Detecting lysosomal storage disorders by glycomic profiling using liquid chromatography mass spectrometry.

BACKGROUND: Urine and plasma biomarker testing for lysosomal storage disorders by liquid chromatography mass spectrometry (LC-MS) currently requires multiple analytical methods to detect the abnormal accumulation of oligosaccharides, mucopolysaccharides, and glycolipids. To improve clinical testing efficiency, we developed a single LC-MS method to simultaneously identify disorders of oligosaccharide, mucopolysaccharide, and glycolipid metabolism with minimal sample preparation. METHODS: We created a single chromatographic method for separating free glycans and glycolipids in their native form, using an amide column and high pH conditions. We used this glycomic profiling method both in untargeted analyses of patient and control urines using LC ion-mobility high-resolution MS (biomarker discovery), and targeted analyses of urine, serum, and dried blood spot samples by LC-MS/MS (clinical validation). RESULTS: Untargeted glycomic profiling revealed twenty biomarkers that could identify and subtype mucopolysaccharidoses. We incorporated these with known oligosaccharide and glycolipid biomarkers into a rapid test that identifies at least 27 lysosomal storage disorders, including oligosaccharidoses, mucopolysaccharidoses, sphingolipidoses, glycogen storage disorders, and congenital disorders of glycosylation and de-glycosylation. In a validation set containing 115 urine samples from patients with lysosomal storage disorders, all were unambiguously distinguished from normal controls, with correct disease subtyping for 88% (101/115) of cases. Glucosylsphingosine was reliably elevated in dried blood spots from Gaucher disease patients with baseline resolution from galactosylsphingosine. CONCLUSION: Glycomic profiling by liquid chromatography mass spectrometry identifies a range of lysosomal storage disorders. This test can be used in clinical evaluations to rapidly focus a diagnosis, as well as to clarify or support additional gene sequencing and enzyme studies.

Spondyloepimetaphyseal dysplasia with elevated plasma lysosomal enzymes caused by homozygous variant in MBTPS1.

Variants in MBTPS1 (membrane-bound transcription factor peptidase, site 1) encoding the protein convertase site-1 protease (S1P) were recently reported in a single individual with skeletal dysplasia and elevated plasma lysosomal enzymes. Here, we report the second individual with this newly described autosomal recessive spondyloepiphyseal dysplasia (OMIM #618392), presenting severe growth retardation, cataract and dysmorphic features, mainly retromicrognathia. Epilepsy and craniosynostosis were novel findings in our proband. She was found to be homozygous for a novel nonsense variant p.Trp983Ter in MBTPS1. In addition, she had normal levels of lysosomal enzyme activity in leukocytes but elevated levels in plasma. Our description confirms the existence of this new skeletal dysplasia and expands the phenotype and genotype of the disease.

Plasma and dried blood spot lysosphingolipids for the diagnosis of different sphingolipidoses: a comparative study.

Background Lysosphingolipids, the N-deacylated forms of sphingolipids, have been identified as potential biomarkers of several sphingolipidoses, such as Gaucher, Fabry, Krabbe and Niemann-Pick diseases and in GM1 and GM2 gangliosidoses. To date, different methods have been developed to measure various lysosphingolipids (LysoSLs) in plasma. Here, we present a novel liquid chromatography tandem mass spectrometry (LC-MS/MS) assay for a simultaneous quantification of LysoSLs (HexSph, LysoGb3, LysoGM1, LysoGM2, LysoSM and LysoSM509) in dried blood spot (DBS). This LC-MS/MS method was used to compare the levels of LysoSLs in DBS and plasma in both affected patients and healthy controls. Methods Lysosphingolipids were extracted from a 3.2 mm diameter DBS with a mixture of methanol:acetonitrile:water (80:15:5, v/v) containing internal stable isotope standards. Chromatographic separation was performed using a C18 column with a gradient of water and acetonitrile both with 0.1% formic acid in a total run time of 4 min. The compounds were detected in the positive ion mode electrospray ionization (ESI)-MS/MS by multiple reaction monitoring (MRM). Results The method was validated on DBS to demonstrate specificity, linearity, lowest limit of quantification, accuracy and precision. The reference ranges were determined in pediatric and adult populations. The elevated levels of LysoSLs were identified in Gaucher disease (HexSph), Fabry disease (LysoGb3), prosaposin deficiency (HexSph and LysoGb3) and Niemann-Pick disease types A/B and C (LysoSM and LysoSM509). The correlation in the levels between DBS and plasma was excellent for LysoGb3 and HexSph but poor for LysoSM and LysoSM509. Conclusions Despite the fact that plasma LysoSLs determination remains the gold standard, our LC-MS/MS method allows a rapid and reliable quantification of lysosphingolipids in DBS. The method is a useful tool for the diagnosis of different sphingolipidoses except for Niemann-Pick type C.

