Enzyme analysis for Pompe disease in leukocytes; superior results with natural substrate compared with artificial substrates

Abstract

Enzyme analysis for Pompe disease in leukocytes has been greatly improved by the introduction of acarbose, a powerful inhibitor of interfering alpha-glucosidases, which are present in granulocytes but not in lymphocytes. Here we show that the application of acarbose in the enzymatic assay employing the artificial substrate 4-methylumbelliferyl-alpha-D: -glucoside (MU-alphaGlc) is insufficient to clearly distinguish patients from healthy individuals in all cases. Also, the ratios of the activities without/with acarbose only marginally discriminated Pompe patients and healthy individuals. By contrast, when the natural substrate glycogen is used, the activity in leukocytes from patients (n = 82) with Pompe disease is at most 17% of the lowest control value. The use of artificial substrate in an assay with isolated lymphocytes instead of total leukocytes is a poor alternative as blood samples older than one day invariably yield lymphocyte preparations that are contaminated with granulocytes. To diagnose Pompe disease in leukocytes we recommend the use of glycogen as substrate in the presence of acarbose. This assay unequivocally excludes Pompe disease. To also exclude pseudo-deficiency of acid alpha-glucosidase caused by the sequence change c.271G>A (p.D91N or GAA2; homozygosity in approximately 1:1000 caucasians), a second assay employing MU-alphaGlc substrate plus acarbose or DNA analysis is required.

Urine analysis of glucose tetrasaccharide by HPLC; a useful marker for the investigation of patients with Pompe and other glycogen storage diseases

“…However, the existence of pseudo deficiency mutations increases the likeliness of false positive results being obtained (Diggelen et al 2009). The finding that Glc 4 is only marginally elevated when compared to untreated Pompe patients raises the possibility that urinary Glc 4 measurements may be a useful adjunct to a diagnostic enzyme service.…”

“…However, for some patients significant residual GAA activity can make the definitive diagnosis of Pompe disease difficult (van Diggelen et al 2009). In recent years, a new method has been developed to allow the rapid determination of GAA (+/− acarbose) in dried blood spots.…”

PAS‐positive lymphocyte vacuoles can be used as diagnostic screening test for Pompe disease

“…The test result should always be considered in combination with other results—for example, creatine kinase (CK), alanine aminotransferase (ALAT), and aspartate aminotransferase (ASAT) (Winkel et al 2005)—and a careful clinical examination. On the other hand, when a high percentage of PAS-positive lymphocyte vacuoles is found, further testing by measuring acid α-glucosidase activity in leukocytes (van Diggelen et al 2009) is always indicated. This fits into previously proposed diagnostic algorithms for childhood- and adult-onset Pompe disease (Straub 2008b).…”

“…Milder forms of the disease may present at any age, with limb-girdle weakness as the most prominent clinical feature (Hirschhorn and Reuser 2000; van der Ploeg and Reuser 2008). The diagnosis of Pompe disease is confirmed by measuring acid α-glucosidase activity in leukocytes using the latest optimized procedures, or in cultured fibroblasts (Hirschhorn and Reuser 2000; van Diggelen et al 2009; Winchester et al 2008). These tests, however, are carried out in a specialized laboratory and are usually not applied unless there is already a strong suspicion for Pompe disease.…”

Remarkably low fibroblast acid α-glucosidase activity in three adults with Pompe disease

“…The GAA activity in leukocytes was measured with glycogen as substrate in the presence of acarbose to inhibit the glucoamylase activity (Glyc + ACAB) in accordance with previously described procedures [17,18]. The GAA activity in fibroblasts and muscle was measured with 4-methylumbelliferyl-α-D-glucopyranoside (4-MU) and/or glycogen as substrates [8].…”