Clinica Chimica Acta 430 (2014) 24–27
Contents lists available at ScienceDirect
Clinica Chimica Acta journal homepage: www.elsevier.com/locate/clinchim
Identification of mucopolysaccharidosis I heterozygotes based on biochemical characteristics of L-iduronidase from dried blood spots Derbis Campos a,⁎, Madelyn Monaga a, Ernesto C. González b, Darlenis Herrera a,1 a b
Department of Biochemical Genetics, National Center of Medical Genetics, 146 Street No. 3102, Havana, Cuba Neonatal Research Laboratory, Center of Immunoassays, 134 Street and 25 Ave, Havana, Cuba
a r t i c l e
i n f o
Article history: Received 14 May 2013 Received in revised form 20 December 2013 Accepted 23 December 2013 Available online 31 December 2013 Keywords: Lysosomal storage diseases MPS I IDUA Dried blood spots Mucopolysaccharidosis I heterozygotes
a b s t r a c t Background: Mucopolysaccharidosis I (MPS I) is a genetic disorder caused by deficiency of L-iduronidase (IDUA) activity. Heterozygote screening is a highly requested service by risk families; however, determination of IDUA activity alone is not sufficient to discriminate between heterozygotes and normal individuals because a significant overlap occurs between them. The aim of this study was to characterize the enzyme eluted from heterozygote's dried blood samples and determine if there are differences with that of normal individuals. Methods: We determined Km, Vmax and the thermal stability of the enzyme at 50 °C. Results: Vmax from heterozygotes (7.28 ± 2.72 μmol/lblood/h) was significantly different than the obtained in controls (10.52 ± 2.05 μmol/lblood/h), while their Km were similar: 0.633 ± 0.339 mmol/l and 0.672 ± 0.246 mmol/l, respectively. After a 12 h pre-incubation period, IDUA activity in controls was significantly lower compared to heterozygotes. Conclusions: IDUA eluted from dried blood spots of heterozygotes differs from that of controls in terms of Vmax and thermal stability. These parameters can be used as an important tool for the detection of carriers for MPS I. This is the first report describing a differential behavior of these parameters for a lysosomal enzyme obtained from dried blood. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Mucopolysaccharidosis I (MPS I) (OMIM ID: 607014, 607015, 607016), produced by deficient activity of the lysosomal enzyme L-iduronidase (IDUA) (EC 3.2.1.76) is one of the most prevalent lysosomal storage diseases (LSD) worldwide [1]. It has a wide clinical heterogeneity in relation to their debut, progression and severity; as well as a high morbidity and mortality. Currently, it's possible to actively treat this disease by hematopoietic stem cell transplantation and enzyme replacement therapy. However, their effectiveness varies depending on the time of diagnosis and the phenotype [2,3]. Identification of heterozygotes is one of the most frequently requested services by members of families at risk since it allows the genetic counseling for future reproductive decisions. MPS I is diagnosed by testing a deficient IDUA activity in a sample of the individual under clinical suspicion. However, heterozygotes present enzyme activity Abbreviations: MPS I, mucopolysaccharidosis I; IDUA, L-iduronidase; LSD, lysosomal storage diseases; DBS, dried blood spots; 4-MU-ID, 4-methylumbelliferyl-α-L-iduronide; TCA, trichloroacetic acid; 4-MU, 4-methylumbelliferone. ⁎ Corresponding author at: Department of Biochemical Genetics, National Center of Medical Genetics, 146 Street No. 3102, Postal Code 1600, Havana, Cuba. Tel.: +53 7 208 9991. E-mail address: [email protected] (D. Campos). 1 Present address: Department of Science and Technological Innovation, Institute for Scientific and Technological Information, 18 A Street, Havana, Cuba. 0009-8981/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.cca.2013.12.035
levels which are overlapped with those of normal subjects. This overlap between the two populations is an innate feature of IDUA activity and is given by the high allelic heterogeneity that has been described for the IDUA gene due to the high number of mutations and polymorphisms of this gen, as is true for most of the lysosomal enzymes [1]. Furthermore, carriers' identification through mutation analysis is a complex, time consuming and expensive task due to the high polymorphism of the IDUA gen [4] (NCBI dbVar/dbSNP ID: IDUA). Studies in fibroblast [5], plasma [6] and leukocyte [7] samples show a differential biochemical behavior of the IDUA obtained from heterozygotes compared to normal individuals. This could be used as an alternative approach for the identification of carriers. However, the differential behavior of IDUA or any other lysosomal enzyme has not been described using samples of dried blood spots (DBS) on filter paper; which would be very useful given the advantages of this type of sample relative to its collection, handling, transportation and storage. 2. Materials and methods 2.1. Collecting DBS In order to describe the biochemical behavior of IDUA eluted from DBS, whole blood samples from 25 heterozygotes and 25 normal individuals were collected. Samples were dispensed on filter paper (Schleicher and Schuell 903) and allowed to dry for 24 h at 20–25 °C.