Zinc Homeostasis in Platelet-Related Diseases.

Zn2+ deficiency in the human population is frequent in underdeveloped countries. Worldwide, approximatively 2 billion people consume Zn2+-deficient diets, accounting for 1-4% of deaths each year, mainly in infants with a compromised immune system. Depending on the severity of Zn2+ deficiency, clinical symptoms are associated with impaired wound healing, alopecia, diarrhea, poor growth, dysfunction of the immune and nervous system with congenital abnormalities and bleeding disorders. Poor nutritional Zn2+ status in patients with metastatic squamous cell carcinoma or with advanced non-Hodgkin lymphoma, was accompanied by cutaneous bleeding and platelet dysfunction. Forcing Zn2+ uptake in the gut using different nutritional supplementation of Zn2+ could ameliorate many of these pathological symptoms in humans. Feeding adult rodents with a low Zn2+ diet caused poor platelet aggregation and increased bleeding tendency, thereby attracting great scientific interest in investigating the role of Zn2+ in hemostasis. Storage protein metallothionein maintains or releases Zn2+ in the cytoplasm, and the dynamic change of this cytoplasmic Zn2+ pool is regulated by the redox status of the cell. An increase of labile Zn2+ pool can be toxic for the cells, and therefore cytoplasmic Zn2+ levels are tightly regulated by several Zn2+ transporters located on the cell surface and also on the intracellular membrane of Zn2+ storage organelles, such as secretory vesicles, endoplasmic reticulum or Golgi apparatus. Although Zn2+ is a critical cofactor for more than 2000 transcription factors and 300 enzymes, regulating cell differentiation, proliferation, and basic metabolic functions of the cells, the molecular mechanisms of Zn2+ transport and the physiological role of Zn2+ store in megakaryocyte and platelet function remain elusive. In this review, we summarize the contribution of extracellular or intracellular Zn2+ to megakaryocyte and platelet function and discuss the consequences of dysregulated Zn2+ homeostasis in platelet-related diseases by focusing on thrombosis, ischemic stroke and storage pool diseases.

A metabolomic map of Zellweger spectrum disorders reveals novel disease biomarkers.

PURPOSE: Peroxisome biogenesis disorders-Zellweger spectrum disorders (PBD-ZSD) are metabolic diseases with multisystem manifestations. Individuals with PBD-ZSD exhibit impaired peroxisomal biochemical functions and have abnormal levels of peroxisomal metabolites, but the broader metabolic impact of peroxisomal dysfunction and the utility of metabolomic methods is unknown. METHODS: We studied 19 individuals with clinically and molecularly characterized PBD-ZSD. We performed both quantitative peroxisomal biochemical diagnostic studies in parallel with untargeted small molecule metabolomic profiling in plasma samples with detection of >650 named compounds. RESULTS: The cohort represented intermediate to mild PBD-ZSD subjects with peroxisomal biochemical alterations on targeted analysis. Untargeted metabolomic profiling of these samples revealed elevations in pipecolic acid and long-chain lysophosphatidylcholines, as well as an unanticipated reduction in multiple sphingomyelin species. These sphingomyelin reductions observed were consistent across the PBD-ZSD samples and were rare in a population of >1,000 clinical samples. Interestingly, the pattern or "PBD-ZSD metabolome" was more pronounced in younger subjects suggesting studies earlier in life reveal larger biochemical changes. CONCLUSION: Untargeted metabolomics is effective in detecting mild to intermediate cases of PBD-ZSD. Surprisingly, dramatic reductions in plasma sphingomyelin are a consistent feature of the PBD-ZSD metabolome. The use of metabolomics in PBD-ZSD can provide insight into novel biomarkers of disease.

