- Email: [email protected]
Increased substrate concentration boosts enzyme activity levels of fluorometric α-l-iduronidase enzyme activity assay
S48
Abstracts
96 A multi-national, randomized, double-blind, placebo-controlled study to evaluate the efficacy and safety of BMN 110 treatment for mucopolysaccharidosis IVA (Morquio syndrome type A) Christian Hendriksza, Barbara K. Burtonb, Thomas Flemingc, Roberto Giuglianid, Paul Harmatze, Derralynn Hughesf, Simon Jonesg, Shuan-Pei Linh, Karl Eugen Mengeli, Maurizio Scarpaj, Vassili Valayannopoulosk, a University of Manchester, Salford Royal NHS Foundation Trust, Birmingham Children's Hospital NHS Foundation Trust, Salford, Lancashire, UK, bAnn and Robert H. Lurie Children's Hospital and Northwestern University Feinberg School of Medicine, Chicago, IL, USA, cSchool of Public Health, University of Washington, WA, USA, dHospital de Clinicas de Porto Alegre, Porto Alegre, Brazil, eChildren's Hospital and Research Center, Oakland, CA, USA, f University College London, London, UK, gCentral Manchester University Hospital, Manchester, United Kingdom, hMacKey Memorial Hospital, Taipei, Taiwan, iUniversity of Mainz, Mainz, Germany, jUniversity of Padova, Padova, Italy, kChu Paris, Hôpital Necker-Enfants Malades, Paris France Mucopolysaccharidosis IVA is caused by a deficiency in Nacetylgalactosamine-6 sulfatase (GALNS) activity resulting in pathologic lysosomal accumulation of keratan sulfate (KS), leading to multiple clinical manifestations. A placebo-controlled, multinational randomized trial was conducted to evaluate efficacy and safety of recombinant human GALNS (BMN110) in 176 patients with mucopolysaccharidosis IVA, ≥5 years of age. For 6 minute walk distance at 24 weeks, relative to the change in placebo, the 2 mg/kg/wk regimen provided a statistically significant increase in mean distance of 22.5 m (95% CI: 4.0, 40.9; p = 0.0174), but the 2 mg/kg/qow regimen provided an increase of only 1.4 m (95% CI: −17.8, 18.9). For 3 minute stair climb at 24 weeks, relative to placebo, the 2 mg/kg/wk regimen had a mean increase of 1.1 stairs/minute (95% CI: −2.1, 4.4) while the 2 mg/kg/qow regimen had a mean decrease of 0.5 stairs/minute (95% CI: −3.7, 2.8). For both regimens, urinary KS was substantially reduced. Adverse events (AE) to BMN110 were similar to those seen in clinical trials of other enzyme replacement therapies. Serious AE related to study drug occurred in 3.4% on weekly dosing of BMN110 and 0% on placebo. In the weekly dosing group, 17 (1.3%) of 1345 infusions were interrupted due to AE. All patients subsequently resumed dosing. doi:10.1016/j.ymgme.2012.11.110
97 Increased substrate concentration boosts enzyme activity levels of fluorometric α-l-iduronidase enzyme activity assay Tyler Herzog, Li Ou, Chester Whitley, University of Minnesota, Minneapolis, Minnesota, USA Background: Mucopolysaccharidosis type I (MPS I) is a lysosomal disease due to deficiency of the enzyme α-L-iduronidase which results in the accumulation of the glycosaminoglycans dermatan sulfate and heparan sulfate. Current efforts in assessing the severity of this disease employ a fluorometric assay involving the fluorescent substrate 4-methylumbelliferyl-α-L-iduronide (4MU-iduronide) to determine enzyme activity present in samples. An early assay used a final reaction concentration of 0.2 μM and in this same study the Michaelis-Menten constants (Km) for fibroblasts and leukocytes were 0.147 μM and 0.179 μM, respectively (Hopwood et al. 1978). Km is the concentration of 4MU-iduronide which produces a reaction velocity of ½-maximal velocity (Vmax), but higher concentrations closer to enzyme saturation are generally recommended for reliable assays. At lower substrate concentrations, variations in substrate concentration result in relatively large errors in measured
activity because the lower substrate concentrations are on the steepest part of the curve. The highest substrate concentration cited in the literature is 2.85 mM (Whitley et al. 1987), but is relatively expensive for high-volume studies. In other assays, final substrate concentrations have included 25 M (Kakkis et al. 1993; Zheng et al. 2003), 50 M (Liu et al. 2004), 2 mM (Dimenico et al. 2005), and 2.85 mM (Whitley et al. 1987; Hartung et al. 2004) Hypothesis: Given that 4MU-iduronide concentrations for enzyme activity assays in the literature are varied, we hypothesized that increasing the substrate concentration towards the Km and correcting for Km using the Michaelis–Menten equation would lead to more accurate enzyme activity values. Materials and Methods: Blood samples were collected from MPS I mice and heterozygotic littermates. Plasma and serum was obtained. An equal amount of homogenized sample and 4MU-iduronide, with a final reaction concentration of 25 M, 50 M, or 180 M, were mixed and incubated for 60 minutes