, the risk of kidney failure and death depends on the severity of proteinuria, which correlates with the extent of podocyte loss and glomerular scarring. We investigated whether proteinuria contributes directly to progressive glomerulosclerosis through the suppression of podocyte regeneration and found that individual components of proteinuria exert distinct effects on renal progenitor survival and differentiation toward a podocyte lineage. In particular, albumin prevented podocyte differentiation from human renal progenitors in vitro by sequestering retinoic acid, thus impairing retinoic acid response element (RARE)-mediated transcription of podocyte-specific genes. In mice with Adriamycin nephropathy, a model of human FSGS, blocking endogenous retinoic acid synthesis increased proteinuria and exacerbated glomerulosclerosis. This effect was related to a reduction in podocyte number, as validated through genetic podocyte labeling in NPHS2.Cre;mT/mG transgenic mice. In RARE-lacZ transgenic mice, albuminuria reduced retinoic acid bioavailability and impaired RARE activation in renal progenitors, inhibiting their differentiation into podocytes. Treatment with retinoic acid restored RARE activity and induced the expression of podocyte markers in renal progenitors, decreasing proteinuria and increasing podocyte number, as demonstrated in serial biopsy specimens. These results suggest that albumin loss through the damaged filtration barrier impairs podocyte regeneration by sequestering retinoic acid and promotes the generation of FSGS lesions. Our findings may explain why reducing proteinuria delays CKD progression and provide a biologic rationale for the clinical use of pharmacologic modulators to induce regression of glomerular diseases.

A rapid liquid chromatography tandem mass spectrometry-based method for measuring propranolol on dried blood spots.

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.

Tandem mass spectrometry, but not T-cell receptor excision circle analysis, identifies newborns with late-onset adenosine deaminase deficiency.

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.

Screening of lysosomal storage disorders: application of the online trapping-and-cleanup liquid chromatography/mass spectrometry method for mucopolysaccharidosis I.

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.

Development of an UPLC-MS/MS method for the determination of antibiotic ertapenem on dried blood spots.

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.

Vitamin D3 quantification in a cod liver oil-based supplement.

A reliable, accurate and reproducible method to quantify vitamin D3 (Vit. D3) in oily dietary supplements was developed after three Vit. D3 intoxications were diagnosed as reasonably resulting from a dietary administration of a cod liver oil based supplement. Liquid chromatography coupled to mass spectrometry operating in atmospheric pressure chemical ionization conditions (LC-APCI) and by using a deuterium labelled internal standard resulted to be an effective technique to reach the analytical aim. Due to the complexity of the oily matrix, the new analytical approach required a solid phase extraction step prior to analysis. The amount of Vit. D3 declared on the label of the cod liver oil based supplement for each soft capsule is 1.5µg. Consequently, the method was developed to quantify Vit. D3 amounts in the range 1-5µg/mL. To improve reliability of obtained data, both MS and MS/MS acquisition methods were employed. The method was evaluated by measuring the characteristic parameters such as linearity, precision, accuracy and robustness and cross checked against a certified pharmaceutical preparation. The LC-APCI-MS and MS/MS methods were applied in order to assess the Vit. D3 content in the dietary supplements taken by the intoxicated patients, found about three order of magnitude higher than that declared. The Vit. D3 content of other batches of the same commercial product was found as declared.

Spatially ordered surfactant assemblies in the gas phase: negatively charged bis(2-ethylhexyl)sulfosuccinate-alkaline metal ion aggregates.

The formation and structural features of negatively charged aggregates of sodium bis(2-ethylhexyl)sulfosuccinate (AOTNa) surfactant molecules in the gas phase have been investigated by electrospray ionization mass spectrometry (ESI-MS) and density functional theory calculations. Mainly driven by the interactions of alkali metal ions both with the oxygen atoms of the sulfonate group and with the succinate moiety of the AOT- anion, spatially ordered supramolecular assemblies, characterized by an internal core composed of surfactant counterions and hydrophilic head groups surrounded by the surfactant alkyl chains pointing outwards, are formed. Calculations have shown that surfactant self-organization in the gas phase is energetically favoured, the energy of formation of negatively charged aggregates from isolated AOTNa and AOT- being linearly related to the aggregation number. Information on the chelating properties of AOTNa towards clusters of inorganic salts was achieved by infusion of solutions at various AOTNa/metal salt (NaCl, NaBr, NaI, LiI, KCl, CsI, RbI) ratios in the ESI source of a mass spectrometer. A wide variety of negatively charged AOT-metal aggregates, some of them also incorporating halide (X-) ions, has been observed. Calculations have shown that the capture of a halide anion to give the AOTMX- species is favoured but the energetics of the process depends on the alkali metal and halide types. The use of energy-resolved mass spectrometry has allowed us to evaluate the stability of different complexes and to evaluate the role played by the metal ion. Overall, the present investigation supports the idea that, in the gas phase, mainly driven by electrostatic interactions, surfactant molecules are present as molecular aggregates characterized by a reverse micelle-like organization with an internal core formed by the surfactant counterions and head groups surrounded by the surfactant alkyl chains. These peculiar aggregates are able to incorporate ionic clusters in their hydrophilic core.