Detection of mucopolysaccharidosis III-A (Sanfilippo Syndrome-A) in dried blood spots (DBS) by tandem mass spectrometry.

BACKGROUND: With ongoing efforts to develop improved treatments for Sanfilippo Syndrome Type A (MPS-IIIA), a disease caused by the inability to degrade heparan sulfate in lysosomes, we sought to develop an enzymatic activity assay for the relevant enzyme, sulfamidase, that uses dried blood spots (DBS). METHODS: We designed and synthesized a new sulfamidase substrate that can be used to measure sulfamidase activity in DBS using liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS: Sulfamidase activity was readily detected in DBS using the new substrate and LC-MS/MS. Sulfamidase activity showed acceptable linearity proportional to the amount of enzyme and reaction time. Sulfamidase activity in 238 random newborns was well elevated compared to the range of activities measured in DBS from 8 patients previously confirmed to have MPS-IIIA. CONCLUSIONS: This is the first report of an assay capable of detecting sulfamidase in DBS. The new assay could be useful in diagnosis and potentially for newborn screening of MPS-IIIA.

Large-scale study of clinical and biochemical characteristics of Chinese patients diagnosed with Krabbe disease.

Krabbe disease (KD) is a rare disease caused by the deficiency of ß-galactocerebrosidase. This study investigated 22 unrelated Chinese patients, including their clinical presentations, plasma psychosine levels and ß-galactocerebrosidase gene mutations. We found the late-onset form of KD present in 82% of the patients in our study, which was more prevalent than in patients from other populations. Plasma psychosine levels were elevated in KD, which were correlated with the severity of clinical presentations. Sanger sequencing identified 8 novel mutations, including 7 missense mutations, p.H253Y, p.S259L, p.P318L, p.F350V, p.T428A, p.L530P, p.G586D, and 1 splicing mutation, c.1251+1G>A. Quantitative real-time polymerase chain reaction (PCR) and multiplex ligation-dependent probe amplification identified a novel exon 12 and 14 deletion, separately. Next generation sequencing, applied at the final step, revealed 2 missense mutant alleles missed using Sanger sequencing. The most common mutation in Chinese population is p.P154H, which accounts for 20.5% of alleles. Consistent with the higher prevalence of the late-onset form of KD, missense mutations predominated in our study, different with the common mutation types in Europe and Japan. This work was the first large-scale study of Chinese KD patients describing their clinical, biochemical and genetic characteristics, which furthered our understanding of this classical neurological lysosomal storage disease.

Newborn screening for lysosomal storage disorders by tandem mass spectrometry in North East Italy.

