Publications by authors named "Michael Gelb"

281 Publications

Exercise-Induced Alterations in Phospholipid Hydrolysis, Airway Surfactant and Eicosanoids and their Role in Airway Hyperresponsiveness in Asthma.

Am J Physiol Lung Cell Mol Physiol 2021 Feb 3. Epub 2021 Feb 3.

Division of Pulmonary & Critical Care, University of Washington.

The mechanisms responsible for driving endogenous airway hyperresponsiveness (AHR) in the form of exercise-induced bronchoconstriction (EIB) are not fully understood. We examined alterations in airway phospholipid hydrolysis, surfactant degradation, and lipid mediator release in relation to AHR severity and changes induced by exercise challenge. Paired induced sputum (n=18) and bronchoalveolar lavage (BAL) fluid (n=11) were obtained before and after exercise challenge in asthmatic subjects. Samples were analyzed for phospholipid structure, surfactant function and levels of eicosanoid and secreted phospholipase A group 10 (sPLA-X). A primary epithelial cell culture model was used to model effects of osmotic stress on sPLA-X. Exercise challenge resulted in increased surfactant degradation, phospholipase activity, and eicosanoid production in sputum samples of all patients. Subjects with EIB had higher levels of surfactant degradation and phospholipase activity in BAL fluid. Higher basal sputum levels of cysteinyl leukotrienes (CysLTs) and prostaglandin D (PGD) were associated with direct AHR and both the post-exercise and absolute change in CysLTs and PGD levels were associated with EIB severity. Surfactant function was either abnormal at baseline or became abnormal after exercise challenge. Baseline levels of sPLA-X in sputum and the absolute change in amount of sPLA-X with exercise were positively correlated with EIB severity. Osmotic stress ex vivo resulted in movement of water and release of sPLA-X to the apical surface. In summary, exercise challenge promotes changes in phospholipid structure and eicosanoid release in asthma, providing two mechanisms that promote bronchoconstriction, particularly in individuals with EIB who have higher basal levels phospholipid turnover.
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http://dx.doi.org/10.1152/ajplung.00546.2020DOI Listing
February 2021

Family Attitudes regarding Newborn Screening for Krabbe Disease: Results from a Survey of Leukodystrophy Registries.

Int J Neonatal Screen 2020 Aug 20;6(3). Epub 2020 Aug 20.

Duke University PBMT and Cellular Therapy Program, Durham, NC 27705, USA.

Newborn screening (NBS) for Krabbe disease (KD) is currently underway in eight states in the USA, and there is continued discussion of whether to implement KD NBS in additional states. Workgroup members sought to survey a large number of families affected by KD. Families in KD and leukodystrophy family registries were contacted to seek their participation in . The 170 respondents are comprised of the following: 138 family members with a KD individual diagnosed after development of symptoms, 20 notified about KD via NBS, and 12 with a KD individual diagnosed through family history of KD. The key results are that all NBS families with an early-infantile KD family member elected to pursue hematopoietic stem cell transplantation therapy. Of the 170 responders, 165 supported the implementation of KD NBS in all states in the USA.
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http://dx.doi.org/10.3390/ijns6030066DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7570074PMC
August 2020

Toward newborn screening of metachromatic leukodystrophy: results from analysis of over 27,000 newborn dried blood spots.

Genet Med 2020 Nov 20. Epub 2020 Nov 20.

Department of Chemistry, University of Washington, Seattle, WA, USA.

Purpose: Metachromatic leukodystrophy (MLD) is a lysosomal storage disorder caused by the deficiency of arylsulfatase A (ARSA), which results in the accumulation of sulfatides. Newborn screening for MLD may be considered in the future as innovative treatments are advancing. We carried out a research study to assess the feasibility of screening MLD using dried blood spots (DBS) from de-identified newborns.

Methods: To minimize the false-positive rate, a two-tier screening algorithm was designed. The primary test was to quantify C16:0-sulfatide in DBS by ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The screening cutoff was established based on the results from 15 MLD newborns to achieve 100% sensitivity. The secondary test was to measure the ARSA activity in DBS from newborns with abnormal C16:0-sulfatide levels. Only newborns that displayed both abnormal C16:0-sulfatide abundance and ARSA activity were considered screen positives.

Results: A total of 27,335 newborns were screened using this two-tier algorithm, and 2 high-risk cases were identified. ARSA gene sequencing identified these two high-risk subjects to be a MLD-affected patient and a heterozygote.

Conclusion: Our study demonstrates that newborn screening for MLD is highly feasible in a real-world scenario with near 100% assay specificity.
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http://dx.doi.org/10.1038/s41436-020-01017-5DOI Listing
November 2020

Zoltan Lukacs Passed Away.

Int J Neonatal Screen 2020 Nov 3;6(4). Epub 2020 Nov 3.

Office of the International Society for Neonatal Screening, Reigerskamp 273, 3607 HP Maarssen, The Netherlands.

It is with great sadness that we have to inform you of the passing on 13 August 2020, of Dr [...].
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http://dx.doi.org/10.3390/ijns6040086DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7711949PMC
November 2020

Evaluation of Multiple Methods for Quantification of Glycosaminoglycan Biomarkers in Newborn Dried Blood Spots from Patients with Severe and Attenuated Mucopolysaccharidosis-I.

Int J Neonatal Screen 2020 Sep 26;6(3):69. Epub 2020 Aug 26.

