Enzyme Substrates for the Diagnosis of
Lysosomal Storage Disorders (LSDs)

Tools for the diagnosis of Fabry Disease, Fucosidosis, Gaucher Disease, Mannosidosis, Mucopolysaccharidosis type I, type IV, type VII, and Pompe Disease

Fabry Disease

Fabry disease is an x-linked lysosomal disorder caused by a deficient activity of the hydrolase alpha-galactosidase A, which cleaves terminal alpha-galactosyl moieties from various glycoconjugates. The enzyme deficiency causes a progressive lysosomal deposition of globotriaosylceramide in cells throughout the body. The clinical findings are represented by pain and paresthesias in the extremities, angiokeratoma in skin and mucous membranes and impaired sweating during childhood or adolescence. Corneal and lenticular opacities may be present. Proteinuria, renal impairment, cardiac and neurological lesions and hypertension develop with time. Fabry disease most often affects males, although clinial manifestations in female heterozygotes can range from asymptotic to a disease as severe as that suffered by hemizygous affected males.
Enzyme replacement therapy has become available and has been shown to reduce lipid depositions in tissue biopsies.


Initially, the diagnosis of Fabry disease is based on the presence of the symptoms. In males, the most efficient and reliable method for confirmation of the diagnosis is the demonstration of deficient alpha-galactosidase A enzyme activity in plasma, isolated leukocytes, and/or cultured cells. The test is a fluorometric assay using the artificial substrate 4-methylumbelliferyl-alpha-D-galactopyranoside (M-5544). Males with classic Fabry disease have less than 1% alpha-galactosidase A activity. In females, measurement of alpha-galactosidase A activity is unreliable, although demonstration of decreased alpha-galactosidase activity is diagnostic of the carrier state, many carrier females have normal alpha-galactosidase A activity. Molecular genetic testing of the GLA gene using complete gene sequencing is the most reliable method for the diagnosis of carrier females.

1. Fuller M, Lovejoy M, Brooks DA, Harkin ML, Hopwood JJ, Meikle PJ. Immunoquantification of alpha-galactosidase: evaluation for the diagnosis of Fabry disease. Clin Chem. 2004;50:1979-85
2. Mayes JS, Scheerer JB, Sifers RN, Donaldson ML. Differential assay for lysosomal alpha-galactosidases in human tissues and its application to Fabry's disease. Clin Chim Acta. 1981;112:247-51


Fucosidosis is an autosomal recessive lysosomal storage disease caused by defective alpha-L-fucosidase resulting in accumulation of certain fucose-containing complex compounds (i.e., glycosphingolipids, glycolipids, and glycoproteins) in many tissues of the body. Different phenotypes include clinical features such as neurologic deterioration, growth retardation, visceromegaly, seizures in a severe early form and coarse facial features.


Reduced alpha-L-fucosidase activity is measured using the synthetic substrate, 4-methylumbelliferyl-alpha-L-fucoside (M-5539). The asssay is performed on white blood cells or on cultured skin fibroblasts. Prenatal diagnosis can be performed on cultured amniotic fluid cells or chorionic villus cells.

1. Beratis NG, Turner BM, Hirschhorn K. Fucosidosis: detection of the carrier state in peripheral blood leukocytes. J Pediatr. 1975;87:1193-8.
2. Beratis NG, Hirschhorn K. Alpha-L-fucosidase in cultured bone marrow fibroblasts from fucosidosis patients. Am J Med Genet. 1978;2:23-9.

Gaucher Disease

Gaucher disease is a rare autosomal recessive disorder characterized by defective function of the catabolic enzyme beta-glucocerebrosidase leading to an accumulation of glucocerebroside in cells of the monocyte-macrophage system. Although rare, Gaucher disease is the most common inherited (genetic) lysosomal storage disease. Gaucher disease is characterized by a wide array of different symptoms and the severity of the disease ranges from undetectable to lethal. Symptoms include hepatomegaly and splenomegaly, anemia, neutropenia and thrombocytopenia and osteoporosis. Neurological symptoms occur only in some types of Gaucher's. Enzyme replacement therapy is available for type 1 and most type 3 patients and can decrease or reverse many of the complications.


