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ABSTRACT: Recent technical advances have re-invigorated the study of sphingolipid metabolism in general, and helped to highlight the varied and important roles that sphingolipids play in pancreatic β-cells. Sphingolipid metabolites such as ceramide, glycosphingolipids, sphingosine 1-phosphate and gangliosides modulate many β-cell signaling pathways and processes implicated in β-cell diabetic disease such as apoptosis, β-cell cytokine secretion, ER-to-golgi vesicular trafficking, islet autoimmunity and insulin gene expression. They are particularly relevant to lipotoxicity. Moreover, the de novo synthesis of sphingolipids occurs on many subcellular membranes, in parallel to secretory vesicle formation, traffic and granule maturation events. Indeed, the composition of the plasma membrane, determined by the activity of neutral sphingomyelinases, affects β-cell excitability and potentially insulin exocytosis while another glycosphingolipid, sulfatide, determines the stability of insulin crystals in granules. Most importantly, sphingolipid metabolism on internal membranes is also strongly implicated in regulating β-cell apoptosis.
Islets 05/2012; 4(3):177-87.
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ABSTRACT: Glycogen storage in the α-glucosidase knockout((6neo/6neo)) mouse recapitulates the biochemical defect that occurs in the human condition; as such, this mouse serves as a model for the inherited metabolic deficiency of lysosomal acid α-glucosidase known as Pompe disease. Although this model has been widely used for the assessment of therapies, the time course of glycogen accumulation that occurs as untreated Pompe mice age has not been reported. To address this, we developed a quantitative method involving amyloglucosidase digestion of glycogen and quantification of the resulting free glucose by liquid chromatography/electrospray ionization-tandem mass spectrometry. The method was sensitive enough to measure as little as 0.1 μg of glycogen in tissue extracts with intra- and interassay coefficients of variation of less than 12%. Quantification of glycogen in tissues from Pompe mice from birth to 26 weeks of age showed that, in addition to the accumulation of glycogen in the heart and skeletal muscle, glycogen also progressively accumulated in the brain, diaphragm, and skin. Glycogen storage was also evident at birth in these tissues. This method may be particularly useful for longitudinal assessment of glycogen reduction in response to experimental therapies being trialed in this model.
Analytical Biochemistry 02/2012; 421(2):759-63. · 3.00 Impact Factor
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ABSTRACT: Lysosomal protein profiling is being developed as a high throughput method to screen populations for lysosomal storage disorders (LSD).
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.
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.
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.
Journal of Medical Genetics 03/2011; 48(6):422-5. · 6.36 Impact Factor
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ABSTRACT: Saturated fatty acids promote lipotoxic ER (endoplasmic reticulum) stress in pancreatic β-cells in association with Type 2 diabetes. To address the underlying mechanisms we employed MS in a comprehensive lipidomic screen of MIN6 β-cells treated for 48 h with palmitate. Both the overall mass and the degree of saturation of major neutral lipids and phospholipids were only modestly increased by palmitate. The mass of GlcCer (glucosylceramide) was augmented by 70% under these conditions, without any significant alteration in the amounts of either ceramide or sphingomyelin. However, flux into ceramide (measured by [3H]serine incorporation) was augmented by chronic palmitate, and inhibition of ceramide synthesis decreased both ER stress and apoptosis. ER-to-Golgi protein trafficking was also reduced by palmitate pre-treatment, but was overcome by overexpression of GlcCer synthase. This was accompanied by increased conversion of ceramide into GlcCer, and reduced ER stress and apoptosis, but no change in phospholipid desaturation. Sphingolipid alterations due to palmitate were not secondary to ER stress since they were neither reproduced by pharmacological ER stressors nor overcome using the chemical chaperone phenylbutyric acid. In conclusion, alterations in sphingolipid, rather than phospholipid, metabolism are more likely to be implicated in the defective protein trafficking and enhanced ER stress and apoptosis of lipotoxic β-cells.