D. Campos et al. / Clinica Chimica Acta 430 (2014) 24–27
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DBS samples were stored in closed plastic bags with silica gel at −20 °C until use. Collection and handling of all samples were consistent with the Declaration of Helsinki of the World Health Organization [8]. Informed consent was obtained from all individuals who participated in this study. The protocol was approved by the Ethics Committee and the Scientific Council belonging to the National Center of Medical Genetics, Cuba. 2.2. IDUA activity assay Enzyme activity was assayed by an ultramicroassay developed in our laboratory [9], as a modification to the original method described by Chamoles et al. [10]. A 3-mm diameter disc (~ 3.6 μl of blood) was punched from each DBS sample and placed in a 96-well microplate. We added 40 μl of formate buffer 50 mmol/l (pH 2.8) containing d-saccharic acid-1,4-lactone 0.04 mmol/l and 20 μl of the substrate 4-MU-α-L-iduronide 2.00 mmol/l in distilled water. After vortex-mixing for 10 min, the microplate was incubated for 20 h at 37 °C in a humid chamber. Next, we added 5 μl of trichloroacetic acid (TCA) 6.1 mol/l for protein precipitation, vortex-mixing briefly and then let the microplate kept for 30 min at room temperature. Finally, to 5 μl of the supernatant, transferred directly to a well of a fluorescence reading ultramicroplate, we added 25 μl of glycine-carbonate buffer 85 mmol/l (pH 10.5) and measured the fluorescence (excitation: 365 nm; emission: 450 nm) of the enzyme product: 4-methylumbelliferone (4-MU) in a fluorimeterphotometer PR-521 (Tecnosuma International S.A). One blank was run for each sample applying the same protocol than for samples but adding the substrate, which had been incubated separately, just before adding the volume of TCA 6.1 mol/l. IDUA activity was expressed as micromoles of product formed per liter of blood per hour, using a calibration curve of 4-MU. Each sample was analyzed in triplicate. To obtain the kinetic parameters: Vmax and Km, a substrate concentration of 0.05–3.50 mmol/l was used. The Michaelis–Menten curve was plotted using the program GraphPad Prism 5 (GraphPad Software Inc) and both kinetic parameters were obtained by direct calculation through this program. Thermal stability of the enzyme was assayed by pre-incubating the samples, after adding the formate buffer solution 50 mmol/l (pH 2.8), for 0.25, 0.5, 1, 6, 12 and 24 h at 50 °C before adding 20 μl of the substrate solution at 2 mmol/l. This was followed by incubation at 37 °C for 20 h and the procedure continued as described above. 2.3. Statistical analysis Statistical analysis was performed using GraphPad Prism 5. Values for IDUA activity, Km and Vmax are expressed as the mean ± SD and the confidence interval for the mean with 95% of probability (IC95). Comparison between multiple groups was performed using the nonparametric Kruskal–Wallis test followed by Dunn's test for post-hoc comparisons. Comparisons between two groups were performed using the nonparametric Mann–Whitney test. Receiveroperator characteristic (ROC) curves were used to calculate specific cutoffs values for controls and heterozygotes. A p b 0.05 was taken for all statistical tests.
Fig. 1. IDUA activity in DBS from MPS I heterozygotes and controls. Blank circles: heterozygotes (n = 25), black circles: normal controls (n = 25). Each individual value obtained is represented. The line inside each group represents the median. The p value obtained indicates no significant differences between groups (Mann–Whitney test).
3.1. Km and Vmax determination Fig. 2 shows the Michaelis–Menten curve obtained for the IDUA eluted from DBS samples from heterozygotes and controls. The Km for heterozygotes was 0.633 ± 0.339 mmol/l (IC95: 0.216 – 1.050 mmol/l) with a range from 0.110 to 1.280 mmol/l; while controls had a Km of 0.672 ± 0.246 mmol/l (IC95: 0.481 – 0.862 mmol/l) with a range from 0.256 to 1.100 mmol/l. The Vmax of the reaction for the IDUA from heterozygotes was 7.28 ± 2.72 μmol/lblood/h (IC95: 5.80 – 8.77 μmol/lblood/h), range: 4.00 to 9.45 μmol/lblood/h. The Vmax for the enzyme from controls was 10.52 ± 2.05 μmol/lblood/h (IC95: 9.57 – 11.47 μmol/lblood/h), range: 8.03 to 13.01 μmol/lblood/h. As shown in Fig. 3, significant differences for Vmax between the two groups were obtained, but not for Km. According to these results, we calculated a tentative cutoffs value for Vmax of 9.51 μmol/lblood/h, with 100% sensitivity and 92% specificity. 3.2. Heat stability studies The residual IDUA activity had an exponential behavior (controls: R2 = 0.970; heterozygotes: R2 = 0.990), with values for half-life of 4.5 h (IC95: 3.68 – 5.70 h) for controls and 4.4 h (IC95: 3.97– 5.07 h) for heterozygotes. There was a significant decrease in IDUA activity
3. Results Fig. 1 shows the individual values of IDUA activity obtained for each group of samples. Controls had a mean value of 6.24 ± 2.23 μmol/lblood/h (IC95: 5.32 – 7.16 μmol/lblood/h) with a range from 2.55 to 10.78 μmol/lblood/h; while IDUA activity in heterozygotes was 5.11 ± 2.05 μmol/lblood/h (IC95: 4.27 – 5.95 μmol/lblood/h) with a range from 1.06 to 8.95 μmol/lblood/h. As shown in Fig. 1, no significant differences were found between the two groups since most heterozygotes had IDUA activity values which overlapped with controls values.