Department of Chemistry, University of Washington, Seattle, WA 98195, USA;

All newborn screening (NBS) for mucopolysaccharidosis-I (MPS-I) is carried out by the measurement of α-iduronidase (IDUA) enzymatic activity in dried blood spots (DBS). The majority of low enzyme results are due to pseudodeficiencies, and studies from the Mayo Clinic have shown that the false positive rate can be greatly reduced by including a second-tier analysis of glycosaminoglycans (GAGs) in DBS as part of NBS. In the present study, we obtained newborn DBS from 13 patients with severe MPS-I and 2 with attenuated phenotypes. These samples were submitted to four different GAG mass spectrometry analyses in a comparative study: (1) internal disaccharide; (2) endogenous disaccharide; (3) Sensi-Pro; (4) Sensi-Pro Lite (a variation of Sensi-Pro with a simplified workflow). Patients with attenuated MPS-I show less GAG elevation than those with severe disease, and all MPS-I patients were separated from the reference range using all four methods. The minimal differential factor (lowest GAG marker level in MPS-I samples divided by highest level in the reference range of 30 random newborns) was about two for internal disaccharide, Sensi-Pro, and Sensi-Pro Lite methods. The endogenous disaccharide was clearly the best method with a minimal differential of 16-fold. This study supports use of second-tier GAG analysis of newborn DBS, especially the endogenous disaccharide method, as part of NBS to reduce the false positive rate.
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http://dx.doi.org/10.3390/ijns6030069DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7570209PMC
September 2020

Tandem Mass Spectrometry Enzyme Assays for Multiplex Detection of 10-Mucopolysaccharidoses in Dried Blood Spots and Fibroblasts.

Anal Chem 2020 09 13;92(17):11721-11727. Epub 2020 Aug 13.

Department of Chemistry, University of Washington, Seattle, Washington 98195, United States.

The mucopolysaccharidoses (MPSs) are a class of inborn errors of metabolism caused by deficiency of each of the enzymes involved in the lysosomal degradation of mucopolysaccharides. Newborn screening panels worldwide have been recently expanded to include one or more MPS disorders, as treatments are available and are most efficacious if initiated early in life. Here we report the first multiplex assay of 10 enzymatic activities in dried blood spots and fibroblast lysates that allow newborn screening and diagnosis of all MPS disorders except the ultrarare MPS-IX. The assay consists of incubation of enzyme-specific substrates with dried blood spot punches or fibroblast lysate followed by quantification of enzymatic products using liquid chromatography-tandem mass spectrometry (LC-MS/MS) together with internal standards. Assay of all MPS enzymes using fluorimetric or other methods has not been possible. The steps of the LC-MS/MS assay are sufficiently simple and rapid to be used in newborn screening and diagnostic laboratories. Assays showed acceptable precision, and enzymatic activities measured in confirmed MPS samples are well below the reference range.
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http://dx.doi.org/10.1021/acs.analchem.0c01750DOI Listing
September 2020

A Case of Lysosomal Acid Lipase Deficiency Confirmed by Response to Sebelipase Alfa Therapy.

J Pediatr Gastroenterol Nutr 2020 12;71(6):726-730

Department of Pathology, University of California San Francisco, San Francisco, CA.

Lysosomal acid lipase (LAL) deficiency, or cholesterol ester storage disease, is a disorder affecting the breakdown of cholesterol esters and triglycerides within lysosomes. Clinical findings include hepatomegaly, hepatic dysfunction, and dyslipidemia with a wide range of phenotypic variability and age of onset. The available clinical and molecular information of the patient presented herein was consistent with a diagnosis of LAL deficiency, but her LAL activity assay repeatedly showed normal or borderline low results. Her response to enzyme replacement therapy and demonstrable deficiency on a newer specific enzymatic assay ultimately confirmed her diagnosis of LAL deficiency.
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http://dx.doi.org/10.1097/MPG.0000000000002870DOI Listing
December 2020

Toward newborn screening of cerebrotendinous xanthomatosis: results of a biomarker research study using 32,000 newborn dried blood spots.

Genet Med 2020 Oct 10;22(10):1606-1612. Epub 2020 Jun 10.

Department of Chemistry, University of Washington, Seattle, WA, USA.

Purpose: Cerebrotendinous xanthomatosis (CTX) is a treatable hereditary disorder caused by the deficiency of sterol 27-hydroxylase, which is encoded by the CYP27A1 gene. Different newborn screening biomarkers for CTX have been described, including 7α,12α-dihydroxy-4-cholesten-3-one (7α12αC4), 5β-cholestane-3α,7α,12α,25-tetrol glucuronide (GlcA-tetrol), the ratio of GlcA-tetrol to tauro-chenodeoxycholic acid (t-CDCA) (GlcA-tetrol/t-CDCA), and the ratio of tauro-trihydroxycholestanoic acid (t-THCA) to GlcA-tetrol (t-THCA/GlcA-tetrol). We set out to evaluate these screening methods in a research study using over 32,000 newborn dried blood spots (DBS).

Methods: Metabolites were extracted from DBS with methanol containing internal standard, which was then quantified by ultraperformance liquid chromatography tandem mass spectrometry (UPLC-MS/MS).

Results: The measurement of 7α12αC4 was complicated by isobaric interferences and was discontinued. A total of 32,737 newborns were screened based on the GlcA-tetrol concentration in DBS. GlcA-tetrol/t-CDCA and t-THCA/GlcA-tetrol ratios were also calculated. Newborns displaying both elevated GlcA-tetrol and GlcA-tetrol/t-CDCA ratio were considered to be screen positives. The t-THCA/GlcA-tetrol ratio was used to further distinguish CTX screen positives from Zellweger Spectrum Disorder (ZSD) screen positives. Only one newborn displayed both elevated GlcA-tetrol concentration in DBS and a typical CTX biochemical profile. This newborn was interpreted as a CTX-affected patient as CYP27A1 gene sequencing identified two known pathogenic variants.

Conclusion: The results indicate that both GlcA-tetrol and the GlcA-tetrol/t-CDCA ratio are excellent CTX biomarkers suitable for newborn screening. By characterizing the relationship of GlcA-tetrol, t-CDCA, and t-THCA as secondary markers, 100% assay specificity can be achieved.
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http://dx.doi.org/10.1038/s41436-020-0846-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7529987PMC
October 2020

Detection of GM1-gangliosidosis in newborn dried blood spots by enzyme activity and biomarker assays using tandem mass spectrometry.