Biochemical Journal 01/2011; 435(1):267-76. · 4.90 Impact Factor
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Clinical Chemistry 12/2008; 54(11):1925-7. · 7.91 Impact Factor
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ABSTRACT: The catabolism of dermatan sulfate (DS) commences with endohydrolysis of the polysaccharide to oligosaccharides by proposed endo-beta-N-acetylhexosaminidase and endohexuronidase activities. To investigate the substrate specificities of these activities, we developed an assay to measure specific products of their action upon oligosaccharide substrates. Tetra- to tetradecasaccharides, rich in glucuronic acid (GlcA) or iduronic acid (IdoA), were obtained from chondroitinase ABC digests of chondroitin sulfate (CS)-A and DS, respectively, separated by gel-filtration chromatography and characterized by electrospray ionization-tandem mass spectrometry (ESI-MS/MS). Endo-beta-N-acetylhexosaminidase and endohexuronidase cleavage of these oligosaccharides was then assessed by incubating with cell homogenate (source of endoglycosidase activity) and measuring di- to octasaccharide products derived from the nonreducing end of the substrate by ESI-MS/MS. We found that both activities preferentially degraded the GlcA-rich substrate, with minor activity toward the IdoA-rich substrate and that a minimum of four and five monosaccharides were required on the reducing side of the target glycosidic linkage for endo-beta-N-acetylhexosaminidase and endohexuronidase cleavage, respectively. Thus, the minimum-sized substrates were a hexasaccharide for endo-beta-N-acetylhexosaminidase and an octasaccharide for endohexuronidase. We observed that endo-beta-N-acetylhexosaminidase sequentially removed tetrasaccharides from the nonreducing end of oligosaccharides when unrestricted by substrate length, whereas endohexuronidase activity was random and comparatively low. The activities displayed acidic pH optima and were shown by subcellular fractionation to reside in lysosomes and late endosomes. We suggest that these activities represent the known Hyal-1 and endo-beta-glucuronidase enzymes and that these enzymes act in concert to degrade GlcA-rich domains of DS but are less active toward regions containing IdoA.
Glycobiology 10/2008; 18(12):1119-28. · 3.58 Impact Factor
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ABSTRACT: Gaucher disease (GD) is an inborn error of glycosphingolipid metabolism resulting from a deficiency of the lysosomal enzyme beta-glucosidase leading to the accumulation of glucosylceramide (GC) in lysosomes of affected cells. In order to determine the effect of GC accumulation on intracellular lipid content in fibroblasts from patients with GD, we measured individual species of ceramide, di- and trihexosylceramide, sphingomyelin, phosphatidylcholine, phosphatidylinositol and phosphatidylglycerol using electrospray ionisation-tandem mass spectrometry. The different subspecies of each lipid class correlated with each other and were summed to give total lipid concentrations. In addition to GC, we also noted secondary elevations in other lipids, especially in type 2 GD. Sub-cellular fractionation showed that GC was not confined to the lysosome but increased throughout the cell. The sequelae of extra-lysosomal accumulation may have implications in the pathogenic mechanisms of GD by interaction with biochemical and metabolic pathways located outside the lysosome. The elevation of ceramide in confluent type 2 GD fibroblasts redistributed from its primary site of accumulation in the lysosome to the endosomal region at four-weeks post-confluence. The accumulation of lipids in the endosome and lysosome suggests both impaired trafficking of lipids and reduced capacity of the lysosome to degrade lipids.