Fig. 2. Michaelis–Menten plot for the IDUA eluted from DBS from MPS I heterozygotes and controls. Blank circles/dashed line: heterozygotes (n = 25), black circles/continuous line: normal controls (n = 25). Each symbol represents the mean value along with its standard deviation.
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D. Campos et al. / Clinica Chimica Acta 430 (2014) 24–27
Fig. 4. Decrease of IDUA activity in DBS as a function of pre-incubation time at 50 °C. Blank circles/dashed line: heterozygotes (n = 25), black circles/continuous line: normal controls (n = 25). Each symbol represents the mean value with its standard deviation. The first 3 incubation times are indicated inside parentheses. Different letters indicate significant differences (p b 0.05) for both groups in respect to pre-incubation times (Kruskal–Wallis and Dunn's test).
Fig. 3. Biochemical properties of IDUA activity in DBS from MPS I heterozygotes and controls. A: individual values of Km for each sample. B: individual values of Vmax for each sample. Blank circles: heterozygotes (n = 25), black circles: normal controls (n = 25). The line inside each group represents the median. The p values obtained indicate significant differences between groups for Vmax but not for Km (Mann–Whitney test).
IDUA species from leukocytes, as well as the ones present in plasma, must contribute to the enzyme activity value obtained in DBS; so there must be similarities with the differential biochemical behavior between controls and the heterozygotes described by Mandelli et al. [6,7] for these 2 types of samples. However, since Km and Vmax depend on the characteristics of the activity assay used (pH, temperature, enzyme concentration); it is not possible to compare the nominal values obtained among the three types of samples. These authors obtained differences in Vmax for the enzyme from leukocytes between heterozygotes and normal controls, similar to our study, but not for the IDUA from plasma [6,7]. Regarding Km, results in leukocytes by Mandelli et al. [7] are not homogeneous. Differential behavior was observed for the subgroup of carriers with IDUA activity values very similar to the control group, while those with lower activity values did not differ in their Km values compared with controls. This latter finding is similar to our results. For the enzyme from plasma, Mandelli et al. [6] obtained a differential behavior, although a great variability with respect to the controls was observed. In this case, they did not perform the stratification of heterozygotes.
following the period between 30 min and an hour of pre-incubation at 50 °C for both heterozygotes and controls samples (Fig. 4). However, IDUA activity in heterozygotes was more stable under long periods of pre-incubation compared to the control (Fig. 5). Taking 24 h as a more convenient period of time according to standard working hours on a routine laboratory, we calculated a tentative cutoffs value for residual IDUA activity of 3.76%, with 100% sensitivity and 92% specificity.
4. Discussion Results presented here describe the behavior of the kinetic parameters Vmax and Km of a lysosomal enzyme eluted from DBS and the use of these parameters to detect MPS I heterozygotes due to the inability of using IDUA activity values by itself for this purpose. These parameters have previously been determined in leukocytes and serum/plasma, since this type of samples has traditionally been used to detect deficiencies of lysosomal enzymes activities and thus to diagnose different LSD. However, at present a widespread use of DBS as an ideal sample for these diagnoses has occurred due to its benefits, including their potential use in neonatal screening programs for some LSD. Therefore, the use of this type of sample for carriers' detection would also be very advantageous.
Fig. 5. Differential thermal stability of IDUA activity in DBS from MPS I heterozygotes compared to controls. Blank circles: heterozygotes (n = 25), black circles: normal controls (n = 25). Each individual value obtained is represented. The line inside each group represents the median. The p values obtained indicate significant differences between groups for both 12 and 24 h of pre-incubation at 50 °C (Mann–Whitney test).