Leukocyte and beta-glucocerebrosidase enzyme assay is done to assess glucocerebrosidase activity in an assay using a synthetic substrate, 4-methylumbelliferyl-beta-D-glucoside (M-5650). 30% lower activity at pH 4.0 compared to normal is indicative of Gaucher. Identifying patient's enzyme activity is also necessary to introduce an appropriate therapy. Further diagnostic tests are bone marrow biopsy and genetic testing.

1. Daniels LB, Glew RH. beta-Glucosidase assays in the diagnosis of Gaucher's disease. Clin Chem. 1982;28:569-77
2. Hakansson G, Dreborg S, Lindsten J, Svennerholm L. Assay of the beta-glucosidase activity with natural labelled and artificial substrates in cultivated skin fibroblasts from homozygotes and heterozygotes with the Norrbottnian type of Gaucher disease. Clin Genet. 1980;18:268-73
3. Peters SP, Lee RE, Glew RH. A microassay for Gaucher's disease. Clin Chim Acta. 1975;60:391-6
4. Wenger DA, Clark C, Sattler M, Wharton C. Synthetic substrate beta-glucosidase activity in leukocytes: a reproducible method for the identification of patients and carriers of Gaucher's disease. Clin Genet. 1978;13:145-53


Alpha-mannosidosis is an autosomal recessive lysosomal storage disease caused by a deficiency of lysosomal alpha-mannosidase activity. Because of this deficiency, glycopeptides are accumulated within the lysosomes leading to the impairment of the cell or organ function. The disorder shows a wide range of clinical phenotypes, ranging from very serious to very mild forms. Symptoms include coarse facial features, mental retardation, immune deficiency, skeletal malformation and muscular pain and weakness. There are also rare cases of beta-mannosidosis caused by beta-mannosidase deficiency.


Impaired alpha-mannosidase or beta-mannosidase activity can be assessed using the synthetic fluorogenic substrate, 4-methylumbelliferyl-alpha-D-mannoside or 4-methylumbelliferyl-beta-D-mannoside, respectively (M-5730,M-5738). The asssay is performed on white blood cells or on cultured skin fibroblasts. Prenatal diagnosis can be performed on cultured amniotic fluid cells or chorionic villus cells.

1. Petushkova NA, Ivleva TS, Vozniy YaM. Human chorionic beta-mannosidase: comparison with beta-mannosidase from human cultured fibroblasts.Prenat Diagn. 1992;12:835-9.
2. Prence EM, Natowicz MR. Diagnosis of alpha-mannosidosis by measuring alpha-mannosidase in plasma. Clin Chem. 1992;38:501-3.

Mucopolysaccharidosis type I

l Mucopolysaccharidoses (MPS disorders) are a group of rare genetic disorders of polysaccharide metabolism caused by the deficiency of one of ten specific lysosomal enzymes. The accumulation of undegraded mucopolysaccharides in the cells causes a number of physical abnormalities.
Mucopolysaccharidosis type I (MPS I) is caused by a deficiency of the enzyme alpha-L-iduronidase, an enzyme which is involved in the breakdown of dermatan and heparan sulphates. The leading symptoms of the most severe form of MPS I, often called Hurler syndrome, are skeletal deformities and a delay in motor and mental development. Patients with the milder form (Scheie disease) are of almost normal height and do not show mental retardation. Typical symptoms are stiff joints, corneal opacities, carpal tunnel syndrome and mild skeletal changes. A great number of mutations of the alpha-L-iduronidase gene has been detected, explaing the broad phenotypic heterogeneity of the disease.
Enzyme replacement therapy using recombinant human alpha-L-iduronidase has become available and is effective in arresting progression of Hurler syndrome.


Increased urinary excretion of dermatan- and heparan sulphate are indicative for alpha-L-iduronidase deficiency. For confirmation of the diagnosis, alpha-L-iduronidase activity in leukocytes or cultured fibroblasts is measured. Alpha-L-iduronidase activity is determined in a fluorometric enzyme assay measuring 4-MU released by hydrolysis of the fluorogenic substrate 4-methylumbelliferyl-alpha-L-iduronide (M-5536). This sensitive enzyme substrate is also appropriate for purification and characterization of alpha-L-iduronidase.