Molecular Genetics and Metabolism 05/2008; 93(4):437-43. · 3.19 Impact Factor
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ABSTRACT: BMP [bis(monoacylglycero)phosphate] is an acidic phospholipid and a structural isomer of PG (phosphatidylglycerol), consisting of lysophosphatidylglycerol with an additional fatty acid esterified to the glycerol head group. It is thought to be synthesized from PG in the endosomal/lysosomal compartment and is found primarily in multivesicular bodies within the same compartment. In the present study, we investigated the effect of lysosomal storage on BMP in cultured fibroblasts from patients with eight different LSDs (lysosomal storage disorders) and plasma samples from patients with one of 20 LSDs. Using ESI-MS/MS (electrospray ionization tandem MS), we were able to demonstrate either elevations or alterations in the individual species of BMP, but not of PG, in cultured fibroblasts. All affected cell lines, with the exception of Fabry disease, showed a loss of polyunsaturated BMP species relative to mono-unsaturated species, and this correlated with the literature reports of lysosomal dysfunction leading to elevations of glycosphingolipids and cholesterol in affected cells, processes thought to be critical to the pathogenesis of LSDs. Plasma samples from patients with LSDs involving storage in macrophages and/or with hepatomegaly showed an elevation in the plasma concentration of the C(18:1)/C(18:1) species of BMP when compared with control plasmas, whereas disorders involving primarily the central nervous system pathology did not. These results suggest that the release of BMP is cell/tissue-specific and that it may be useful as a biomarker for a subset of LSDs.
Biochemical Journal 05/2008; 411(1):71-8. · 4.90 Impact Factor
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ABSTRACT: Glucosylceramide (GC) is a metabolic intermediate derived from the cellular turnover of membrane gangliosides and globosides. The catabolism of GC is impaired in Gaucher disease (GD) and consequently GC accumulates in affected cells leading to clinical manifestations of GD. The primary cell type affected in GD is the macrophage, and we investigated what effect excess GC has on the spatial coordination of other sphingolipids and phospholipids in a macrophage model of GD. A THP-1 macrophage model of GD was established by supplementation of the culture media with conduritol B epoxide, a specific irreversible inhibitor of acid beta-glucosidase. This cell model accumulated up to 12-fold more GC compared with untreated cells. Sub-cellular fractionation showed that, initially, the primary site of GC accumulation was the lysosome but as more GC accumulated it distributed relatively evenly across the cell and was present in all sub-cellular fractions. We also observed secondary elevations in the concentrations of ceramide, di- and trihexosylceramide and phosphatidylglycerol, which all had similar sub-cellular distributions to that of GC, initially increasing in the lysosome and then throughout the sub-cellular compartments. Our results suggest that with excess GC accumulation, the pathway trafficking GC to the lysosome becomes saturated and GC as well as other sphingolipids are shunted to other parts of the cell. The presence of these sphingolipids at non-physiological concentrations is likely to interfere with other biochemical pathways outside the lysosome, leading to cell dysfunction and ultimately pathological mechanisms apparent in GD, such as macrophage activation.
Molecular Genetics and Metabolism 01/2008; 92(4):336-45. · 3.19 Impact Factor
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ABSTRACT: Lysosomal storage disorders are a group of genetic diseases, each with a broad spectrum of clinical presentation that ranges from attenuated to severe. The immunochemical analysis of patient samples is aimed at several key aspects of patient management, including early detection of the disorder, prediction of clinical severity, determining the most appropriate therapeutic regimen, and monitoring of patients on therapy.
In this study, we review the current and emerging technology available to achieve these assessments.
Immune assays have direct practical application for the early detection, diagnosis and prognosis of lysosomal storage disorder patients. Multiplexing of these assays may provide a platform to allow newborn screening for multiple lysosomal storage disorders.
We have reviewed the immunochemical techniques available for the analysis of lysosomal storage disorder patient samples and advise that these may be used in conjunction with other technologies for effective patient management.