BACKGROUND: Lysosomal storage diseases (LSDs) are inborn errors of metabolism resulting from 50 different inherited disorders. The increasing availability of treatments and the importance of early intervention have stimulated newborn screening (NBS) to diagnose LSDs and permit early intervention to prevent irreversible impairment or severe disability. We present our experience screening newborns in North East Italy to identify neonates with Mucopolysaccharidosis type I (MPS I) and Pompe, Fabry, and Gaucher diseases. METHODS: Activities of acid ß-glucocerebrosidase (ABG; Gaucher), acid α-glucosidase (GAA; Pompe), acid α-galactosidase (GLA; Fabry), and acid α-L-iduronidase (IDUA; MPS-I) in dried blood spots (DBS) from all newborns during a 17-month period were determined by multiplexed tandem mass spectrometry (MS/MS) using the NeoLSD® assay system. Enzymatic activity cutoff values were determined from 3500 anonymous newborn DBS. In the screening study, samples were retested if the value was below cutoff and a second spot was requested, with referral for confirmatory testing and medical evaluation if a low value was obtained. RESULTS: From September 2015 to January 2017, 44,411 newborns were screened for the four LSDs. We recalled 40 neonates (0.09%) for collection of a second DBS. Low activity was confirmed in 20, who had confirmatory testing. Ten of 20 had pathogenic mutations: two Pompe, two Gaucher, five Fabry, and one MPS-I. The incidences of Pompe and Gaucher diseases were similar (1/22,205), with Fabry disease the most frequent (1/8882) and MPS-I the rarest (1/44411). The combined incidence of the four disorders was 1/4411 births. CONCLUSIONS: Simultaneously determining multiple enzyme activities by MS/MS, with a focus on specific biochemical markers, successfully detected newborns with LSDs. The high incidence of these disorders supports this screening program.

Multiplex Tandem Mass Spectrometry Enzymatic Activity Assay for Newborn Screening of the Mucopolysaccharidoses and Type 2 Neuronal Ceroid Lipofuscinosis.

BACKGROUND: We expanded the use of tandem mass spectrometry combined with liquid chromatography (LC-MS/MS) for multiplex newborn screening of seven lysosomal enzymes in dried blood spots (DBS). The new assays are for enzymes responsible for the mucopolysaccharidoses (MPS-I, -II, -IIIB, -IVA, -VI, and -VII) and type 2 neuronal ceroid lipofuscinosis (LINCL). METHODS: New substrates were prepared and characterized for tripeptidyl peptidase 1 (TPP1), α-N-acetylglucosaminidase (NAGLU), and lysosomal ß-glucuronidase (GUSB). These assays were combined with previously developed assays to provide a multiplex LC-MS/MS assay of 7 lysosomal storage diseases. Multiple reaction monitoring of ion dissociations for enzyme products and deuterium-labeled internal standards was used to quantify the enzyme activities. RESULTS: Deidentified DBS samples from 62 nonaffected newborns were analyzed to simultaneously determine (run time 2 min per DBS) the activities of TPP1, NAGLU, and GUSB, along with those for α-iduronidase (IDUA), iduronate-2-sulfatase (I2S), N-acetylgalactosamine-6-sulfatase (GALNS), and N-acetylgalactosamine-4-sulfatase (ARSB). The activities measured in the 7-plex format showed assay response-to-blank-activity ratios (analytical ranges) of 102-909 that clearly separated healthy infants from affected children. CONCLUSIONS: The new multiplex assay provides a robust comprehensive newborn screening assay for the mucopolysaccharidoses. The method has been expanded to include additional lysosomal storage diseases.

Simultaneous Testing for 6 Lysosomal Storage Disorders and X-Adrenoleukodystrophy in Dried Blood Spots by Tandem Mass Spectrometry.

BACKGROUND: Newborn screening for lysosomal storage disorders (LSD) has revealed that late-onset variants of these conditions are unexpectedly frequent and therefore may evade diagnosis. We developed an efficient and cost-effective multiplex assay to diagnose six LSDs and several peroxisomal disorders in patients presenting with diverse phenotypes at any age. METHODS: Three 3-mm dried blood spot (DBS) punches were placed into individual microtiter plates. One disc was treated with a cocktail containing acid sphingomyelinase-specific substrate and internal standard (IS). To the second DBS we added a cocktail containing substrate and IS for ß-glucosidase, acid α-glucosidase, α-galactosidase A, galactocerebrosidase, and α-L-iduronidase. The third DBS was extracted with methanol containing d4-C26 lysophosphatidylcholine as IS and stored until the enzyme plates were combined and purified by liquid-liquid and solid-phase extraction. The extracts were evaporated, reconstituted with the extract from the lysophosphatidylcholine plate, and analyzed by flow injection tandem mass spectrometry. RESULTS: Reference intervals were determined by analysis of 550 samples from healthy controls. DBS from confirmed patients with 1 of the 6 LSDs (n = 33), X-adrenoleukodystrophy (n = 9), or a peroxisomal biogenesis disorder (n = 5), as well as carriers for Fabry disease (n = 17) and X-adrenoleukodystrophy (n = 5), were analyzed for assay validation. Prospective clinical testing of 578 samples revealed 25 patients affected with 1 of the detectable conditions. CONCLUSIONS: Our flow injection tandem mass spectrometry approach is amenable to high-throughput population screening for Hurler disease, Gaucher disease, Niemann-Pick A/B disease, Pompe disease, Krabbe disease, Fabry disease, X-adrenoleukodystrophy, and peroxisomal biogenesis disorder in DBS.