1. Beesley CE, Meaney CA, Greenland G, Adams V, Vellodi A, Young EP, Winchester BG. Identification and characterization of 13 new mutations in mucopolysaccharidosis type I patients. Hum Genet. 2001;109(5):503-11.
2. Butterworth J, Broadhead DM. Comparison of the substrate 4-methylumbelliferyl-alpha-l-iduronide with phenyl-alpha-L-iduronide for the diagnosis of Hurler's disease in cultured cells. J Inherit Metab Dis. 1980;2(3):71-4.
3. Den Tandt WR, Scharpe S. Characteristics of leukocyte and plasma methylumbelliferyl-alpha-L-iduronide iduronidase. Biochem Med Metab Biol. 1989;41(1):18-24.
4. Hopwood JJ, Muller V, Smithson A, Baggett N. A fluorometric assay using 4-methylumbelliferyl alpha-L-iduronide for the estimation of alpha-L-iduronidase activity and the detection of Hurler and Scheie syndromes. Clin Chim Acta. 1979;92(2):257-65.
5. Isemura M, Kosaka H, Ikenaka T, Kido R, Yoshimura T. Fluorometric measurement of urinary alpha-L-iduronidase activity. J Biochem (Tokyo). 1978;84(3):627-32.
6. Minami R, Watanabe Y, Kudoh T, Oyanagi K, Nakao T. Fluorometric measurement of alpha-L-iduronidase activity using 4-methylumbelliferyl-alpha-L-iduronide. Tohoku J Exp Med. 1980;130(4):381-4.
7. Schuchman EH, Guzman NA, Desnick RJ. Human alpha-L-iduronidase. I. Purification and properties of the high uptake (higher molecular weight) and the low uptake (processed) forms. J Biol Chem. 1984;259(5):3132-40.
8. Tsvetkova IV, Karpova EA, Voznyi YV, Zolotukhina TV, Biryukov VV, Semyachkina AN. Use of 4-trifluoromethylumbelliferyl-alpha-L-iduronide as a new substrate for detection of alpha-L-iduronidase deficiency in human tissues and for rapid prenatal diagnosis of Hurler disease. J Inherit Metab Dis. 1991;14(2):134-9.

Mucopolysaccharidosis type IV

Mucopolysaccharidosis type IV (Morquio syndrome) is the most common MPS disorder. Two forms of mucopolysaccharidosis type IV are recognized, type A and type B. Type A disease (Morquio syndrome type A) is characterized by the absence of the enzyme galactose-6-sulphate sulphatase. Mucopolysaccharidosis type IV B (Morquio syndrome type B) results from a deficiency of the enzyme beta-galactosidase. The symptoms that are caused by an abnormal accumulation of keratan sulfate in the body include growth retardation, mildly coarse facial features, hearing loss, corneal opacities, valvular heart disease, frequent upper respiratory infections, restrictive lung disease and mild hepatomegaly.


Beta-galactosidase activity can be measured using the artificial fluorogenic substrate 4-methylumbelliferyl-beta-D-galactopyranoside (M-5550). 4-methylumbelliferyl-beta-D-galactopyranoside-6-sulfate (M-5558) can be used to assess galactose-6-sulphate sulphatase activity.