Clinical Chemistry 10/2006; 52(9):1660-8. · 7.91 Impact Factor
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ABSTRACT: alpha-Glucosidase (EC 3.2.1.3) is a lysosomal enzyme that hydrolyses alpha-1,4- and alpha-1,6-linkages of glycogen to produce free glucose. A deficiency in alpha-glucosidase activity results in glycogen storage disorder type II (GSD II), also called Pompe disease. Here, d-glucose was shown to be a competitive inhibitor of alpha-glucosidase and when added to culture medium at 6.0 g/L increased the production of this protein by CHO-K1 expression cells and stabilised the enzyme activity. D-Glucose also prevented alpha-glucosidase aggregation/precipitation and increased protein yield in a modified purification scheme. In fibroblast cells, from adult-onset GSD II patients, D-glucose increased the residual level of alpha-glucosidase activity, suggesting that a structural analogue of d-glucose may be used for enzyme enhancement therapy.
FEBS Letters 09/2006; 580(18):4365-70. · 3.54 Impact Factor
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Peter J Meikle,
Dallas J Grasby,
Caroline J Dean,
Debbie L Lang,
Michelle Bockmann,
Alison M Whittle,
Michael J Fietz,
Henrik Simonsen,
Maria Fuller,
Douglas A Brooks,
John J Hopwood
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ABSTRACT: Lysosomal storage disorders (LSD) are chronic progressive diseases that have a devastating impact on the patient and family. Most patients are clinically normal at birth but develop symptoms early in childhood. Despite no curative treatment, a number of therapeutic options are available to improve quality of life. To achieve this, there is a pressing need for newborn screening to identify affected individuals early, before the onset of severe irreversible pathology. We have developed a multiplexed immune-quantification assay of 11 different lysosomal proteins for the identification of individuals with an LSD and evaluated this assay in a retrospective study using blood-spots from; newborns subsequently diagnosed with an LSD (n=19, six different LSD), individuals sampled after diagnosis of an LSD (n=92, 11 different LSD), newborn controls (n=433), and adult controls (n=200). All patients with mucopolysaccharidosis type I (MPS I), MPS II, MPS IIIA, MPS VI, metachromatic leukodystrophy, Niemann-Pick disease type A/B, and multiple sulfatase deficiency could be identified by reduced enzyme levels compared to controls. All mucolipidosis type II/III patients were identified by the elevation of several lysosomal enzymes, above the control range. Most Fabry, Pompe, and Gaucher disease patients were identified from either single protein differences or profiles of multiple protein markers. Newborn screening for multiple LSD is achievable using multiplexed immune-quantification of a panel of lysosomal proteins. With further validation, this method could be readily incorporated into existing screening laboratories and will have a substantial impact on patient management and counseling of families.
Molecular Genetics and Metabolism 09/2006; 88(4):307-14. · 3.19 Impact Factor
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ABSTRACT: Heparan sulfate is a linear glycosaminoglycan with considerable structural diversity that binds a myriad of growth factors and proteins that play pivotal roles in a variety of biological processes. We have investigated the structural complexity of partially degraded fragments of heparan sulfate in mucopolysaccharidosis type IIIA in which there is a defect in heparan sulfate catabolism. Mono- to hexadecasaccharides were isolated from the urine of a mucopolysaccharidosis IIIA patient and shown to have non-reducing end glucosamine N-sulfate residues, reflecting the catabolic deficiency in heparan N-sulfatase (sulfamidase) activity. The use of nitrous acid digestion (pH 1.5) combined with separation by reverse-phase high-performance liquid chromatography and analysis by electrospray ionization-mass spectrometry identified multiple forms of these oligosaccharides with some N-acetylated glucosamine residues and one to three sulfates per disaccharide. Furthermore, we demonstrated that each oligosaccharide existed in multiple sulfated forms. Many structural isomers were present, suggesting a complex mixture of oligosaccharides present in the urine as a consequence of a defect in heparan sulfate degradation.
Analytical Chemistry 08/2006; 78(13):4534-42. · 5.86 Impact Factor
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ABSTRACT: Mucopolysaccharidosis type II (MPS II) is a lysosomal storage disorder related to a deficiency in the enzyme iduronate-2-sulfatase (IDS). Clinical trials of enzyme replacement therapy are in progress, but effective treatment will require screening assays to enable early detection and diagnosis of MPS II. Our study evaluated the diagnostic accuracy of IDS protein and enzyme activity measurements in dried blood spots and plasma.