at 37 °C in an Applied Biosystems 2720 Thermal Cycler. The reaction was stopped using a glycine carbonate buffer, and fluorescence was determined on a BioTek SynergyMx 96well plate reader. In addition, the reaction between a sample and 180 M 4MUI substrate was stopped every 10 minutes for 2 hours to determine when the reaction was linear. The Michaelis-Menten equation was used to calculate Vmax based on the Km, fluorescent substrate concentration, and initial velocity from our studies. This Vmax was used for enzyme activity calculations. Results: IDUA activity increased with each successively higher substrate concentration (1.17 U/ml, 1.75 U/ml, and 3.83 U/ml for plasma) in a linear manner. For the kinetic reading, the reaction velocity was steady through 30 min. Conclusion: A reaction time of 30 minutes is sufficient to achieve a inear reaction velocity using a 4MU-iduronide final reaction concentration of 180 M. These results indicate a potentially more accurate method of determining enzyme activity than those currently published. doi:10.1016/j.ymgme.2012.11.111
98 The impact of Fabry on pregnancy (IFOP) Alexandrea Holmes, Dawn Laney, Suma Shankar, Emory University, Decatur, GA, USA Fabry disease (FD) is an X-linked genetic lysosomal storage disorder caused by a deficiency of the lysosomal enzyme alpha-galactosidase A (alpha-gal A; EC 3.2.1.22). The lack of alpha-galactosidase A enzyme results in the storage of globotriaosylceramide (GL3), a fatty substance, in the lysosomes of cells throughout the body. As Fabry disease is a microvascular disease and storage of lipids are present in the placenta, theoretical concerns have been raised about a possibility of increased pregnancy complications in affected women. This study has been designed as a retrospective survey to better understand the actual risks in the past pregnancies of women affected by Fabry disease. Survey questions include queries about prenatal medications, teratogenic exposures, prenatal testing, common pregnancy complications, Fabry symptom status during pregnancy, obstetrical history, and immediate neonatal history. The survey is designed as patient response followed by investigator follow up and inquiry with survey respondents. Medical records will be obtained for the curation of pregnancy complication information. Based on statistical power calculations, the target numbers of subjects is 200. Preliminary data will be presented with interim discussion and conclusions. doi:10.1016/j.ymgme.2012.11.112
Abstracts
96 A multi-national, randomized, double-blind, placebo-controlled study to evaluate the efficacy and safety of BMN 110 treatment for mucopolysaccharidosis IVA (Morquio syndrome type A) Christian Hendriksza, Barbara K. Burtonb, Thomas Flemingc, Roberto Giuglianid, Paul Harmatze, Derralynn Hughesf, Simon Jonesg, Shuan-Pei Linh, Karl Eugen Mengeli, Maurizio Scarpaj, Vassili Valayannopoulosk, a University of Manchester, Salford Royal NHS Foundation Trust, Birmingham Children's Hospital NHS Foundation Trust, Salford, Lancashire, UK, bAnn and Robert H. Lurie Children's Hospital and Northwestern University Feinberg School of Medicine, Chicago, IL, USA, cSchool of Public Health, University of Washington, WA, USA, dHospital de Clinicas de Porto Alegre, Porto Alegre, Brazil, eChildren's Hospital and Research Center, Oakland, CA, USA, f University College London, London, UK, gCentral Manchester University Hospital, Manchester, United Kingdom, hMacKey Memorial Hospital, Taipei, Taiwan, iUniversity of Mainz, Mainz, Germany, jUniversity of Padova, Padova, Italy, kChu Paris, Hôpital Necker-Enfants Malades, Paris France Mucopolysaccharidosis IVA is caused by a deficiency in Nacetylgalactosamine-6 sulfatase (GALNS) activity resulting in pathologic lysosomal accumulation of keratan sulfate (KS), leading to multiple clinical manifestations. A placebo-controlled, multinational randomized trial was conducted to evaluate efficacy and safety of recombinant human GALNS (BMN110) in 176 patients with mucopolysaccharidosis IVA, ≥5 years of age. For 6 minute walk distance at 24 weeks, relative to the change in placebo, the 2 mg/kg/wk regimen provided a statistically significant increase in mean distance of 22.5 m (95% CI: 4.0, 40.9; p = 0.0174), but the 2 mg/kg/qow regimen provided an increase of only 1.4 m (95% CI: −17.8, 18.9). For 3 minute stair climb at 24 weeks, relative to placebo, the 2 mg/kg/wk regimen had a mean increase of 1.1 stairs/minute (95% CI: −2.1, 4.4) while the 2 mg/kg/qow regimen had a mean decrease of 0.5 stairs/minute (95% CI: −3.7, 2.8). For both regimens, urinary KS was substantially reduced. Adverse events (AE) to BMN110 were similar to those seen in clinical trials of other enzyme replacement therapies. Serious AE related to study drug occurred in 3.4% on weekly dosing of BMN110 and 0% on placebo. In the weekly dosing group, 17 (1.3%) of 1345 infusions were interrupted due to AE. All patients subsequently resumed dosing. doi:10.1016/j.ymgme.2012.11.110
