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Diagnosis of Morquio Syndrome in Dried Blood Spots Based on a New MRM-MS Assay.

Cozma C, Eichler S, Wittmann G, Flores Bonet A, Kramp GJ, Giese AK, Rolfs A - PLoS ONE (2015)

Bottom Line: Mucopolysaccharidosis IVA (MPS IVA; Morquio A disease) is an autosomal recessive disease caused and characterized by a decreased activity of N-acetylgalactosamine-6-sulfate sulfatase (GALNS), resulting in accumulation of keratan sulfate and chondroitin-6-sulfate in tissues and secondary organ damage.The material extracted from DBS was incubated with a 4-methylumbelliferyl-β-D-galactopyranoside-6-sulfate as a specific substrate.Final enzymatic product, 4-methylumbelliferone, obtained after adding exogenous beta-galactosidase, was quantified by LC/MRM-MS (liquid-chromatography/multiple-reaction-monitoring mass-spectrometry). 4-propyl-5-hydroxy-7-methyl-2h-chromen-2-one was used as internal standard, a compound with a similar molecular structure and fragmentation pattern in negative ion mode as 4-methylumbelliferone.

View Article: PubMed Central - PubMed

Affiliation: Centogene AG, Rostock, Germany.

ABSTRACT

Background: Mucopolysaccharidosis IVA (MPS IVA; Morquio A disease) is an autosomal recessive disease caused and characterized by a decreased activity of N-acetylgalactosamine-6-sulfate sulfatase (GALNS), resulting in accumulation of keratan sulfate and chondroitin-6-sulfate in tissues and secondary organ damage. Recently approved enzyme replacement therapy renders the easy and early identification of MPS IVA of out-most importance.

Methodology: We propose a completely new assay for the stable and reproducible detection of GALNS deficiency in dry blood spots (DBS). For the validation blood samples were taken from 59 healthy individuals and 24 randomly selected genetically confirmed MPS IVA patients. The material extracted from DBS was incubated with a 4-methylumbelliferyl-β-D-galactopyranoside-6-sulfate as a specific substrate. Final enzymatic product, 4-methylumbelliferone, obtained after adding exogenous beta-galactosidase, was quantified by LC/MRM-MS (liquid-chromatography/multiple-reaction-monitoring mass-spectrometry). 4-propyl-5-hydroxy-7-methyl-2h-chromen-2-one was used as internal standard, a compound with a similar molecular structure and fragmentation pattern in negative ion mode as 4-methylumbelliferone.

Findings: The enzymatic assay yielded a positive and negative predictive value of 1.0 for genetically confirmed MPS IVA patients (GALNS activity of 0.35 ± 0.21 μmol/L/h) and for controls with normal GALNS activity (23.1 ± 5.3 μmol/L /h). With present enzymatic conditions, the reaction yield in dried blood spots is at least 20 fold higher than any previously reported data with other assays.

Interpretation: The present LC/MRM-MS based assay for MPS IVA diagnosis provides an easy, highly-standardized, accurate and innovative quantification of the enzymatic product in vitro and distinguishes perfectly between MPS IVA affected patients and normal controls. This technique will significantly simplify the early detection of MPS IVA patients.

No MeSH data available.


Related in: MedlinePlus

TripleQuad MRM-MS detection of 4-MU.Hydroxyl-chromen-2H-one compounds can be detected in Q1 scan as a (M-H)- ion and, under specific collision energy, the hetero-cycle is broken with a neutral loss of the fragment containing–COO•. We propose that the remaining fragment undergoes a molecular rearrangement to obtain a more stable structure. For enzymatic product detection and quantification, two transitions are monitored: 175/119 (4-MU) and 217/160 (internal standard). A. Collision induced dissociation fragmentation spectrum (MS2) of the analyte (4-MU), obtained using an ABSciex 5500, and MRM-MS transition spectrum monitored during the MPS IV assays (175/119); B. Collision induced dissociation fragmentation spectrum (MS2) of the internal standard (4-propyl-5-hydroxy-7-methyl-2H-chromen-2-one), obtained using an ABSciex 5500, and MRM-MS transition spectrum monitored during the MPS IV assays (217/160).
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pone.0131228.g001: TripleQuad MRM-MS detection of 4-MU.Hydroxyl-chromen-2H-one compounds can be detected in Q1 scan as a (M-H)- ion and, under specific collision energy, the hetero-cycle is broken with a neutral loss of the fragment containing–COO•. We propose that the remaining fragment undergoes a molecular rearrangement to obtain a more stable structure. For enzymatic product detection and quantification, two transitions are monitored: 175/119 (4-MU) and 217/160 (internal standard). A. Collision induced dissociation fragmentation spectrum (MS2) of the analyte (4-MU), obtained using an ABSciex 5500, and MRM-MS transition spectrum monitored during the MPS IV assays (175/119); B. Collision induced dissociation fragmentation spectrum (MS2) of the internal standard (4-propyl-5-hydroxy-7-methyl-2H-chromen-2-one), obtained using an ABSciex 5500, and MRM-MS transition spectrum monitored during the MPS IV assays (217/160).