D. Campos et al. / Clinica Chimica Acta 430 (2014) 24–27
Concerning the thermal stability of the IDUA, after a period of 12 h of pre-incubation at 50 °C the enzyme from heterozygous DBS retained a significantly higher percentage of their original activity compared with controls. This effect is very similar to that reported by Mandelli et al. [6] for plasma samples, although in our case this decrease was not linear but exponential. In leukocyte this effect is obtained only after 15 min of pre-incubation, reaching almost complete denaturation of the enzyme at the end of an hour [7]. The presence of protective elements in plasma and thus in DBS may promote greater stability to the enzyme. An important observation to the studies made by Mandelli et al. [6,7] and applicable to ours, is that they did not identify any of the mutations present in heterozygous individuals. For this reason, it would be important in future studies to estimate IDUA functionality in terms of Km and Vmax in relation with the specific mutation present on each heterozygote. Therefore, the results could be extrapolated to any carrier, regardless of the mutation present in one of its alleles. Currently there is a growing interest in the application of tandem mass spectrometry (MS/MS) for the diagnosis of LSD, mainly as part as newborn screening programs. This is due to the possibility it offers to simultaneously and quickly quantify the specific products of many enzyme activity assays [11]. However, results using MS/MS as a carrier detection method also show a level of overlap between the values of enzymatic activity of the heterozygotes and controls [12], as it occurs with traditional fluorescent methods. As we previously stated, this is a feature of the majority of lysosomal enzymes, including IDUA, due to the high allelic heterogeneity. For this reason, the determination of IDUA activity by itself, regardless of the method used, would not allow a high sensitivity and specificity in the detection of carriers. Nevertheless, as the number or laboratories with this technology is increasing; it would be interesting, although the technological possibilities of our laboratory do not allow it yet, to carry out a similar analysis to the one fulfilled in our case to characterize the biochemical properties of IDUA eluted from DBS but applying MS/MS. 5. Conclusions Based on the results shown here, determining Vmax and the percentage of residual IDUA activity after 24 h of pre-incubation at
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50 °C in DBS can be used to discriminate completely between samples from normal individuals and heterozygotes. Especially due to its simplicity, the second proposed method is much more feasible and practical for studies of carriers in families at risk.
Acknowledgment We thank José L. Hernandez Sierra for his valuable help in the final language revision of the manuscript.
References [1] Neufeld EF, Muenzer J. The mucopolysaccharidoses. In: Scriver C, Beaudet A, Valle D, editors. The metabolic and molecular bases of inherited disease. Ney York, New York: McGraw Hill; 2001. p. 3421–52. [2] de Ru MH, Boelens JJ, Das AM, et al. Enzyme replacement therapy and/or hematopoietic stem cell transplantation at diagnosis in patients with mucopolysaccharidosis type I: results of a European consensus procedure. Orphanet J Rare Dis 2011;6:55. [3] Tolar J, Orchard PJ. Alpha-L-iduronidase therapy for mucopolysaccharidosis type I. Biologics, 2; 2008 743–51. [4] The Human Gene Mutation Database. Institute of Medical Genetics in Cardiff, United Kingdom. BIOBASE Databases, ID: IDUA. http://www.hgmd.cf.ac.uk (acceded November 12, 2012). [5] Clements PR, Yogalingam G, Brooks DA, Muller VJ, Hopwood JJ. α-L-iduronidase genotype/protein function relationships in MPS I fibroblasts. J Inherit Metab Dis 2000;23:237. [6] Mandelli J, Wajner A, Pires R, Giugliani R, Coelho JC. Detection of mucopolysaccharidosis type I heterozygotes on the basis of the biochemical properties of plasma alpha-Liduronidase. Clin Chim Acta 2001;312:81–6. [7] Mandelli J, Wajner A, Pires RF, Giugliani R, Coelho JC. Detection of mucopolysaccharidosis type I heterozygotes based on the biochemical characteristics of leukocyte alpha-L-iduronidase. Arch Med Res 2002;33:20–4. [8] World Medical Association Declaration of Helsinki: ethical principles for medical research involving human subjects. JAMA 2000;284:3043–5. [9] Campos D, Monaga M, González EC, Herrera D, de la Peña D. Optimization of enzymatic diagnosis for mucopolysaccharidosis I in dried blood spots on filter paper. Clin Biochem 2013;46:805–9. [10] Chamoles NA, Blanco M, Gaggioli D. Diagnosis of alpha-L-iduronidase deficiency in dried blood spots on filter paper: the possibility of newborn diagnosis. Clin Chem 2001;47:780–1. [11] Spacil Z, Tatipaka H, Barcenas M, Scott CR, Turecek F, Gelb MH. High-throughput assay of 9 lysosomal enzymes for newborn screening. Clin Chem 2013;59:502–11. [12] Li Y, Scott CR, Chamoles NA, et al. Direct multiplex assay of lysosomal enzymes in dried blood spots for newborn screening. Clin Chem 2004;50:1785–96.