J Inherit Metab Dis 2021 Jan 26;44(1):264-271. Epub 2020 Aug 26.

Departments of Chemistry, University of Washington, Seattle, Washington, USA.

GM1-gangliosidosis is a rare autosomal recessive lysosomal storage disease caused by deficiency of β-galactosidase (GLB1). Newborn screening (NBS) may be warranted in the near future given the initiation of a number of gene therapy clinical trials. Here, we report a tandem mass spectrometry (MS/MS) enzymatic assay of GLB1 using dried blood spots (DBS), and the demonstration that GLB1 activities in newborn DBS from seven GM1-gangliosidosis patients are well below those measured in random newborn DBS. MS/MS analysis of two glycan biomarkers, dp5 and A2G2, shows high elevation in newborn DBS from GM1-gangliosidosis compared to the levels in the nonaffected reference range.
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http://dx.doi.org/10.1002/jimd.12269DOI Listing
January 2021

Macrophages Expressing GALC Improve Peripheral Krabbe Disease by a Mechanism Independent of Cross-Correction.

Neuron 2020 07 5;107(1):65-81.e9. Epub 2020 May 5.

Hunter James Kelly Research Institute, Departments of Biochemistry and Neurology, Jacobs School of Medicine and Biomedical Sciences, University at Buffalo, Buffalo, NY 14203, USA. Electronic address:

Many therapies for lysosomal storage disorders rely on cross-correction of lysosomal enzymes. In globoid cell leukodystrophy (GLD), mutations in GALC cause psychosine accumulation, inducing demyelination, a neuroinflammatory "globoid" reaction and neurodegeneration. The efficiency of GALC cross-correction in vivo, the role of the GALC substrate galactosylceramide, and the origin of psychosine are poorly understood. Using a novel GLD model, we show that cross-correction does not occur efficiently in vivo and that Galc-deficient Schwann cells autonomously produce psychosine. Furthermore, macrophages require GALC to degrade myelin, as Galc-deficient macrophages are transformed into globoid cells by exposure to galactosylceramide and produce a more severe GLD phenotype. Finally, hematopoietic stem cell transplantation in patients reduces globoid cells in nerves, suggesting that the phagocytic response of healthy macrophages, rather than cross-correction, contributes to the therapeutic effect. Thus, GLD may be caused by at least two mechanisms: psychosine-induced demyelination and secondary neuroinflammation from galactosylceramide storage in macrophages.
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http://dx.doi.org/10.1016/j.neuron.2020.03.031DOI Listing
July 2020

Achieving Congruence among Reference Laboratories for Absolute Abundance Measurement of Analytes for Rare Diseases: Psychosine for Diagnosis and Prognosis of Krabbe Disease.

Int J Neonatal Screen 2020 Jun 31;6(2). Epub 2020 Mar 31.

Departments of Chemistry and Biochemistry, University of Washington, Seattle, WA 98195, USA.

Measurement of the absolute concentration of the biomarker psychosine in dried blood spots (DBS) is useful for diagnosis and prognosis of Krabbe disease and to support newborn screening of this leukodystrophy. As for assays for more common diseases, it is important to achieve congruence when multiple clinical laboratories provide testing. Four clinical laboratories, one research laboratory, and a commercial vendor collaborated with the goal to achieve congruence in quantitative psychosine measurement in DBS. A set of DBS calibrators was prepared by a single vendor and used in each reference laboratory to make a standard curve of measured psychosine in DBS versus the stated calibrator psychosine level. Congruence between the participating five laboratories was achieved using the psychosine DBS calibrators. This allowed application of disease-specific reference ranges obtained by the reference laboratory with the most extensive data by the other reference laboratories. Congruence between multiple reference laboratories in the measurement of the absolute concentration of biomarkers in DBS (and by extension other samples) is possible by the use of a common set of DBS calibrators.
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http://dx.doi.org/10.3390/ijns6020029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7199469PMC
June 2020

Secreted Phospholipase A Group X Acts as an Adjuvant for Type 2 Inflammation, Leading to an Allergen-Specific Immune Response in the Lung.

J Immunol 2020 06 27;204(12):3097-3107. Epub 2020 Apr 27.

Division of Pulmonary, Critical Care and Sleep, Department of Medicine, University of Washington, Seattle, WA 98109;

Secreted phospholipase A (sPLA) enzymes release free fatty acids, including arachidonic acid, and generate lysophospholipids from phospholipids, including membrane phospholipids from cells and bacteria and surfactant phospholipids. We have shown that an endogenous enzyme sPLA group X (sPLA-X) is elevated in the airways of asthmatics and that mice lacking the sPLA-X gene () display attenuated airway hyperresponsiveness, innate and adaptive immune responses, and type 2 cytokine production in a model of airway sensitization and challenge using a complete allergen that induces endogenous adjuvant activity. This complete allergen also induces the expression of sPLA-X/ In the periphery, an sPLA found in bee venom (bee venom PLA) administered with the incomplete Ag OVA leads to an Ag-specific immune response. In this study, we demonstrate that both bee venom PLA and murine sPLA-X have adjuvant activity, leading to a type 2 immune response in the lung with features of airway hyperresponsiveness and Ag-specific type 2 airway inflammation following peripheral sensitization and subsequent airway challenge with OVA. Further, the adjuvant effects of sPLA-X that result in the type 2-biased OVA-specific adaptive immune response in the lung were dependent upon the catalytic activity of the enzyme, as a catalytically inactive mutant form of sPLA-X does not elicit the adaptive component of the immune response, although other components of the immune response were induced by the inactive enzyme, suggesting receptor-mediated effects. Our results demonstrate that exogenous and endogenous sPLAs play an important role in peripheral sensitization, resulting in airway responses to inhaled Ags.
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http://dx.doi.org/10.4049/jimmunol.2000102DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7375513PMC
June 2020

A highly multiplexed biochemical assay for analytes in dried blood spots: application to newborn screening and diagnosis of lysosomal storage disorders and other inborn errors of metabolism.