Mentions: The most important improvement introduced by the present assay, is the development of a stable, reproducible multiple reaction monitoring mass spectrometry method for the detection and quantification of 4-methylumbelliferone (4-MU). Although previous studies on flavonoid compounds refer to 4-MU as an internal standard [21; 22; 23], there are no published studies on the routine quantification of 4-MU by mass spectrometry with an application in clinical chemistry. 4-MU, as other hydroxyl-chromen-2H-one derivatives, is a very stable molecule, fluorescent in UV light and thus used routinely in fluorimetric enzyme assays [24]. However, in the presence of blood elements, especially hemoglobin, fluorescence quenching was reported by different studies. This phenomenon cannot be precisely quantified for each DBS sample separately, rendering a quantification error inevitable. Also, although sensitive, fluorometry uses only a relative quantification through external standard curve. With the development of new instrumentation, multiple reaction monitoring mass spectrometry rivals the sensitivity and the high through-put capacity of the fluorometry; however, by using an internal standard, the selectivity and precision of the quantification is highly superior to the fluorimetric method. Here, we report a new quantification method for 4-MU using an TripleQuad 5500 mass spectrometer (ABSciex, Germany) using a standard curve of nine 4-MU dilutions from 0 μg/mL to 1 μg/mL and an internal standard with a fixed concentration of 0.5 μg/mL. As internal standards for 4-MU quantification, several hydroxyl-chromen-2H-one derivatives were tested and all were found suitable having similar fragmentation pattern with 4-MU under similar collision induced dissociation (CID) conditions. However, 4-propyl-5-hydroxy-7-methyl-2H-chromen-2-one was chosen due to its different parent/daughter transitions (217/160—internal standard and 175/119–4-MU) and similar fragmentation pattern (see Fig 1); also due to its different HPLC retention time compared to 4-MU. The standard curve was linear and reproducible for the concentration range tested (that includes the analytical range for the method).


Diagnosis of Morquio Syndrome in Dried Blood Spots Based on a New MRM-MS Assay.

Cozma C, Eichler S, Wittmann G, Flores Bonet A, Kramp GJ, Giese AK, Rolfs A - PLoS ONE (2015)

TripleQuad MRM-MS detection of 4-MU.Hydroxyl-chromen-2H-one compounds can be detected in Q1 scan as a (M-H)- ion and, under specific collision energy, the hetero-cycle is broken with a neutral loss of the fragment containing–COO•. We propose that the remaining fragment undergoes a molecular rearrangement to obtain a more stable structure. For enzymatic product detection and quantification, two transitions are monitored: 175/119 (4-MU) and 217/160 (internal standard). A. Collision induced dissociation fragmentation spectrum (MS2) of the analyte (4-MU), obtained using an ABSciex 5500, and MRM-MS transition spectrum monitored during the MPS IV assays (175/119); B. Collision induced dissociation fragmentation spectrum (MS2) of the internal standard (4-propyl-5-hydroxy-7-methyl-2H-chromen-2-one), obtained using an ABSciex 5500, and MRM-MS transition spectrum monitored during the MPS IV assays (217/160).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4492791&req=5