Pilot study of newborn screening for six lysosomal storage diseases using Tandem Mass Spectrometry.

BACKGROUND: There is current expansion of newborn screening (NBS) programs to include lysosomal storage disorders because of the availability of treatments that produce an optimal clinical outcome when started early in life. OBJECTIVE: To evaluate the performance of a multiplex-tandem mass spectrometry (MS/MS) enzymatic activity assay of 6 lysosomal enzymes in a NBS laboratory for the identification of newborns at risk for developing Pompe, Mucopolysaccharidosis-I (MPS-I), Fabry, Gaucher, Niemann Pick-A/B, and Krabbe diseases. METHODS AND RESULTS: Enzyme activities (acid α-glucosidase (GAA), galactocerebrosidase (GALC), glucocerebrosidase (GBA), α-galactosidase A (GLA), α-iduronidase (IDUA) and sphingomyeline phosphodiesterase-1 (SMPD-1)) were measured on ~43,000 de-identified dried blood spot (DBS) punches, and screen positive samples were submitted for DNA sequencing to obtain genotype confirmation of disease risk. The 6-plex assay was efficiently performed in the Washington state NBS laboratory by a single laboratory technician at the bench using a single MS/MS instrument. The number of screen positive samples per 100,000 newborns were as follows: GAA (4.5), IDUA (13.6), GLA (18.2), SMPD1 (11.4), GBA (6.8), and GALC (25.0). DISCUSSION: A 6-plex MS/MS assay for 6 lysosomal enzymes can be successfully performed in a NBS laboratory. The analytical ranges (enzyme-dependent assay response for the quality control HIGH sample divided by that for all enzyme-independent processes) for the 6-enzymes with the MS/MS is 5- to 15-fold higher than comparable fluorimetric assays using 4-methylumbelliferyl substrates. The rate of screen positive detection is consistently lower for the MS/MS assay compared to the fluorimetric assay using a digital microfluidics platform.

Newborn screening for lysosomal storage diseases.

BACKGROUND: There is worldwide interest in newborn screening for lysosomal storage diseases because of the development of treatment options that give better results when carried out early in life. Screens with high differentiation between affected and nonaffected individuals are critical because of the large number of potential false positives. CONTENT: This review summarizes 3 screening methods: (a) direct assay of enzymatic activities using tandem mass spectrometry or fluorometry, (b) immunocapture-based measurement of lysosomal enzyme abundance, and (c) measurement of biomarkers. Assay performance is compared on the basis of small-scale studies as well as on large-scale pilot studies of mass spectrometric and fluorometric screens. SUMMARY: Tandem mass spectrometry and fluorometry techniques for direct assay of lysosomal enzymatic activity in dried blood spots have emerged as the most studied approaches. Comparative mass spectrometry vs fluorometry studies show that the former better differentiates between nonaffected vs affected individuals. This in turn leads to a manageable number of screen positives that can be further evaluated with second-tier methods.

Reference intervals of α-glycosidase, ß-glycosidase, and α-galactosidase in dried blood spot in a Turkish newborn population.

