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1 Laboratory of Neurochemistry, Uriarte 2383, 1425 Buenos Aires, Argentina.
aAuthor for correspondence. Fax 5411-4774-5920; e-mail nachamoles{at}fibertel.com.ar.
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Abstract |
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Methods: To test tubes containing 3-mm blood spots, we added elution liquid and fluorescent or radioactive substrate solution. After incubation at 37 °C, the reaction was terminated by the addition of a stop buffer. The amount of hydrolyzed product was compared with a calibrator to allow the quantification of enzyme activity. Sample stability was studied during storage for 21 days and during shipment of samples. We measured enzyme activities in 85 healthy controls (35 newborn, 50 adult), 57 patients suffering from 11 lysosomal storage diseases, and 46 obligate carriers.
Results: Intra- and interassay CVs were <9% and <15%, respectively. Mean activity losses during transportation or storage for up to 21 days at 4 °C were 
27%. Enzyme activities in all patients were outside the ranges of values seen for carriers and controls.
Conclusions: The described methodology distinguishes between patients and controls with samples that are sufficiently stable to be mailed to the testing laboratory.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We describe methods for the assay of eight lysosomal enzymes in dried blood spots on filter paper (DBFP), that allow the screening of 12 lysosomal storage diseases manifested with a Hurler-like phenotype. The enzymes included in this protocol, the disorders produced by their dysfunction, the number of patients and carriers examined, and the references for the assay methods that have been adapted to DBFP are shown in Table 1
. MPS III, or Sanfilippo syndrome type A, B, C, and D, and MPS IV A, or Morquio syndrome type A, can present with Hurler-like symptoms, particularly in the early-onset variants. The leukocyte diagnostic methods for these disorders could not be adapted to DBFP samples.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). DBFP samples from 50 healthy adults (18–54 years) and 35 newborns (3–7 days postpartum) were used as controls. DBFP samples were stored at 4 °C in plastic bags until analysis. The assays were performed no more than 72 h after blood sampling.
chemicals
4-Methylumbelliferone, 4-methyl-umbelliferyl (4MU)-
-L-iduronide, D-saccharic acid-1, 4-lactone, 4MU-sulfate, 4MU-ß-D-glucuronic acid, 4MU-ß-D-galactoside, 4MU--L-fucoside, 4MU--D-mannoside, 4MU-ß-D-mannoside, and 4MU-2-acetamido-2-deoxi-ß-D-glucosaminide were obtained from Sigma Co. Tritiated iduronosyl sulfate anhydro-mannitol sulfate and Cellex E (anion-exchange resin) were supplied by Toronto Research Chemicals.
fluorescence enzyme assays in dbfp samples
The detailed assay of -L-iduronidase in DBFP samples has been published elsewhere (2). Briefly, to duplicate 2-mL disposable test tubes containing a 3-mm blood spot (
3.6 µL of blood) obtained with a standard paper punch, we added elution liquid and substrate. After gentle mixing, the tubes were incubated at 37 °C for different times in a slowly oscillating shaking water bath. The tubes were then placed in ice, and 300 µL of glycine-carbonate buffer (0.085 mol/L, pH 10.5) was added to stop the reaction. The filter paper did not need to be removed during the analysis. One blank tube was assayed for each sample. Blanks were prepared by adding 300 µL of stop buffer to a mixture of the paper punch in the elution liquid and the substrate, which had been incubated separately. The elution liquid, substrate, and the incubation time, represented as "EL", "S", and "IT", respectively, for each enzyme assay were as follows.
(a) -L-Iduronidase: IT, 20 h; EL, 40 µL of formate buffer (0.05 mol/L, pH 2.8), containing 0.3 µg of D-saccharic acid-1,4-lactone; S, 20 µL of 2 mmol/L 4MU--L-iduronide in distilled water.
(b) Arylsulfatase B: IT, 20 h; EL, 30 µL of distilled water and 20 µL of 15 mmol/L lead acetate in sodium acetate buffer (0.05 mol/L, pH 5.0); S, 50 µL of 10 mmol/L 4MU-sulfate in sodium acetate buffer (0.05 mol/L, pH 5.0).
(c) ß-D-Glucuronidase: IT, 4 h; EL, 50 µL of distilled water; S, 50 µL of 10 mmol/L 4MU-ß-D-glucuronic acid in sodium acetate buffer (0.1 mol/L, pH 4.8).
(d) ß-D-Galactosidase: IT, 3 h; EL, 40 µL of citrate-phosphate buffer (0.05 mol/L, pH 4.4) in 45 g/L NaCl; S, 20 µL of 0.8 mmol/L 4MU-ß-D-galactoside in distilled water.
(e) -L-Fucosidase: IT, 20 h; EL, 30 µL of sodium citrate buffer (0.17 mol/L, pH 4.5); S, 50 µL of 1 mmol/L 4MU--L-fucoside in distilled water.
(f) ß-Hexosaminidase: IT, 2 h; EL, 50 µL of citrate-phosphate buffer (0.022 mol/L, pH 4.4); S, 100 µL of 3 mmol/L 4MU-2-acetamido-2-deoxy-ß-D-glucosaminide in citrate-phosphate buffer (0.022 mol/L, pH 4.4).