pone.0131228.g001: TripleQuad MRM-MS detection of 4-MU.Hydroxyl-chromen-2H-one compounds can be detected in Q1 scan as a (M-H)- ion and, under specific collision energy, the hetero-cycle is broken with a neutral loss of the fragment containing–COO•. We propose that the remaining fragment undergoes a molecular rearrangement to obtain a more stable structure. For enzymatic product detection and quantification, two transitions are monitored: 175/119 (4-MU) and 217/160 (internal standard). A. Collision induced dissociation fragmentation spectrum (MS2) of the analyte (4-MU), obtained using an ABSciex 5500, and MRM-MS transition spectrum monitored during the MPS IV assays (175/119); B. Collision induced dissociation fragmentation spectrum (MS2) of the internal standard (4-propyl-5-hydroxy-7-methyl-2H-chromen-2-one), obtained using an ABSciex 5500, and MRM-MS transition spectrum monitored during the MPS IV assays (217/160).
Mentions: The most important improvement introduced by the present assay, is the development of a stable, reproducible multiple reaction monitoring mass spectrometry method for the detection and quantification of 4-methylumbelliferone (4-MU). Although previous studies on flavonoid compounds refer to 4-MU as an internal standard [21; 22; 23], there are no published studies on the routine quantification of 4-MU by mass spectrometry with an application in clinical chemistry. 4-MU, as other hydroxyl-chromen-2H-one derivatives, is a very stable molecule, fluorescent in UV light and thus used routinely in fluorimetric enzyme assays [24]. However, in the presence of blood elements, especially hemoglobin, fluorescence quenching was reported by different studies. This phenomenon cannot be precisely quantified for each DBS sample separately, rendering a quantification error inevitable. Also, although sensitive, fluorometry uses only a relative quantification through external standard curve. With the development of new instrumentation, multiple reaction monitoring mass spectrometry rivals the sensitivity and the high through-put capacity of the fluorometry; however, by using an internal standard, the selectivity and precision of the quantification is highly superior to the fluorimetric method. Here, we report a new quantification method for 4-MU using an TripleQuad 5500 mass spectrometer (ABSciex, Germany) using a standard curve of nine 4-MU dilutions from 0 μg/mL to 1 μg/mL and an internal standard with a fixed concentration of 0.5 μg/mL. As internal standards for 4-MU quantification, several hydroxyl-chromen-2H-one derivatives were tested and all were found suitable having similar fragmentation pattern with 4-MU under similar collision induced dissociation (CID) conditions. However, 4-propyl-5-hydroxy-7-methyl-2H-chromen-2-one was chosen due to its different parent/daughter transitions (217/160—internal standard and 175/119–4-MU) and similar fragmentation pattern (see Fig 1); also due to its different HPLC retention time compared to 4-MU. The standard curve was linear and reproducible for the concentration range tested (that includes the analytical range for the method).

Bottom Line: Mucopolysaccharidosis IVA (MPS IVA; Morquio A disease) is an autosomal recessive disease caused and characterized by a decreased activity of N-acetylgalactosamine-6-sulfate sulfatase (GALNS), resulting in accumulation of keratan sulfate and chondroitin-6-sulfate in tissues and secondary organ damage.The material extracted from DBS was incubated with a 4-methylumbelliferyl-β-D-galactopyranoside-6-sulfate as a specific substrate.Final enzymatic product, 4-methylumbelliferone, obtained after adding exogenous beta-galactosidase, was quantified by LC/MRM-MS (liquid-chromatography/multiple-reaction-monitoring mass-spectrometry). 4-propyl-5-hydroxy-7-methyl-2h-chromen-2-one was used as internal standard, a compound with a similar molecular structure and fragmentation pattern in negative ion mode as 4-methylumbelliferone.

View Article: PubMed Central - PubMed

Affiliation: Centogene AG, Rostock, Germany.

ABSTRACT

Background: Mucopolysaccharidosis IVA (MPS IVA; Morquio A disease) is an autosomal recessive disease caused and characterized by a decreased activity of N-acetylgalactosamine-6-sulfate sulfatase (GALNS), resulting in accumulation of keratan sulfate and chondroitin-6-sulfate in tissues and secondary organ damage. Recently approved enzyme replacement therapy renders the easy and early identification of MPS IVA of out-most importance.

Methodology: We propose a completely new assay for the stable and reproducible detection of GALNS deficiency in dry blood spots (DBS). For the validation blood samples were taken from 59 healthy individuals and 24 randomly selected genetically confirmed MPS IVA patients. The material extracted from DBS was incubated with a 4-methylumbelliferyl-β-D-galactopyranoside-6-sulfate as a specific substrate. Final enzymatic product, 4-methylumbelliferone, obtained after adding exogenous beta-galactosidase, was quantified by LC/MRM-MS (liquid-chromatography/multiple-reaction-monitoring mass-spectrometry). 4-propyl-5-hydroxy-7-methyl-2h-chromen-2-one was used as internal standard, a compound with a similar molecular structure and fragmentation pattern in negative ion mode as 4-methylumbelliferone.

Findings: The enzymatic assay yielded a positive and negative predictive value of 1.0 for genetically confirmed MPS IVA patients (GALNS activity of 0.35 ± 0.21 μmol/L/h) and for controls with normal GALNS activity (23.1 ± 5.3 μmol/L /h). With present enzymatic conditions, the reaction yield in dried blood spots is at least 20 fold higher than any previously reported data with other assays.

Interpretation: The present LC/MRM-MS based assay for MPS IVA diagnosis provides an easy, highly-standardized, accurate and innovative quantification of the enzymatic product in vitro and distinguishes perfectly between MPS IVA affected patients and normal controls. This technique will significantly simplify the early detection of MPS IVA patients.

No MeSH data available.


Related in: MedlinePlus