1. van Diggelen OP, Zhao H, Kleijer WJ, Janse HC, Poorthuis BJ, van Pelt J, Kamerling JP, Galjaard H. A fluorimetric enzyme assay for the diagnosis of Morquio disease type A (MPS IV A). Clin Chim Acta. 1990;187(2):131-9.
2. Groebe H, Krins M, Schmidberger H, von Figura K, Harzer K, Kresse H, Paschke E, Sewell A, Ullrich K. Morquio syndrome (mucopolysaccharidosis IV B) associated with beta-galactosidase deficiency. Report of two cases. Am J Hum Genet. 1980;32(2):258-72.
3. van der Horst GT, Kleijer WJ, Hoogeveen AT, Huijmans JG, Blom W, van Diggelen OP. Morquio B syndrome: a primary defect in beta-galactosidase. Am J Med Genet. 1983;16(2):261-75.
4. Kleijer WJ, Geilen GC, Garritsen V, Huijmans JG, Los FJ, Voznyi YV, van Diggelen OP. First-trimester diagnosis of Morquio disease type A. Prenat Diagn. 2000;20(3):183-5.
5. Okumiya T, Sakuraba H, Kase R, Sugiura T. Imbalanced substrate specificity of mutant beta-galactosidase in patients with Morquio B disease. Mol Genet Metab. 2003;78(1):51-8.
7. Zhao H, Van Diggelen OP, Thoomes R, Huijmans J, Young E, Mazurczak T, Kleijer WJ. Prenatal diagnosis of Morquio disease type A using a simple fluorometric enzyme assay. Prenat Diagn. 1990;10(2):85-91.
8. Zhao H, van Diggelen OP, Kleijer WJ, Li P. Enzymatic diagnosis of Morquio A syndrome with a new fluorimetric substrate. Chin Med Sci J. 1991;6(1):9-13.

Mucopolysaccharidosis type VII

Mucoploysaccaridosis type VII (Sly syndrome) is a very rare autosomal recessive disorder characterized by defective function of the enzyme beta-glucuronidase that leads to an accumulation of dermatan sulfate, heparan sulfate and chondroitin sulfate in many tissues and organs of the body including the central nervous system.
The symptoms of mucopolysaccharidosis type VII are similar to those of type I and may include mental retardation, short stature with an unusually short trunk, abnormalities of the intestines, corneas of the eyes, and the skeletal system.


Beta-glucuronidase activity can be measured using the artificial fluorogenic substrate 4-methylumbelliferyl-beta-D-glucuronide (M-5700,M-5704).

1. Glaser JH, Sly WS. Beta-glucuronidase deficiency mucopolysaccharidosis: methods for enzymatic diagnosis. J Lab Clin Med. 1973;82(6):969-77.

Pompe Disease

Pompe disease is an autosomal recessive disorder of glycogen metabolism caused by a deficiency or dysfunction of the lysosomal hydrolase acid alpha-glucosidase (GAA). GAA is an enzyme found in tissues that is responsible for the degradation of a small percentage of glycogen. A deficiency of GAA enzyme activity results in the excessive storage of glycogen in lysosomes in multiple tissues, with cardiac and skeletal muscle tissues most seriously affected. Eventually this accumulation leads to significant cardiomyopathy (in infantile-onset cases) and generalized muscle weakness.


Given the wide range of clinical presentations for Pompe disease, the clinical paths to diagnosis tend to vary. A conclusive diagnosis generally requires an enzyme assay test to demonstrate reduced or absent activity for the lysosomal enzyme acid alpha-glucosidase in cultured skin fibroblasts, lymphocytes, or muscle tissue. GAA activity can be assessed using the artificial fluorogenic substrate 4-Methylumbelliferyl-alpha-D-glucopyranoside (M-5600). Patients with infantile-onset Pompe disease generally demonstrate less than 1%, patients with late-onset less than 40% of normal GAA enzyme activity.

1. Okumiya T, Keulemans JL, Kroos MA, Van der Beek NM, Boer MA, Takeuchi H, Van Diggelen OP, Reuser AJ. A new diagnostic assay for glycogen storage disease type II in mixed leukocytes. Mol Genet Metab. 2006;88:22-8.
2. Umapathysivam K, Hopwood JJ, Meikle PJ. Determination of acid alpha-glucosidase activity in blood spots as a diagnostic test for Pompe disease. Clin Chem. 2001;47:1378-83.
3. Zhang H, Kallwass H, Young SP, Carr C, Dai J, Kishnani PS, Millington DS, Keutzer J, Chen YT, Bali D. Comparison of maltose and acarbose as inhibitors of maltase-glucoamylase activity in assaying acid alpha-glucosidase activity in dried blood spots for the diagnosis of infantile Pompe disease. Genet Med. 2006;8:302-6.

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