Genet Med 2020 Jul 20;22(7):1262-1268. Epub 2020 Apr 20.

Department of Chemistry, University of Washington, Seattle, WA, USA.

Purpose: To develop a multiplexed assay for the newborn screening of lysosomal storage disorders and additional inborn errors in a flexible, comprehensive, and affordable manner to keep up with the expansion of the newborn screening panel.

Methods: Ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was chosen as the detection platform for its superiority compared to traditional flow-injection MS/MS.

Results: A high-throughput, 18-plex UPLC-MS/MS assay was developed for screening purposes with a sample turnaround time of 2.7 minutes. The assay was consolidated such that only four dried blood spot punches were required, and it displayed good precision and reproducibility.

Conclusion: We report a highly multiplexed UPLC-MS/MS assay that is appropriate for the newborn screening of 15 lysosomal storage diseases and 3 additional inborn errors. It can be further expanded to include additional conditions for which presymptomatic diagnosis may facilitate optimum treatment outcome.
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http://dx.doi.org/10.1038/s41436-020-0790-9DOI Listing
July 2020

Validation of Liquid Chromatography-Tandem Mass Spectrometry-Based 5-Plex Assay for Mucopolysaccharidoses.

Int J Mol Sci 2020 Mar 16;21(6). Epub 2020 Mar 16.

Department of Pediatrics, Shimane University Faculty of Medicine, Izumo 693-8501, Japan.

Mucopolysaccharidoses (MPSs) are rare lysosomal storage diseases caused by the accumulation of undegraded glycosaminoglycans in cells and tissues. The effectiveness of early intervention for MPS has been reported. Multiple-assay formats using tandem mass spectrometry have been developed. Here, we developed a method for simultaneous preparation and better measurement of the activities of five enzymes involved in MPSs, i.e., MPS I, MPS II, MPS IIIB, MPS IVA, and MPS VI, which were validated using 672 dried blood spot samples obtained from healthy newborns and 23 patients with MPS. The mean values of the enzyme activities and standard deviations in controls were as follows: α-iduronidase (IDUA), 4.19 ± 1.53 µM/h; iduronate-2-sulfatase (I2S), 8.39 ± 2.82 µM/h; -acetyl-α-glucosaminidase (NAGLU), 1.96 ± 0.57 µM/h; -acetylgalactosamine-6-sulfatase (GALNS), 0.50 ± 0.20 µM/h; and -acetylgalactosamine-4-sulfatase (ARSB), 2.64 ± 1.01 µM/h. All patients displayed absent or low enzyme activity. In MPS I, IIIB, and VI, each patient group was clearly separated from controls, whereas there was some overlap between the control and patient groups in MPS II and IVA, suggesting the occurrence of pseudo-deficiencies. Thus, we established a multiplex assay for newborn screening using liquid chromatography tandem mass spectrometry, allowing simultaneous pretreatment and measurement of five enzymes relevant to MPSs.
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http://dx.doi.org/10.3390/ijms21062025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7139616PMC
March 2020

The critical role of psychosine in screening, diagnosis, and monitoring of Krabbe disease.

Genet Med 2020 Jun 24;22(6):1108-1118. Epub 2020 Feb 24.

Biochemical Genetics Laboratory, Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, USA.

Purpose: Newborn screening (NBS) for Krabbe disease (KD) is performed by measurement of galactocerebrosidase (GALC) activity as the primary test. This revealed that GALC activity has poor specificity for KD. Psychosine (PSY) was proposed as a disease marker useful to reduce the false positive rate for NBS and for disease monitoring. We report a highly sensitive PSY assay that allows identification of KD patients with minimal PSY elevations.

Methods: PSY was extracted from dried blood spots or erythrocytes with methanol containing d-PSY as internal standard, and measured by liquid chromatography-tandem mass spectrometry.

Results: Analysis of PSY in samples from controls (N = 209), GALC pseudodeficiency carriers (N = 55), GALC pathogenic variant carriers (N = 27), patients with infantile KD (N = 26), and patients with late-onset KD (N = 11) allowed for the development of an effective laboratory screening and diagnostic algorithm. Additional longitudinal measurements were used to track therapeutic efficacy of hematopoietic stem cell transplantion (HSCT).

Conclusion: This study supports PSY quantitation as a critical component of NBS for KD. It helps to differentiate infantile from later onset KD variants, as well as from GALC variant and pseudodeficiency carriers. Additionally, this study provides further data that PSY measurement can be useful to monitor KD progression before and after treatment.
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http://dx.doi.org/10.1038/s41436-020-0764-yDOI Listing
June 2020

Phenotypic Drug Discovery for Human African Trypanosomiasis: A Powerful Approach.

Trop Med Infect Dis 2020 Feb 5;5(1). Epub 2020 Feb 5.

Department of Chemistry, University of Washington, Seattle, WA 98195, USA.

