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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: 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.This technique will significantly simplify the early detection of MPS IVA patients.

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

Influence of storage conditions on GALNS activity in DBS.Stability tests show rthat filtercards with DBS stored in zip bags maintain their enzymatic activity when stored at– 20°C and 4°C up to 35 days, while samples stored at a temperature of 27°C and above lose over 40% of their activity in the first 5 days. Ideal sample transport should be at a maximum of 22°C and storage at 4°C or below.
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pone.0131228.g004: Influence of storage conditions on GALNS activity in DBS.Stability tests show rthat filtercards with DBS stored in zip bags maintain their enzymatic activity when stored at– 20°C and 4°C up to 35 days, while samples stored at a temperature of 27°C and above lose over 40% of their activity in the first 5 days. Ideal sample transport should be at a maximum of 22°C and storage at 4°C or below.

Mentions: Compared with other lysosomal enzymes that are stable for longer time periods (months) in DBS stored at room temperature, our experiments showed that GALNS activity is dependent on the transport and storage conditions of the filtercards. Stability tests performed on the same blood sample at -20°C, + 4°C, room temperature, and 37°C, on sealed and air exposed filtercards, showed a decrease of enzymatic activity in the unsealed filtercards compared with the ones stored in plastic bags for the same temperature. The same study proved stability of GALNS activity for the samples stored at -20°C and at 4°C for at least 6 weeks in sealed plastic bags (Fig 4).


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)

Influence of storage conditions on GALNS activity in DBS.Stability tests show rthat filtercards with DBS stored in zip bags maintain their enzymatic activity when stored at– 20°C and 4°C up to 35 days, while samples stored at a temperature of 27°C and above lose over 40% of their activity in the first 5 days. Ideal sample transport should be at a maximum of 22°C and storage at 4°C or below.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0131228.g004: Influence of storage conditions on GALNS activity in DBS.Stability tests show rthat filtercards with DBS stored in zip bags maintain their enzymatic activity when stored at– 20°C and 4°C up to 35 days, while samples stored at a temperature of 27°C and above lose over 40% of their activity in the first 5 days. Ideal sample transport should be at a maximum of 22°C and storage at 4°C or below.
Mentions: Compared with other lysosomal enzymes that are stable for longer time periods (months) in DBS stored at room temperature, our experiments showed that GALNS activity is dependent on the transport and storage conditions of the filtercards. Stability tests performed on the same blood sample at -20°C, + 4°C, room temperature, and 37°C, on sealed and air exposed filtercards, showed a decrease of enzymatic activity in the unsealed filtercards compared with the ones stored in plastic bags for the same temperature. The same study proved stability of GALNS activity for the samples stored at -20°C and at 4°C for at least 6 weeks in sealed plastic bags (Fig 4).

Bottom Line: 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.This technique will significantly simplify the early detection of MPS IVA patients.

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