We collected dried-blood-spot and plasma samples from unaffected control individuals and from MPS II patients. We measured IDS protein concentration with a 2-step time-delayed dissociation-enhanced lanthanide fluorescence immunoassay. To measure enzyme activity, we immobilized anti-IDS antibody on microtiter plates to capture the enzyme and measured its activity with the fluorogenic substrate 4-methylumbelliferyl sulfate.
Dried-blood-spot samples from MPS II patients showed an almost total absence of IDS activity (0-0.075 micromol x h(-1) x L(-1)) compared with control blood spots (0.5-4.7 micromol x h(-1) x L(-1)) and control plasma (0.17-8.1 micromol x h(-1) x L(-1)). A dried-blood-spot sample from only 1 of 12 MPS II patients had detectable concentrations of IDS protein (24.8 microg/L), but no IDS protein was detected in plasma from MPS II patients. Ranges for IDS protein in control samples were 25.8-153 microg/L for blood spots and 22.8-349.4 microg/L for plasma.
Measurement of the IDS protein concentration and enzyme activity (as measured by a simple fluorogenic assay with an immune capture technique) enables identification of the majority of MPS II patient samples from both dried blood spots and plasma samples.
Clinical Chemistry 05/2006; 52(4):643-9. · 7.91 Impact Factor
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ABSTRACT: Within cells, dermatan sulfate (DS) and heparan sulfate (HS) are degraded in two steps. The initial endohydrolysis of these polysaccharides is followed by the sequential action of lysosomal exoenzymes to reduce the resulting oligosaccharides to monosaccharides and inorganic sulfate. Mucopolysaccharidosis (MPS) type II is a lysosomal storage disorder caused by a deficiency of the exoenzyme iduronate-2-sulfatase (I2S). Consequently, partially degraded fragments of DS and HS have been shown to accumulate in the lysosomes of affected cells and are excreted in the urine. Di- to hexadecasaccharides, isolated from the urine of a MPS II patient using anion exchange and gel filtration chromatography, were identified using electrospray ionization-tandem mass spectrometry (ESI-MS/MS). These oligosaccharides were shown to have non-reducing terminal iduronate-2-sulfate residues by digestion with recombinant I2S. A pattern of growing oligosaccharide chains composed of alternating uronic acid and N-acetylhexosamine residues was identified and suggested to originate from DS. A series of oligosaccharides consisting of hexosamine/N-acetylhexosamine alternating with uronic acid residues was also identified and on the basis of the presence of unacetylated hexosamine; these oligosaccharides are proposed to derive from HS. The presence of both odd and even-length oligosaccharides suggests both endo-beta-glucuronidase and endo-N-acetylhexosaminidase activities toward both glycosaminoglycans. Furthermore, the putative HS oligosaccharide structures identified indicate that heparanase activities are directed toward regions of both low and high sulfation, while the N-acetylhexosaminidase activity acted only in regions of low sulfation in this polysaccharide.
Glycobiology 05/2006; 16(4):318-25. · 3.58 Impact Factor
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ABSTRACT: Enzyme replacement therapy (ERT) has proven to be an effective therapy for some lysosomal storage disorder (LSD) patients. A potential complication during ERT is the generation of an immune response against the replacement protein. We have investigated the antigenicity of two distantly related glycosidases, alpha-glucosidase (Pompe disease or glycogen storage disease type II, GSD II), and alpha-L-iduronidase (Hurler syndrome, mucopolysaccharidosis type I, MPS I). The linear sequence epitope reactivity of affinity purified polyclonal antibodies to recombinant human alpha-glucosidase and alpha-L-iduronidase was defined, to both glycosidases. The polyclonal antibodies exhibited some cross-reactive epitopes on the two proteins. Moreover, a monoclonal antibody to the active site of alpha-glucosidase showed cross-reactivity with a catalytic structural element of alpha-L-iduronidase. In a previous study, in MPS I patients who developed an immune response to ERT, this same site on alpha-L-iduronidase was highly antigenic and the last to tolerise following repeated enzyme infusions. We conclude that glycosidases can exhibit cross-reactive epitopes, and infer that this may relate to common structural elements associated with their active sites.