The work began with the screening of a library of 700,000 small molecules for inhibitors of growth (a phenotypic screen). The resulting set of 1035 hit compounds was reviewed by a team of medicinal chemists, leading to the nomination of 17 chemically distinct scaffolds for further investigation. The first triage step was the assessment for brain permeability (looking for brain levels at least 20% of plasma levels) in order to optimize the chances of developing candidates for treating late-stage human African trypanosomiasis. Eleven scaffolds subsequently underwent hit-to-lead optimization using standard medicinal chemistry approaches. Over a period of six years in an academic setting, 1539 analogs to the 11 scaffolds were synthesized. Eight scaffolds were discontinued either due to insufficient improvement in antiparasitic activity (5), poor pharmacokinetic properties (2), or a slow (static) antiparasitic activity (1). Three scaffolds were optimized to the point of curing the acute and/or chronic infection model in mice. The progress was accomplished without knowledge of the mechanism of action (MOA) for the compounds, although the MOA has been discovered in the interim for one compound series. Studies on the safety and toxicity of the compounds are planned to help select candidates for potential clinical development. This research demonstrates the power of the phenotypic drug discovery approach for neglected tropical diseases.
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http://dx.doi.org/10.3390/tropicalmed5010023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7157241PMC
February 2020

Newborn screening for Morquio disease and other lysosomal storage diseases: results from the 8-plex assay for 70,000 newborns.

Orphanet J Rare Dis 2020 02 3;15(1):38. Epub 2020 Feb 3.

Department of Medical Genetics, National Taiwan University Hospital, Taipei, Taiwan.

Background: The necessity of early treatment for lysosomal storage diseases (LSDs) has triggered the development of newborn screening for LSDs in recent years. Here we report the first 70,000 newborns screened for Mucopolysaccharidosis (MPS) type 4A (Morquio syndrome) and other LSDs by an 8-plex assay including the original 4-plex LSD screening tandem mass spectrometry (MS/MS) assay for Pompe disease, Fabry disease, Gaucher disease, and MPS I disease.

Methods: The additional reaction for MPS II, MPS 3B, MPS 4A, and MPS 6 enzymes was performed separately from the 4-plex reaction. The two reactions were quenched and extracted, then combined before carrying out a single 2-min UPLC-MS/MS analysis.

Results: From Mar. 2018 to Apr. 2019, 73,743 newborns were screened with the 8-plex LSD screening assay. The 8-plex assay revealed a better analytical precision than the previous 4-plex assay possibly because the 8-plex was carried out using UPLC-MS/MS. Six newborns were found to have low MPS-4A enzyme (N-acetylgalactosamine-6-sulfatase) activity and biallelic GALNS pathogenic mutations in trans; these patients are presumably affected with MPS4A, making an incidence of one in 12,291 (95% confident interval (CI): 5633-26,817). One mutation, c.857C > T (p.T286 M) of the GALNS gene, accounted 5 of the 12 mutated alleles. These newborns had immature vertebral bodies at 1 month of age, and one case was treated with elosulfase alfa 2 mg/kg/week starting from 4 months of age. Among other MPSs screened, one case of MPS I, 3 cases of MPS II, and 3 cases of MPS 3B were detected. One case of mucolipidosis type III was also diagnosed. In conjunction with another 9 patients of Pompe disease, Gaucher disease, and classical Fabry disease, making an incidence of LSDs as one in 3206 newborns (95% CI: 2137 - 4811). The one with infantile-onset Pompe disease and the one with Gaucher disease were treated since the age of 8 days and 41 days respectively.

Conclusions: Routine newborn screening of MPS 4A and other LSDs were made possible by the 8-plex LSD screening assay. However, detailed phenotype prediction and the time to start treatment will need further elucidation.
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http://dx.doi.org/10.1186/s13023-020-1322-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6998831PMC
February 2020

Leukocyte and Dried Blood Spot Arylsulfatase A Assay by Tandem Mass Spectrometry.

Anal Chem 2020 05 16;92(9):6341-6348. Epub 2020 Apr 16.

Department of Chemistry, University of Washington, Seattle, Washington 98195, United States.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays were developed to measure arylsulfatase A (ARSA) activity in leukocytes and dried blood spots (DBS) using deuterated natural sulfatide substrate. These new assays were highly specific and sensitive. Patients with metachromatic leukodystrophy (MLD) and multiple sulfatase deficiency (MSD) displayed a clear deficit in the enzymatic activity and could be completely distinguished from normal controls. The leukocyte assay reported here will be important for diagnosing MLD and MSD patients and for monitoring the efficacy of therapeutic treatments. ARSA activity was measured in DBS for the first time without an antibody. This new ARSA DBS assay can serve as a second-tier test following the sulfatide measurement in DBS for newborn screening of MLD. This leads to an elimination of most of the false positives identified by the sulfatide assay.
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http://dx.doi.org/10.1021/acs.analchem.9b05274DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7203001PMC
May 2020

Newborn Screening for Mucopolysaccharidoses: Results of a Pilot Study with 100 000 Dried Blood Spots.

J Pediatr 2020 01 12;216:204-207. Epub 2019 Nov 12.

Department of Chemistry, University of Washington, Seattle, WA.

Objective: To test, in a newborn screening (NBS) laboratory, the performance of liquid chromatography-tandem mass spectrometry (LC-MS/MS) to assay 5 enzymatic activities in dried blood spots (DBS) for NBS of 5 lysosomal storage diseases (mucopolysaccharidosis [MPS]-II, MPS-IIIB, MPS-IVA, MPS-VI, and MPS-VII).

Study Design: Three mm punches from de-identified DBS were obtained from the Washington NBS laboratory and submitted to the 5-plex LC-MS/MS assay. Screen cut-offs were established by analyzing the enzymatic activity in patients confirmed to have the MPS disorder. DNA sequencing of the relevant gene was performed on a second DBS punch for all samples with enzyme activity below 10% of the mean daily activity.

Results: (1) For MPS-II, 18 below cut-off samples, 1 pathogenic genotype, and 2 "high risk" genotypes; (2) For MPS-IIIB, no below cut-off samples; (3) For MPS-IVA, 8 below cut-off samples, 4 non-pathogenic genotypes, 4 genotypes unobtainable; (4) For MPS-VI, 4 below cut-off samples and no high-risk genotypes; (5) For MPS-VII, 1 below cut-off sample confirmed by genotype and clinical report to be affected.