Contents lists available at ScienceDirect
Clinica Chimica Acta journal homepage: www.elsevier.com/locate/clinchim
Identification of mucopolysaccharidosis I heterozygotes based on biochemical characteristics of L-iduronidase from dried blood spots Derbis Campos a,⁎, Madelyn Monaga a, Ernesto C. González b, Darlenis Herrera a,1 a b
Department of Biochemical Genetics, National Center of Medical Genetics, 146 Street No. 3102, Havana, Cuba Neonatal Research Laboratory, Center of Immunoassays, 134 Street and 25 Ave, Havana, Cuba
a r t i c l e
i n f o
Article history: Received 14 May 2013 Received in revised form 20 December 2013 Accepted 23 December 2013 Available online 31 December 2013 Keywords: Lysosomal storage diseases MPS I IDUA Dried blood spots Mucopolysaccharidosis I heterozygotes
a b s t r a c t Background: Mucopolysaccharidosis I (MPS I) is a genetic disorder caused by deficiency of L-iduronidase (IDUA) activity. Heterozygote screening is a highly requested service by risk families; however, determination of IDUA activity alone is not sufficient to discriminate between heterozygotes and normal individuals because a significant overlap occurs between them. The aim of this study was to characterize the enzyme eluted from heterozygote's dried blood samples and determine if there are differences with that of normal individuals. Methods: We determined Km, Vmax and the thermal stability of the enzyme at 50 °C. Results: Vmax from heterozygotes (7.28 ± 2.72 μmol/lblood/h) was significantly different than the obtained in controls (10.52 ± 2.05 μmol/lblood/h), while their Km were similar: 0.633 ± 0.339 mmol/l and 0.672 ± 0.246 mmol/l, respectively. After a 12 h pre-incubation period, IDUA activity in controls was significantly lower compared to heterozygotes. Conclusions: IDUA eluted from dried blood spots of heterozygotes differs from that of controls in terms of Vmax and thermal stability. These parameters can be used as an important tool for the detection of carriers for MPS I. This is the first report describing a differential behavior of these parameters for a lysosomal enzyme obtained from dried blood. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Mucopolysaccharidosis I (MPS I) (OMIM ID: 607014, 607015, 607016), produced by deficient activity of the lysosomal enzyme L-iduronidase (IDUA) (EC 3.2.1.76) is one of the most prevalent lysosomal storage diseases (LSD) worldwide [1]. It has a wide clinical heterogeneity in relation to their debut, progression and severity; as well as a high morbidity and mortality. Currently, it's possible to actively treat this disease by hematopoietic stem cell transplantation and enzyme replacement therapy. However, their effectiveness varies depending on the time of diagnosis and the phenotype [2,3]. Identification of heterozygotes is one of the most frequently requested services by members of families at risk since it allows the genetic counseling for future reproductive decisions. MPS I is diagnosed by testing a deficient IDUA activity in a sample of the individual under clinical suspicion. However, heterozygotes present enzyme activity Abbreviations: MPS I, mucopolysaccharidosis I; IDUA, L-iduronidase; LSD, lysosomal storage diseases; DBS, dried blood spots; 4-MU-ID, 4-methylumbelliferyl-α-L-iduronide; TCA, trichloroacetic acid; 4-MU, 4-methylumbelliferone. ⁎ Corresponding author at: Department of Biochemical Genetics, National Center of Medical Genetics, 146 Street No. 3102, Postal Code 1600, Havana, Cuba. Tel.: +53 7 208 9991. E-mail address: [email protected] (D. Campos). 1 Present address: Department of Science and Technological Innovation, Institute for Scientific and Technological Information, 18 A Street, Havana, Cuba. 0009-8981/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.cca.2013.12.035
levels which are overlapped with those of normal subjects. This overlap between the two populations is an innate feature of IDUA activity and is given by the high allelic heterogeneity that has been described for the IDUA gene due to the high number of mutations and polymorphisms of this gen, as is true for most of the lysosomal enzymes [1]. Furthermore, carriers' identification through mutation analysis is a complex, time consuming and expensive task due to the high polymorphism of the IDUA gen [4] (NCBI dbVar/dbSNP ID: IDUA). Studies in fibroblast [5], plasma [6] and leukocyte [7] samples show a differential biochemical behavior of the IDUA obtained from heterozygotes compared to normal individuals. This could be used as an alternative approach for the identification of carriers. However, the differential behavior of IDUA or any other lysosomal enzyme has not been described using samples of dried blood spots (DBS) on filter paper; which would be very useful given the advantages of this type of sample relative to its collection, handling, transportation and storage. 2. Materials and methods 2.1. Collecting DBS In order to describe the biochemical behavior of IDUA eluted from DBS, whole blood samples from 25 heterozygotes and 25 normal individuals were collected. Samples were dispensed on filter paper (Schleicher and Schuell 903) and allowed to dry for 24 h at 20–25 °C.