(g) -D-Mannosidase: IT, 2 h; EL, 30 µL of sodium citrate buffer (0.17 mol/L, pH 4.4) containing 17.6 µg of zinc acetate; S, 50 µL of 10 mmol/L 4MU--D-mannoside in distilled water.
Fluorescence (excitation, 365 nm; emission, 450 nm) of the enzyme product 4MU was measured with a Farrand fluorometer model RF-2 (Farrand Optical). The fluorescence readings were corrected for blanks, and the results were compared with the fluorescence from a 4-methylumbelliferone calibrator. Enzymatic activities were expressed as micromoles of substrate hydrolyzed per liter of blood per hour.
iduronate sulfatase assay in dbfp samples
To duplicate 2-mL test tubes containing 3-mm blood spots, we added 50 µL of 16 mmol/L lead acetate as the elution liquid. After gentle mixing for 10 min at room temperature, we added 30 µL of tritiated iduronosyl sulfate anhydromannitol sulfate, 10 µCi/34.3 nmol, specific activity 291 mCi/mmol (Toronto Research Chemicals) (3), previously dissolved in 6.8 mL of sodium acetate buffer (0.33 mol/L, pH 4.5). The tubes were incubated for 20 h at 37 °C in a shaking-water bath. The reaction was stopped with 1 mL of 1 mmol/L sodium dibasic phosphate. The reaction mixture was applied to small columns, each containing 0.6 mL of Cellex E (anion-exchange resin) in water. The columns were washed with 2 mL of 1 mmol/L sodium dibasic phosphate and eluted with 5 mL of 70 mmol/L freshly prepared sodium formate. The eluted liquid was transferred to a scintillation vial, and 15 mL of optifluor (Packard Instrument Co) was added. The vials were counted for 5 min in a Tri-Carb Liquid Scintillation Analyzer (Model 1900 TR; Packard Instrument Co). One enzyme-free blank was assayed in duplicate for each assay. The disintegrations per minute (dpm) readings were corrected for blanks. Iduronate sulfatase activity was expressed as nanomoles of substrate hydrolyzed per liter of blood per hour.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. The extended period of incubation provided enough enzyme products to ensure reliable fluorometer readings. Initially, assays were always performed with one and two punched 3-mm blood spots. The determinations were also made at two different incubation times. Because we did not obtain any significant variation, these steps were later omitted. The possibility of an inhibitor of the enzymatic reactions in the filter paper matrix was eliminated by mixing experiments for each one of the enzymes studied (data not shown).
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The enzyme activities of healthy adult and newborn controls, obligate carriers, and patients are reported in Tables 2
and 3
. The newborn enzyme activities were higher than those of control adults for six enzymes (Table 2
). There was no overlap among the results of patients and carriers or controls. The GM1 gangliosidosis and Sandhoff carriers studied in this experiment showed intermediate degrees of enzyme activity. However, there was a variable overlap between the heterozygotes and the control group for the other reported enzymes. The residual enzyme activities found in DBFP samples from homozygotes are shown in Table 3
, expressed as a percentage of the mean of adult controls.
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The intraassay CVs from healthy controls (n = 10) were 6% and 9% for the fluorescent and radioactive methods, respectively. The interassay CVs for DBFP samples studied on five different occasions within 1 month were 9% for the fluorescent methods and 14% for the iduronate sulfatase assay.
A positive correlation was found between the leukocyte count in blood and the degree of enzyme activity measured in DBFP samples. The decreases in enzyme values related to a low leukocyte count can be easily identified by simultaneous assay of the activities of several enzymes with preservation of their ratios. Activities of arylsulfatase B and ß-galactosidase of 18.5 and 42.1 µmol/L of blood per hour (arylsulfatase B/ß-galactosidase ratio, 0.4), respectively, with a leukocyte count of 10 100/mm3, changed to 9.2 and 22.2 µmol/L of blood per hour (arylsulfatase B/ß-galactosidase ratio, 0.4), respectively, with a leukocyte count of 6000/mm3. Similar results have been observed in two samples stored incorrectly.
The different enzyme activities present in plasma and blood cells explain the nonlinear correlation between the enzyme activities from DBFP and leukocyte samples (data not shown).