Inherited lysosomal storage diseases (LSDs) are rare, and diagnosis is often delayed for 7-10 years. Since the therapies have become available for a limited number of LSDs, (Fabry, Gaucher, Pompe, and MPS-1), early diagnosis of treatable LSDs can be lifesaving or ameliorating and allows timely treatment before irreversible damage occurs. Recently, the use of dried blood spot test (DBS) for newborn screening of LSDs has been proposed for newborn screening tests. They are noninvasive, sensitive, and specific assays with the further advantage of a fast turnaround time compared to measurement in leukocyte and/or fibroblast culture. We aimed to determine the reference intervals for lysosomal enzyme activities of newborn babies in our population and to investigate the effect of gestational week on enzyme activity. One hundred thirty healthy newborn babies (70 girls, 60 boys) were included into the study. α-Glycosidase, ß-glycosidase, and α-galactosidase activities in DBS samples of newborns were determined fluorometrically. Reference intervals were calculated using Dixon's rule and percentiles of 2.5-97.5. Cutoff limits (5 %) for α-glycosidase, ß-glycosidase, and α-galactosidase activities were 0.57, 0.92, and 2.18, respectively. α-Galactosidase activity was higher in girls compared to boys (p < 0.05). Interestingly, α-glycosidase and ß-glycosidase activities of newborns who were delivered before 38 weeks were significantly lower than those who were delivered at 39-40 weeks. Conclusion It is of utmost importance to define the reference intervals for lysosomal enzyme activities as well as cutoff limits for newborn babies with regard to gestational age and sex. More studies to clarify the reason for the change in enzyme activity by gestational week will be required.

Screening patients referred to a metabolic clinic for lysosomal storage disorders.

BACKGROUND: Lysosomal protein profiling is being developed as a high throughput method to screen populations for lysosomal storage disorders (LSD). DESIGN: 1415 blood spots from patients referred to a metabolic clinic for LSD were screened using a single multiplex assay for 14 proteins in a dried blood spot. RESULTS: All patients with Pompe disease, metachromatic leukodystrophy, and mucopolysaccharidosis (MPS) type I, IIIA, IIIB and VI were identified by reduced lysosomal protein. Five samples were identified as possible pseudo-arylsulfatase A deficiency; four were confirmed. One multiple sulfatase deficiency patient was identified with multiple reduced sulfatase proteins. There were 10 MPS II patients identified with reduced iduronate 2-sulfatase, and one MPS II patient with iduronate 2-sulfatase in the unaffected range. For Fabry disease, 10 male patients were identified with reduced α-galactosidase and 2/6 female Fabry heterozygotes returned α-galactosidase concentrations in the male Fabry range. All 10 mucolipidosis II/III patients were identified with multiple raised proteins. For 79 blood spots with chitotriosidase >3.4mg/l, a follow-up one-plex chitotriosidase assay enabled identification of all nine Gaucher patients. CONCLUSION: This study demonstrates the sensitivity and specificity of this technology to accurately identify 99% of LSD patients, with the exception of one MPS II false negative.

The use of dried blood spot samples in the diagnosis of lysosomal storage disorders--current status and perspectives.

Dried blood spot (DBS) methods are currently available for identification of a range of lysosomal storage disorders (LSDs). These disorders are generally characterized by a deficiency of activity of a lysosomal enzyme and by a broad spectrum of phenotypes. Diagnosis of LSD patients is often delayed, which is of particular concern as therapeutic outcomes (e.g. enzyme replacement therapy) are generally more favorable in early disease stages. Experts in the field of LSDs diagnostics and screening programs convened and reviewed experiences with the use of DBS methods, and discuss the diagnostic challenges, possible applications and quality programs in this paper. Given the easy sampling and shipping and stability of samples, DBS has evident advantages over other laboratory methods and can be particularly helpful in the early identification of affected LSD patients through neonatal screening, high-risk population screening or family screening.

Hemoglobin precipitation greatly improves 4-methylumbelliferone-based diagnostic assays for lysosomal storage diseases in dried blood spots.