D. Campos et al. / Clinica Chimica Acta 430 (2014) 24–27
25
DBS samples were stored in closed plastic bags with silica gel at −20 °C until use. Collection and handling of all samples were consistent with the Declaration of Helsinki of the World Health Organization [8]. Informed consent was obtained from all individuals who participated in this study. The protocol was approved by the Ethics Committee and the Scientific Council belonging to the National Center of Medical Genetics, Cuba. 2.2. IDUA activity assay Enzyme activity was assayed by an ultramicroassay developed in our laboratory [9], as a modification to the original method described by Chamoles et al. [10]. A 3-mm diameter disc (~ 3.6 μl of blood) was punched from each DBS sample and placed in a 96-well microplate. We added 40 μl of formate buffer 50 mmol/l (pH 2.8) containing d-saccharic acid-1,4-lactone 0.04 mmol/l and 20 μl of the substrate 4-MU-α-L-iduronide 2.00 mmol/l in distilled water. After vortex-mixing for 10 min, the microplate was incubated for 20 h at 37 °C in a humid chamber. Next, we added 5 μl of trichloroacetic acid (TCA) 6.1 mol/l for protein precipitation, vortex-mixing briefly and then let the microplate kept for 30 min at room temperature. Finally, to 5 μl of the supernatant, transferred directly to a well of a fluorescence reading ultramicroplate, we added 25 μl of glycine-carbonate buffer 85 mmol/l (pH 10.5) and measured the fluorescence (excitation: 365 nm; emission: 450 nm) of the enzyme product: 4-methylumbelliferone (4-MU) in a fluorimeterphotometer PR-521 (Tecnosuma International S.A). One blank was run for each sample applying the same protocol than for samples but adding the substrate, which had been incubated separately, just before adding the volume of TCA 6.1 mol/l. IDUA activity was expressed as micromoles of product formed per liter of blood per hour, using a calibration curve of 4-MU. Each sample was analyzed in triplicate. To obtain the kinetic parameters: Vmax and Km, a substrate concentration of 0.05–3.50 mmol/l was used. The Michaelis–Menten curve was plotted using the program GraphPad Prism 5 (GraphPad Software Inc) and both kinetic parameters were obtained by direct calculation through this program. Thermal stability of the enzyme was assayed by pre-incubating the samples, after adding the formate buffer solution 50 mmol/l (pH 2.8), for 0.25, 0.5, 1, 6, 12 and 24 h at 50 °C before adding 20 μl of the substrate solution at 2 mmol/l. This was followed by incubation at 37 °C for 20 h and the procedure continued as described above. 2.3. Statistical analysis Statistical analysis was performed using GraphPad Prism 5. Values for IDUA activity, Km and Vmax are expressed as the mean ± SD and the confidence interval for the mean with 95% of probability (IC95). Comparison between multiple groups was performed using the nonparametric Kruskal–Wallis test followed by Dunn's test for post-hoc comparisons. Comparisons between two groups were performed using the nonparametric Mann–Whitney test. Receiveroperator characteristic (ROC) curves were used to calculate specific cutoffs values for controls and heterozygotes. A p b 0.05 was taken for all statistical tests.
Fig. 1. IDUA activity in DBS from MPS I heterozygotes and controls. Blank circles: heterozygotes (n = 25), black circles: normal controls (n = 25). Each individual value obtained is represented. The line inside each group represents the median. The p value obtained indicates no significant differences between groups (Mann–Whitney test).
3.1. Km and Vmax determination Fig. 2 shows the Michaelis–Menten curve obtained for the IDUA eluted from DBS samples from heterozygotes and controls. The Km for heterozygotes was 0.633 ± 0.339 mmol/l (IC95: 0.216 – 1.050 mmol/l) with a range from 0.110 to 1.280 mmol/l; while controls had a Km of 0.672 ± 0.246 mmol/l (IC95: 0.481 – 0.862 mmol/l) with a range from 0.256 to 1.100 mmol/l. The Vmax of the reaction for the IDUA from heterozygotes was 7.28 ± 2.72 μmol/lblood/h (IC95: 5.80 – 8.77 μmol/lblood/h), range: 4.00 to 9.45 μmol/lblood/h. The Vmax for the enzyme from controls was 10.52 ± 2.05 μmol/lblood/h (IC95: 9.57 – 11.47 μmol/lblood/h), range: 8.03 to 13.01 μmol/lblood/h. As shown in Fig. 3, significant differences for Vmax between the two groups were obtained, but not for Km. According to these results, we calculated a tentative cutoffs value for Vmax of 9.51 μmol/lblood/h, with 100% sensitivity and 92% specificity. 3.2. Heat stability studies The residual IDUA activity had an exponential behavior (controls: R2 = 0.970; heterozygotes: R2 = 0.990), with values for half-life of 4.5 h (IC95: 3.68 – 5.70 h) for controls and 4.4 h (IC95: 3.97– 5.07 h) for heterozygotes. There was a significant decrease in IDUA activity
3. Results Fig. 1 shows the individual values of IDUA activity obtained for each group of samples. Controls had a mean value of 6.24 ± 2.23 μmol/lblood/h (IC95: 5.32 – 7.16 μmol/lblood/h) with a range from 2.55 to 10.78 μmol/lblood/h; while IDUA activity in heterozygotes was 5.11 ± 2.05 μmol/lblood/h (IC95: 4.27 – 5.95 μmol/lblood/h) with a range from 1.06 to 8.95 μmol/lblood/h. As shown in Fig. 1, no significant differences were found between the two groups since most heterozygotes had IDUA activity values which overlapped with controls values.