There were no significant changes in enzyme activity of DBFP samples after storage for 21 days at -20 °C, 4 °C, or 25 °C. To verify the stability of these enzymes under usual mailing conditions, DBFP samples from four controls, two GM1 gangliosidosis patients, two gangliosidosis carriers, two Maroteaux-Lamy patients, two Maroteaux-Lamy carriers, two Hunter patients, and two Hunter carriers were sent by air mail to the following locations: Sao Paulo, Brazil; Toronto, Canada; Brussels, Belgium; and Christchurch, New Zealand. The samples were sent back to our laboratory during our summer season (temperature, 28–37 °C). The turnaround times were 12, 15, 16, and 18 days, respectively. The mean enzyme activities from DBFP samples obtained from controls, carriers, and patients were reduced by almost the same percentage, which in no case was >27% of the initial activity (Fig. 2
). The reduction did not modify the differentiation of patients from controls and carriers. ß-Galactosidase, ß-glucuronidase, and iduronate sulfatase were the most sensitive to the effect of shipment. Should DBFP samples from a patient under investigation be sent by mail to a specialized laboratory for analysis, it will be necessary to simultaneously send DBFP samples from a nonrelated control and an obligate carrier for comparison. This procedure will point out any unexpected decrease of the enzyme activities produced by transportation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Treatment of some of the genetic disorders expressing a Hurler-like phenotype became possible by bone marrow transplantation (6), enzyme replacement therapy (7), and gene modification (8). The effectiveness of these therapies, particularly for MPS involving the central nervous system, may rely heavily on early diagnosis of the disorders. Early diagnosis of the patient will allow clinicians to take advantage of the period of natural suppression of the immune system of the neonate to maximize the chances for a successful bone marrow engraftment (9). The convenience of treating presymptomatic patients and, at present, the possibility of an early diagnosis may make it reasonable to discuss the use of this methodology for a pilot newborn-screening program. For such a program, microplate adaptation and method automation could be easily performed.
In conclusion, the presented methodology is reliable and sensitive for measuring lysosomal enzyme activities in DBFP samples. DBFP methods can simultaneously measure 50 or more samples in duplicate in each assay, with no need for homogenization or protein assay procedures as is required for isolated leukocytes. All reagents are commercially available, no special or costly equipment is needed, and a quality-control program for the enzyme assays could be easily organized. Our preliminary results suggest that these methods can be applied to screen Hurler-like patients for lysosomal enzymes deficiencies. In abnormal cases, a blood sample for leukocyte isolation or a fibroblast skin culture should be requested for further characterization of the biochemical and molecular phenotypes of the disorder. To further validate this method, it will be important to study a larger population of patients and carriers. However, because of the low frequency of these disorders, such studies will be possible only with the cooperation of several specialized centers around the world.
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Acknowledgments |
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-L-iduronidase deficiency in dried blood spots on filter paper: the possibility of newborn diagnosis. Clin Chem 2001;47:780-781.-L-iduronidase deficiency (Hurler syndrome) in human bone marrow. Proc Natl Acad Sci U S A 1996;93:2025-2030.The following articles in journals at HighWire Press have cited this article:
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  J. Muenzer, J. E. Wraith, L. A. Clarke, and and the International Consensus Panel on the Manag Mucopolysaccharidosis I: Management and Treatment Guidelines Pediatrics, January 1, 2009; 123(1): 19 - 29. [Abstract] [Full Text] [PDF]
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 E. Parkinson-Lawrence, M. Fuller, J. J. Hopwood, P. J. Meikle, and D. A. Brooks Immunochemistry of Lysosomal Storage Disorders Clin. Chem., September 1, 2006; 52(9): 1660 - 1668. [Abstract] [Full Text] [PDF]
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C. J. Dean, M. R. Bockmann, J. J. Hopwood, D. A. Brooks, and P. J. Meikle Detection of Mucopolysaccharidosis Type II by Measurement of Iduronate-2-Sulfatase in Dried Blood Spots and Plasma Samples Clin. Chem., April 1, 2006; 52(4): 643 - 649. [Abstract] [Full Text] [PDF]
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D. Wang, B. Eadala, M. Sadilek, N. A. Chamoles, F. Turecek, C. R. Scott, and M. H. Gelb Tandem Mass Spectrometric Analysis of Dried Blood Spots for Screening of Mucopolysaccharidosis I in Newborns Clin. Chem., May 1, 2005; 51(5): 898 - 900. [Full Text] [PDF]
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Y. Li, K. Brockmann, F. Turecek, C. R. Scott, and M. H. Gelb Tandem Mass Spectrometry for the Direct Assay of Enzymes in Dried Blood Spots: Application to Newborn Screening for Krabbe Disease Clin. Chem., March 1, 2004; 50(3): 638 - 640. [Full Text] [PDF]
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 D. A. Wenger, S. Coppola, and S.-L. Liu Insights Into the Diagnosis and Treatment of Lysosomal Storage Diseases Arch Neurol, March 1, 2003; 60(3): 322 - 328. [Abstract] [Full Text] [PDF]
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Z. Lukacs, P. Santavuori, A. Keil, R. Steinfeld, and A. Kohlschutter Rapid and Simple Assay for the Determination of Tripeptidyl Peptidase and Palmitoyl Protein Thioesterase Activities in Dried Blood Spots Clin. Chem., March 1, 2003; 49(3): 509 - 511. [Full Text] [PDF]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
), y = 0.7089x + 3.52 (R2 = 0.9963); healthy adult (line 2; 
), y = 0.405x + 2.1 (R2 = 0.9957); GM1 carrier (line 3; 
), y = 0.121x + 1.8 (R2 = 0.9661). (B), healthy newborn (line 1; 
, activity in GM1 patient in A and MPS II patient in B.
), samples from Brussels (