97 Increased substrate concentration boosts enzyme activity levels of fluorometric α-l-iduronidase enzyme activity assay Tyler Herzog, Li Ou, Chester Whitley, University of Minnesota, Minneapolis, Minnesota, USA Background: Mucopolysaccharidosis type I (MPS I) is a lysosomal disease due to deficiency of the enzyme α-L-iduronidase which results in the accumulation of the glycosaminoglycans dermatan sulfate and heparan sulfate. Current efforts in assessing the severity of this disease employ a fluorometric assay involving the fluorescent substrate 4-methylumbelliferyl-α-L-iduronide (4MU-iduronide) to determine enzyme activity present in samples. An early assay used a final reaction concentration of 0.2 μM and in this same study the Michaelis-Menten constants (Km) for fibroblasts and leukocytes were 0.147 μM and 0.179 μM, respectively (Hopwood et al. 1978). Km is the concentration of 4MU-iduronide which produces a reaction velocity of ½-maximal velocity (Vmax), but higher concentrations closer to enzyme saturation are generally recommended for reliable assays. At lower substrate concentrations, variations in substrate concentration result in relatively large errors in measured
activity because the lower substrate concentrations are on the steepest part of the curve. The highest substrate concentration cited in the literature is 2.85 mM (Whitley et al. 1987), but is relatively expensive for high-volume studies. In other assays, final substrate concentrations have included 25 M (Kakkis et al. 1993; Zheng et al. 2003), 50 M (Liu et al. 2004), 2 mM (Dimenico et al. 2005), and 2.85 mM (Whitley et al. 1987; Hartung et al. 2004) Hypothesis: Given that 4MU-iduronide concentrations for enzyme activity assays in the literature are varied, we hypothesized that increasing the substrate concentration towards the Km and correcting for Km using the Michaelis–Menten equation would lead to more accurate enzyme activity values. Materials and Methods: Blood samples were collected from MPS I mice and heterozygotic littermates. Plasma and serum was obtained. An equal amount of homogenized sample and 4MU-iduronide, with a final reaction concentration of 25 M, 50 M, or 180 M, were mixed and incubated for 60 minutes at 37 °C in an Applied Biosystems 2720 Thermal Cycler. The reaction was stopped using a glycine carbonate buffer, and fluorescence was determined on a BioTek SynergyMx 96well plate reader. In addition, the reaction between a sample and 180 M 4MUI substrate was stopped every 10 minutes for 2 hours to determine when the reaction was linear. The Michaelis-Menten equation was used to calculate Vmax based on the Km, fluorescent substrate concentration, and initial velocity from our studies. This Vmax was used for enzyme activity calculations. Results: IDUA activity increased with each successively higher substrate concentration (1.17 U/ml, 1.75 U/ml, and 3.83 U/ml for plasma) in a linear manner. For the kinetic reading, the reaction velocity was steady through 30 min. Conclusion: A reaction time of 30 minutes is sufficient to achieve a inear reaction velocity using a 4MU-iduronide final reaction concentration of 180 M. These results indicate a potentially more accurate method of determining enzyme activity than those currently published. doi:10.1016/j.ymgme.2012.11.111
98 The impact of Fabry on pregnancy (IFOP) Alexandrea Holmes, Dawn Laney, Suma Shankar, Emory University, Decatur, GA, USA Fabry disease (FD) is an X-linked genetic lysosomal storage disorder caused by a deficiency of the lysosomal enzyme alpha-galactosidase A (alpha-gal A; EC 3.2.1.22). The lack of alpha-galactosidase A enzyme results in the storage of globotriaosylceramide (GL3), a fatty substance, in the lysosomes of cells throughout the body. As Fabry disease is a microvascular disease and storage of lipids are present in the placenta, theoretical concerns have been raised about a possibility of increased pregnancy complications in affected women. This study has been designed as a retrospective survey to better understand the actual risks in the past pregnancies of women affected by Fabry disease. Survey questions include queries about prenatal medications, teratogenic exposures, prenatal testing, common pregnancy complications, Fabry symptom status during pregnancy, obstetrical history, and immediate neonatal history. The survey is designed as patient response followed by investigator follow up and inquiry with survey respondents. Medical records will be obtained for the curation of pregnancy complication information. Based on statistical power calculations, the target numbers of subjects is 200. Preliminary data will be presented with interim discussion and conclusions. doi:10.1016/j.ymgme.2012.11.112