Fig. 2. Michaelis–Menten plot for the IDUA eluted from DBS from MPS I heterozygotes and controls. Blank circles/dashed line: heterozygotes (n = 25), black circles/continuous line: normal controls (n = 25). Each symbol represents the mean value along with its standard deviation.
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D. Campos et al. / Clinica Chimica Acta 430 (2014) 24–27
Fig. 4. Decrease of IDUA activity in DBS as a function of pre-incubation time at 50 °C. Blank circles/dashed line: heterozygotes (n = 25), black circles/continuous line: normal controls (n = 25). Each symbol represents the mean value with its standard deviation. The first 3 incubation times are indicated inside parentheses. Different letters indicate significant differences (p b 0.05) for both groups in respect to pre-incubation times (Kruskal–Wallis and Dunn's test).
Fig. 3. Biochemical properties of IDUA activity in DBS from MPS I heterozygotes and controls. A: individual values of Km for each sample. B: individual values of Vmax for each sample. Blank circles: heterozygotes (n = 25), black circles: normal controls (n = 25). The line inside each group represents the median. The p values obtained indicate significant differences between groups for Vmax but not for Km (Mann–Whitney test).
IDUA species from leukocytes, as well as the ones present in plasma, must contribute to the enzyme activity value obtained in DBS; so there must be similarities with the differential biochemical behavior between controls and the heterozygotes described by Mandelli et al. [6,7] for these 2 types of samples. However, since Km and Vmax depend on the characteristics of the activity assay used (pH, temperature, enzyme concentration); it is not possible to compare the nominal values obtained among the three types of samples. These authors obtained differences in Vmax for the enzyme from leukocytes between heterozygotes and normal controls, similar to our study, but not for the IDUA from plasma [6,7]. Regarding Km, results in leukocytes by Mandelli et al. [7] are not homogeneous. Differential behavior was observed for the subgroup of carriers with IDUA activity values very similar to the control group, while those with lower activity values did not differ in their Km values compared with controls. This latter finding is similar to our results. For the enzyme from plasma, Mandelli et al. [6] obtained a differential behavior, although a great variability with respect to the controls was observed. In this case, they did not perform the stratification of heterozygotes.
following the period between 30 min and an hour of pre-incubation at 50 °C for both heterozygotes and controls samples (Fig. 4). However, IDUA activity in heterozygotes was more stable under long periods of pre-incubation compared to the control (Fig. 5). Taking 24 h as a more convenient period of time according to standard working hours on a routine laboratory, we calculated a tentative cutoffs value for residual IDUA activity of 3.76%, with 100% sensitivity and 92% specificity.
4. Discussion Results presented here describe the behavior of the kinetic parameters Vmax and Km of a lysosomal enzyme eluted from DBS and the use of these parameters to detect MPS I heterozygotes due to the inability of using IDUA activity values by itself for this purpose. These parameters have previously been determined in leukocytes and serum/plasma, since this type of samples has traditionally been used to detect deficiencies of lysosomal enzymes activities and thus to diagnose different LSD. However, at present a widespread use of DBS as an ideal sample for these diagnoses has occurred due to its benefits, including their potential use in neonatal screening programs for some LSD. Therefore, the use of this type of sample for carriers' detection would also be very advantageous.
Fig. 5. Differential thermal stability of IDUA activity in DBS from MPS I heterozygotes compared to controls. Blank circles: heterozygotes (n = 25), black circles: normal controls (n = 25). Each individual value obtained is represented. The line inside each group represents the median. The p values obtained indicate significant differences between groups for both 12 and 24 h of pre-incubation at 50 °C (Mann–Whitney test).