FEBS Letters 02/2006; 580(1):87-92. · 3.54 Impact Factor
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ABSTRACT: Lysosomal storage diseases (LSDs) comprise a group of at least 50 distinct genetic diseases, each one resulting from a deficiency of a particular lysosomal protein/activity or, in a few cases, from non-lysosomal activities that are involved in lysosomal biogenesis or protein maturation. Fabry disease is the second most common of the LSDs, after Gaucher disease. The reported epidemiological data are likely to be underestimates, due to missed diagnoses of these rare disorders. The positive and negative outcomes of newborn screening for Fabry disease and LSDs in general are considered. Early diagnosis and intervention before the onset of irreversible pathology will provide a substantial benefit to many of these newborns, as well as providing the opportunity for parents to receive genetic counselling. However, there can also be potential harm to the parent/newborn relationship as a consequence of knowing that the baby has an incurable disorder.
01/2006; , ISBN: 190353903X
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Clinical Chemistry 12/2005; 51(11):2200-2. · 7.91 Impact Factor
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ABSTRACT: A method to semiquantify urinary oligosaccharides from patients suffering from oligosaccharidurias is presented. 1-Phenyl-3-methyl-5-pyrazolone has been used to derivatize urinary oligosaccharides prior to analysis by electrospray ionization-tandem mass spectrometry (ESI-MS/MS). Disease-specific oligosaccharides were identified for several oligosaccharidurias, including GM1 gangliosidosis, GM2 gangliosidosis, sialic acid storage disease, sialidase/neuraminidase deficiency, galactosialidosis, I-cell disease, fucosidosis, Pompe and Gaucher diseases, and alpha-mannosidosis. The oligosaccharides were referenced against the internal standard, methyl lactose, to produce ratios for comparison with control samples. Elevations in specific urinary oligosaccharides were indicative of lysosomal disease and the defective catabolic enzyme. This method has been adapted to enable assay of large sample numbers and could readily be extended to other oligosaccharidurias and to monitor oligosaccharide levels in patients receiving treatment. It also has immediate potential for incorporation into a newborn screening program.
Analytical Biochemistry 11/2005; 345(1):30-46. · 3.00 Impact Factor
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ABSTRACT: Pompe disease is an autosomal recessive disorder of glycogen metabolism resulting from a deficiency of acid alpha-glucosidase. Pompe disease can present within a broad clinical spectrum, from the severe infantile to the attenuated adult onset phenotypes. Early diagnosis, in the form of newborn screening has been proposed. However, in the absence of clinical symptoms, prediction of disease severity and progression will be critical to provide appropriate management and treatment of affected individuals.
We have used sensitive immune-assays to measure levels of acid alpha-glucosidase protein and activity in cultured skin fibroblasts and a new glycogen assay to specifically determine the lysosomal accumulation of glycogen in the same cells. These markers were assessed for their ability to predict age of onset.
Acid alpha-glucosidase activity and specific activity as well as lysosomal glycogen showed significant correlations with age of onset, with acid alpha-glucosidase activity having the highest Spearman correlation coefficient (0.887, p<0.001). Lysosomal glycogen accumulated only in cells from infantile and juvenile patients but not from adult-onset patients. However, cells from adult-onset patients had relatively low cytoplasmic glycogen compared to control individuals and other forms of the disease.
Acid-alpha-glucosidase activity and specific activity, and lysosomal glycogen content are useful predictors of age of onset in Pompe disease.
Clinica Chimica Acta 11/2005; 361(1-2):191-8. · 2.54 Impact Factor