Conclusions: These results establish that the number of initial screen positive samples is low and manageable. Thus, population newborn screening for these conditions is feasible in a state newborn screening laboratory.
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http://dx.doi.org/10.1016/j.jpeds.2019.09.036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7159818PMC
January 2020

The Importance of Assay Imprecision near the Screen Cutoff for Newborn Screening of Lysosomal Storage Diseases.

Int J Neonatal Screen 2019 Jun 27;5(2). Epub 2019 Mar 27.

Department of Chemistry, University of Washington, Seattle, WA 98195, USA.

For newborn screening (NBS) of lysosomal storage diseases, programs measure enzymatic activities in dried blood spots (DBS) and, in most cases, act on samples where the measurement is below a specific cutoff value. The rate of false positives and negatives in any NBS program is of critical importance. The measured values across a population of newborns are governed by many factors, and in this article we focus on assay imprecision. Assay parameters including the Analytical Range and the Z-Factor have been discussed as a way to compare assay performance for NBS of lysosomal storage diseases. Here we show that these parameters are not rigorously connected to the rate of false positives and negatives. Rather, it is the assay imprecision near the screen cutoff that is the most important parameter that determines the rate of false positives and negatives. We develop the theoretical treatment of assay imprecision and how it is linked to screen performance. What emerges is a useful type of parametric plot that allows for rigorous assessment of the effect of assay imprecision on the rate of false positives and false negatives that is independent of the choice of screen cutoff value. Such plots are useful in choosing cutoff values. They also show that a high assay imprecision cannot be overcome by changing the cutoff value or by use of postanalysis, statistical tools. Given the importance of assay imprecision near the cutoff, we propose that quality control DBS are most useful if they span a range of analyte values near the cutoff. Our treatment is also appropriate for comparing the performance of multiple assay platforms that each measure the same quantity (i.e., the enzymatic activity in DBS). The analysis shows that it is always best to use the assay platform that gives the lowest imprecision near the cutoff.
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http://dx.doi.org/10.3390/ijns5020017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6641561PMC
June 2019

Tandem mass spectrometry-based multiplex assays for α-mannosidosis and fucosidosis.

Mol Genet Metab 2019 07 10;127(3):207-211. Epub 2019 Jun 10.

Department of Chemistry, University of Washington, Seattle, WA 98195, USA. Electronic address:

Multiplex tandem mass spectrometry (MS/MS)-based enzyme activity assays for newborn screening (NBS) and diagnosis of lysosomal storage diseases (LSDs) in newborns, using dried blood spots (DBS) on newborn screening cards, have garnered much attention due to its sensitivity, high precision, and the capability to screen for an unprecedented number of diseases in a single assay. Herein we report the development of MS/MS-based enzyme assays for the diagnosis of α-mannosidosis and fucosidosis. These new protocols are able to distinguish untreated patients from random newborns, carriers and a post-bone marrow transplant patient. We have successfully multiplexed the α-mannosidosis assay with a multiplex MS/MS assay for the screening and diagnosis of other LSDs, namely Fabry, Pompe, MPS I, Gaucher, Niemann-Pick-A/B, and Krabbe diseases. Additionally, we also multiplexed the fucosidosis NBS assay with a 5-plex assay that tests for MPS-II, MPS-IIIB, MPS-IVA, MPS-VI and MPS-VII.
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http://dx.doi.org/10.1016/j.ymgme.2019.05.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6710107PMC
July 2019

-acyl--phosphocholineserines: structures of a novel class of lipids that are biomarkers for Niemann-Pick C1 disease.

J Lipid Res 2019 08 14;60(8):1410-1424. Epub 2019 Jun 14.

Departments of Medicine, Washington University School of Medicine, St. Louis, MO 63110

Niemann-Pick disease type C1 (NPC1) is a fatal, neurodegenerative, cholesterol storage disorder. With new therapeutics in clinical trials, there is an urgency to improve diagnostics and monitor therapeutic efficacy with biomarkers. In this study, we sought to define the structure of an unknown lipid biomarker for NPC1 with [M + H] ion at 509.3351, previously designated as lysoSM-509. The structure of -palmitoyl--phosphocholineserine (PPCS) was proposed for the lipid biomarker based on the results from mass spectrometric analyses and chemical derivatizations. As no commercial standard is available, authentic PPCS was chemically synthesized, and the structure was confirmed by comparison of endogenous and synthetic compounds as well as their derivatives using liquid chromatography-tandem mass spectrometry (LC-MS/MS). PPCS is the most abundant species among -acyl--phosphocholineserines (APCS), a class of lipids that have not been previously detected in biological samples. Further analysis demonstrated that all APCS species with acyl groups ranging from C14 to C24 were elevated in NPC1 plasma. PPCS is also elevated in both central and peripheral tissues of the NPC1 cat model. Identification of APCS structures provide an opportunity for broader exploration of the roles of these novel lipids in NPC1 disease pathology and diagnosis.
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http://dx.doi.org/10.1194/jlr.RA119000157DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6672039PMC
August 2019

Bioactivity of Farnesyltransferase Inhibitors Against and .

Front Cell Infect Microbiol 2019 29;9:180. Epub 2019 May 29.

Center for Discovery and Innovation in Parasitic Diseases, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, CA, United States.