D. Campos et al. / Clinica Chimica Acta 430 (2014) 24–27
Concerning the thermal stability of the IDUA, after a period of 12 h of pre-incubation at 50 °C the enzyme from heterozygous DBS retained a significantly higher percentage of their original activity compared with controls. This effect is very similar to that reported by Mandelli et al. [6] for plasma samples, although in our case this decrease was not linear but exponential. In leukocyte this effect is obtained only after 15 min of pre-incubation, reaching almost complete denaturation of the enzyme at the end of an hour [7]. The presence of protective elements in plasma and thus in DBS may promote greater stability to the enzyme. An important observation to the studies made by Mandelli et al. [6,7] and applicable to ours, is that they did not identify any of the mutations present in heterozygous individuals. For this reason, it would be important in future studies to estimate IDUA functionality in terms of Km and Vmax in relation with the specific mutation present on each heterozygote. Therefore, the results could be extrapolated to any carrier, regardless of the mutation present in one of its alleles. Currently there is a growing interest in the application of tandem mass spectrometry (MS/MS) for the diagnosis of LSD, mainly as part as newborn screening programs. This is due to the possibility it offers to simultaneously and quickly quantify the specific products of many enzyme activity assays [11]. However, results using MS/MS as a carrier detection method also show a level of overlap between the values of enzymatic activity of the heterozygotes and controls [12], as it occurs with traditional fluorescent methods. As we previously stated, this is a feature of the majority of lysosomal enzymes, including IDUA, due to the high allelic heterogeneity. For this reason, the determination of IDUA activity by itself, regardless of the method used, would not allow a high sensitivity and specificity in the detection of carriers. Nevertheless, as the number or laboratories with this technology is increasing; it would be interesting, although the technological possibilities of our laboratory do not allow it yet, to carry out a similar analysis to the one fulfilled in our case to characterize the biochemical properties of IDUA eluted from DBS but applying MS/MS. 5. Conclusions Based on the results shown here, determining Vmax and the percentage of residual IDUA activity after 24 h of pre-incubation at
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50 °C in DBS can be used to discriminate completely between samples from normal individuals and heterozygotes. Especially due to its simplicity, the second proposed method is much more feasible and practical for studies of carriers in families at risk.
Acknowledgment We thank José L. Hernandez Sierra for his valuable help in the final language revision of the manuscript.
References [1] Neufeld EF, Muenzer J. The mucopolysaccharidoses. In: Scriver C, Beaudet A, Valle D, editors. The metabolic and molecular bases of inherited disease. Ney York, New York: McGraw Hill; 2001. p. 3421–52. [2] de Ru MH, Boelens JJ, Das AM, et al. Enzyme replacement therapy and/or hematopoietic stem cell transplantation at diagnosis in patients with mucopolysaccharidosis type I: results of a European consensus procedure. Orphanet J Rare Dis 2011;6:55. [3] Tolar J, Orchard PJ. Alpha-L-iduronidase therapy for mucopolysaccharidosis type I. Biologics, 2; 2008 743–51. [4] The Human Gene Mutation Database. Institute of Medical Genetics in Cardiff, United Kingdom. BIOBASE Databases, ID: IDUA. http://www.hgmd.cf.ac.uk (acceded November 12, 2012). [5] Clements PR, Yogalingam G, Brooks DA, Muller VJ, Hopwood JJ. α-L-iduronidase genotype/protein function relationships in MPS I fibroblasts. J Inherit Metab Dis 2000;23:237. [6] Mandelli J, Wajner A, Pires R, Giugliani R, Coelho JC. Detection of mucopolysaccharidosis type I heterozygotes on the basis of the biochemical properties of plasma alpha-Liduronidase. Clin Chim Acta 2001;312:81–6. [7] Mandelli J, Wajner A, Pires RF, Giugliani R, Coelho JC. Detection of mucopolysaccharidosis type I heterozygotes based on the biochemical characteristics of leukocyte alpha-L-iduronidase. Arch Med Res 2002;33:20–4. [8] World Medical Association Declaration of Helsinki: ethical principles for medical research involving human subjects. JAMA 2000;284:3043–5. [9] Campos D, Monaga M, González EC, Herrera D, de la Peña D. Optimization of enzymatic diagnosis for mucopolysaccharidosis I in dried blood spots on filter paper. Clin Biochem 2013;46:805–9. [10] Chamoles NA, Blanco M, Gaggioli D. Diagnosis of alpha-L-iduronidase deficiency in dried blood spots on filter paper: the possibility of newborn diagnosis. Clin Chem 2001;47:780–1. [11] Spacil Z, Tatipaka H, Barcenas M, Scott CR, Turecek F, Gelb MH. High-throughput assay of 9 lysosomal enzymes for newborn screening. Clin Chem 2013;59:502–11. [12] Li Y, Scott CR, Chamoles NA, et al. Direct multiplex assay of lysosomal enzymes in dried blood spots for newborn screening. Clin Chem 2004;50:1785–96.