Derivatives of 4-methylumbelliferone (4MU) are favorite substrates for the measurement of lysosomal enzyme activities in a wide variety of cell and tissue specimens. Hydrolysis of these artificial substrates at acidic pH leads to the formation of 4-methylumbelliferone, which is highly fluorescent at a pH above 10. When used for the assay of enzyme activities in dried blood spots the light emission signal can be very low due to the small sample size so that the patient and control ranges are not widely separated. We have investigated the hypothesis that quenching of the fluorescence by hemoglobin leads to appreciable loss of signal and we show that the precipitation of hemoglobin with trichloroacetic acid prior to the measurement of 4-methylumbelliferone increases the height of the output signal up to eight fold. The modified method provides a clear separation of patients' and controls' ranges for ten different lysosomal enzyme assays in dried blood spots, and approaches the conventional leukocyte assays in outcome quality.

Effect of sample collection, temperature and time of storage on ß-galactosidase and total hexosaminidase activities in dried blood collected on filter paper.

BACKGROUND: Dried blood spots (DBS) on filter paper is a valuable sampling technique in clinical chemistry, but the stability of enzymes used in the diagnosis of lysosomal storage diseases (LSDs) needs to be evaluated. METHODS: In a first experiment, blood from 20 subjects was collected using a syringe without additives and distributed into EDTA tubes, heparin tubes, and spotted on filter paper for the comparison of sampling effects. In a second experiment, blood from 30 healthy subjects was spotted on filter paper and analyzed for ß-galactosidase and total hexosaminidase activities after storage of the samples at different temperatures for up to 180 days. RESULTS: Initially, we observed that enzyme activities were the same, independent of the collection method. When DBS was stored at 37°C the activity of ß-galactosidase dropped to 85% of the initial value after 180 days (p<0.05). At all other temperatures (-20°C, 4°C and 25°C), the results were within the methodological error. Total hexosaminidase activity did not change significantly during the entire study period and at different storage temperatures. CONCLUSIONS: The two enzymes investigated in the present study may be stored for up to 17 days (ß-galactosidase) or 180 days (total hexosaminidase) until analysis without loss of activity.

The application of multiplexed, multi-dimensional ultra-high-performance liquid chromatography/tandem mass spectrometry to the high-throughput screening of lysosomal storage disorders in newborn dried bloodspots.

Lysozomal storage disorders are just beginning to be routinely screened using enzyme activity assays involving dried blood spots and tandem mass spectrometry (MS/MS). This paper discusses some of the analytical challenges associated with published assays including complex sample preparation and potential interference from excess residual substrate. Solutions to these challenges are presented in the form of on-line two-dimensional chromatography to eliminate off-line liquid-liquid extraction (LLE) and solid-phase extraction (SPE), the use of ultra-high-performance liquid chromatography (UHPLC) to separate excess substrate from all other analytes and multiplexed sample introduction for higher throughput required of a population screening assay. High sensitivity, specificity and throughput were demonstrated using this novel method.

New strategy for the screening of lysosomal storage disorders: the use of the online trapping-and-cleanup liquid chromatography/mass spectrometry.

The aim of this study was to set up a robust method suitable for large-scale studies (screening) with a minimized preparation process and with reduced running costs, for measuring five enzyme activities on dried blood spots by a new and simplified tandem mass spectrometry-based method. After incubation, all 5 reaction mixtures, carried out separately, were stopped, combined together, and centrifuged. The cleaning-up of the injected mixture was performed through a fast online trapping step preceding a liquid chromatography/tandem mass-spectrometry measurement. This method takes only 4 min as analysis run time and without any purification following the enzymatic reaction. We assessed the effectiveness of this approach in assaying the enzymatic activities on dried blood spots from 10 patients affected by "Pompe", 6 by "Gaucher", 12 by "Fabry", 3 by "Niemann-Pick" A/B, and 2 by "Krabbe" diseases. Reference values were established on 5000 healthy newborns and 300 healthy adults. All affected patients showed enzymatic activities below the normal range. In heterozygous carriers (18 for Fabry, 10 for Pompe, and 4 for Gaucher disease) the activities were slightly lower than in control subjects. The results show that the method set out in its simplicity, low costs, and low processes preparations can be fully applicable to a mass screening.