The protozoan parasite can induce amebic colitis and amebic liver abscess. First-line drugs for the treatment of amebiasis are nitroimidazoles, particularly metronidazole. Metronidazole has side effects and potential drug resistance is a concern. Schistosomiasis, a chronic and painful infection, is caused by various species of the flatworm. There is only one partially effective drug, praziquantel, a worrisome situation should drug resistance emerge. As many essential metabolic pathways and enzymes are shared between eukaryotic organisms, it is possible to conceive of small molecule interventions that target more than one organism or target, particularly when chemical matter is already available. Farnesyltransferase (FT), the last common enzyme for products derived from the mevalonate pathway, is vital for diverse functions, including cell differentiation and growth. Both and genomes encode FT genes. In this study, we phenotypically screened and with the established FT inhibitors, lonafarnib and tipifarnib, and with 125 tipifarnib analogs previously screened against both the whole organism and/or the FT of and . For , we also explored whether synergy arises by combining lonafarnib and metronidazole or lonafarnib with statins that modulate protein prenylation. We demonstrate the anti-amebic and anti-schistosomal activities of lonafarnib and tipifarnib, and identify 17 tipifarnib analogs with more than 75% growth inhibition at 50 μM against . Apart from five analogs of tipifarnib exhibiting activity against both and , 10 additional analogs demonstrated anti-schistosomal activity (severe degenerative changes at 10 μM after 24 h). Analysis of the structure-activity relationship available for the FT suggests that FT may not be the relevant target in and . For , combination of metronidazole and lonafarnib resulted in synergism for growth inhibition. Also, of a number of statins tested, simvastatin exhibited moderate anti-amebic activity which, when combined with lonafarnib, resulted in slight synergism. Even in the absence of a definitive molecular target, identification of potent anti-parasitic tipifarnib analogs encourages further exploration while the synergistic combination of metronidazole and lonafarnib offers a promising treatment strategy for amebiasis.
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http://dx.doi.org/10.3389/fcimb.2019.00180DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6548881PMC
January 2020

Respective contribution of cytosolic phospholipase A2α and secreted phospholipase A IIA to inflammation and eicosanoid production in arthritis.

Prostaglandins Other Lipid Mediat 2019 08 23;143:106340. Epub 2019 May 23.

Centre de Recherche du Centre Hospitalier Universitaire de Québec, Faculté de Médecine de l'Université Laval, Département de microbiologie et immunologie, Québec, QC, G1V 4G2, Canada; Canadian National Transplantation Research Program, Edmonton, Alberta, Canada. Electronic address:

Phospholipase As (PLA) play a key role in generation of eicosanoids. Cytosolic PLAα (cPLAα) is constitutively expressed in most cells, whereas IIA secreted PLA (sPLA-IIA) is induced during inflammation and is present at high levels in the synovial fluid of rheumatoid arthritis patients. In mice, both cPLAα and sPLA-IIA have been implicated in autoimmune arthritis; however, the respective contribution of these two enzymes to the pathogenesis and production of eicosanoids is unknown. We evaluated the respective role of cPLAα and sPLA-IIA with regard to arthritis and eicosanoid profile in an in vivo model of arthritis. While arthritis was most severe in mice expressing both enzymes, it was abolished when both cPLAα and sPLA-IIA were lacking. cPLAα played a dominant role in the severity of arthritis, although sPLA-IIA sufficed to significantly contribute to the disease. Several eicosanoids were modulated during the course of arthritis and numerous species involved sPLA-IIA expression. This study confirms the critical role of PLAs in arthritis and unveils the distinct contribution of cPLAα and sPLA-IIA to the eicosanoid profile in arthritis.
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http://dx.doi.org/10.1016/j.prostaglandins.2019.106340DOI Listing
August 2019

Newborn Screening for Lysosomal Storage Disorders: Methodologies for Measurement of Enzymatic Activities in Dried Blood Spots.

Int J Neonatal Screen 2019 Mar 21;5(1). Epub 2018 Dec 21.

Head Reference Laboratory for Neonatal Screening, Center for Health Protection, National Institute for Public Health and the Environment (RIVM), P. O. Box 1, 3720 BA Bilthoven, The Netherlands.

All worldwide newborn screening (NBS) for lysosomal storage diseases (LSDs) is performed as a first-tier test by measurement of lysosomal enzymatic activities in dried blood spots (DBS). The currently two available methodologies used for measurement of enzymatic activities are tandem mass spectrometry (MS/MS) and digital microfluidics fluorimetry (DMF-F). In this chapter we summarize the workflows for the two platforms. Neither platform is fully automated, but the relative ease of workflow will be dependent upon the specific operation of each newborn screening laboratory on a case-by-case basis. We provide the screen positive rate (the number of below cutoff newborns per 100,000 newborns) from all NBS laboratories worldwide carrying out MS/MS-based NBS of one or more LSDs. The analytical precision of the MS/MS method is higher than that for DMF-F as shown by analysis of a common set of quality control DBS by the Centers for Disease Control and Prevention (CDC). Both the MS/MS and DMF-F platforms enable multiplexing of the LSD enzymes. An advantage of MS/MS over DMF-F is the ability to include assays of enzymatic activities and biomarkers for which no fluorimetric methods exist. Advantages of DMF-F over MS/MS are: 1) Simple to use technology with same-day turn-around time for the lysosomal enzymes with the fastest rates compared to MS/MS requiring overnight analytical runs.; 2) The DMF-F instrumentation, because of its simplicity, requires less maintenance than the MS/MS platform.
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http://dx.doi.org/10.3390/ijns5010001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6448570PMC
March 2019

Newborn Screening for Lysosomal Storage Diseases: Methodologies, Screen Positive Rates, Normalization of Datasets, Second-Tier Tests, and Post-Analysis Tools.

Authors:
Michael H Gelb

Int J Neonatal Screen 2018 Sep 9;4(3). Epub 2018 Jul 9.

Departments of Chemistry, University of Washington, Seattle, WA 98195, USA;

All of the worldwide newborn screening (NBS) for lysosomal storage diseases (LSDs) is done by measurement of lysosomal enzymatic activities in dried blood spots (DBS). Substrates used for these assays are discussed. While the positive predictive value (PPV) is the gold standard for evaluating medical tests, current PPVs for NBS of LSDs cannot be used as a performance metric due to statistical sampling errors and uncertainty in the onset of disease symptoms. Instead, we consider the rate of screen positives as the only currently reliable way to compare LSD NBS results across labs worldwide. It has been suggested that the expression of enzymatic activity data as multiple-of-the-mean is a way to normalize datasets obtained using different assay platforms, so that results can be compared, and universal cutoffs can be developed. We show that this is often not the case, and normalization is currently not feasible. We summarize the recent use of pattern matching statistical analysis together with measurement of an expanded group of enzymatic activities and biomarkers to greatly reduce the number of false positives for NBS of LSDs. We provide data to show that these post-enzymatic activity assay methods are more powerful than genotype analysis for the stratification of NBS for LSDs.
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http://dx.doi.org/10.3390/ijns4030023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6419971PMC
September 2018

Triazolopyrimidines and Imidazopyridines: Structure-Activity Relationships and in Vivo Efficacy for Trypanosomiasis.

ACS Med Chem Lett 2019 Jan 4;10(1):105-110. Epub 2018 Dec 4.

Department of Medicine, University of Washington, Seattle, Washington 98109, United States.

Better therapeutics are greatly needed to treat patients infected with trypanosomatid parasites such as or . This report describes 28 new imidazopyridines and triazolopyrimidines with potent and selective antitrypanosomal activity. Drug-like properties were demonstrated in a number of in vitro assays. In vivo efficacy was observed for and in acute mouse models of infection. Compounds and represent potential leads for new anti-Chagas disease drugs.
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http://dx.doi.org/10.1021/acsmedchemlett.8b00498DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6331164PMC
January 2019

Function of secreted phospholipase A group-X in asthma and allergic disease.

Biochim Biophys Acta Mol Cell Biol Lipids 2019 06 5;1864(6):827-837. Epub 2018 Dec 5.

Department of Medicine, Division of Pulmonary, Critical Care and Sleep, University of Washington, Seattle, WA, United States of America. Electronic address:

Elevated secreted phospholipase A (sPLA) activity in the airways has been implicated in the pathogenesis of asthma and allergic disease for some time. The identity and function of these enzymes in asthma is becoming clear from work in our lab and others. We focused on sPLA group X (sPLA-X) after identifying increased levels of this enzyme in asthma, and that it is responsible for a large portion of sPLA activity in the airways and that the levels are strongly associated with features of airway hyperresponsiveness (AHR). In this review, we discuss studies that implicated sPLA-X in human asthma, and murine models that demonstrate a critical role of this enzyme as a regulator of type-2 inflammation, AHR and production of eicosanoids. We discuss the mechanism by which sPLA-X acts to regulate eicosanoids in leukocytes, as well as effects that are mediated through the generation of lysophospholipids and through receptor-mediated functions. This article is part of a Special Issue entitled Novel functions of phospholipase A2 Guest Editors: Makoto Murakami and Gerard Lambeau.
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http://dx.doi.org/10.1016/j.bbalip.2018.11.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6431270PMC
June 2019

Taiwan National Newborn Screening Program by Tandem Mass Spectrometry for Mucopolysaccharidoses Types I, II, and VI.

J Pediatr 2019 02 6;205:176-182. Epub 2018 Nov 6.

Neonatal Screening Center, The Chinese Foundation of Health, Taipei, Taiwan. Electronic address:

Objective: To evaluate the initial cutoff values, rates of screen positives, and genotypes for the large-scale newborn screening program for multiple mucopolysaccharidoses (MPS) in Taiwan.

Study Design: More than 100 000 dried blood spots were collected consecutively as part of the national Taiwan newborn screening programs. Enzyme activities were measured by tandem mass spectrometry from dried blood spot punches. Genotypes were obtained when a second newborn screening specimen again had a decreased enzyme activity. Additional clinical evaluation was then initiated based on enzyme activity and/or genotype.

Results: Molecular genetic analysis for cases with low enzyme activity revealed 5 newborns with pathogenic alpha-L-iduronidase mutations, 3 newborns with pathogenic iduronate-2-sulfatase mutations, and 1 newborn was a carrier of an arylsulfatase B mutation. Several variants of unknown pathogenic significance were also identified, most likely causing pseudodeficiency.

Conclusions: The highly robust tandem mass spectrometry-based enzyme assays for MPS-I, MPS-II, and MPS-VI allow for high-throughput newborn screening for these lysosomal storage disorders. Optimized cutoff values combined with second tier testing could largely eliminate false-positive results. Accordingly, newborn screening for these lysosomal storage disorders is possible.
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http://dx.doi.org/10.1016/j.jpeds.2018.09.063DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6623979PMC
February 2019

Detection of Infantile Batten Disease by Tandem Mass Spectrometry Assay of PPT1 Enzyme Activity in Dried Blood Spots.

Anal Chem 2018 10 24;90(20):12168-12171. Epub 2018 Sep 24.

Department of Chemistry , University of Washington , Seattle , Washington 98195 , United States.

A new tandem mass spectrometry (MS/MS)-based approach for measurement of the enzymatic activity of palmitoyl protein thioesterase I (PPT1) in dried blood spots (DBS) is presented. Deficiency in this enzyme leads to infantile neuronal ceroid lipofuscinosis (INCL, Infantile Batten disease, CLN1). The assay could distinguish between 80 healthy newborns and three previously diagnosed INCL patients. Unlike the fluorimetric PPT1 assay, the MS/MS assay does not require recombinant β-glucosidase. Furthermore, the assay could be easily combined with a TPP1 enzyme assay (for CLN2 disease) and can be potentially multiplexed with a large panel of additional lysosomal enzyme assays by MS/MS for newborn screening and postscreening analysis.
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http://dx.doi.org/10.1021/acs.analchem.8b03188DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6423508PMC
October 2018