David Millington (Presenter)
Duke University Hospital
Authorship: David S. Millington, Haoyue Zhang, James Beasley and Sarah P. Young
Duke University Health System, Durham, NC 27713, USA
| Short Abstract Dried urine spots (DUS) on filter paper are readily shippable in the mail to a distant laboratory for analysis. We have adapted a previously developed UPLC-MSMS method of analyzing the glycosaminoglycans (GAGs) CS, DS and HS, in liquid urine to extracts of DUS for this purpose. A fixed amount of d3- creatinine is added to a 2 cm diameter disk excised from a DUS, and creatinine and GAGs are co-extracted. An aliquot of the extract is analyzed for creatinine and the remainder is dried, then methanolysed to produce dimeric subunits derived from the principal GAGs. A mixture of isotope-labeled dimers, prepared from standard GAGs by deuteriomethanolysis, enables quantification by pseudo-isotope-dilution. This assay has sufficient sensitivity, precision and accuracy to reliably measure elevated urinary GAGs in DUS from patients with known or suspected mucopolysaccharidoses. |
Long Abstract
Introduction. Glycosaminoglycans (GAGs) are complex proteoglycans that accumulate in the urine of patients with mucopolysaccharidoses (MPS). We recently developed a method for the urinalysis of the major GAGS chondroitin sulphate (CS), dermatan sulphate (DS) and heparan sulphate (HS) using UPLC-MSMS to quantify their methanolysis products [1,2]. The method is useful for both the diagnosis and monitoring of patients on therapy. Here we report a modification of this method to extract and analyze the GAGs from dried urine spots (DUS) on filter paper that facilitates the collection, shipment and analysis of specimens from distant or challenging locations. The main objective was to validate the method
Methods. The collection of urine as DUS was standardized by saturation of PK cards (Whatman 903 Protein saver card) with patient urine at the site of collection and air-drying at ambient temperature. The DUS were shipped by express mail to the laboratory for analysis. Proficiency testing samples from the ERNDIM scheme as well as calibrators and QC materials (prepared by spiking normal urine with GAG standards) were prepared for analysis by making DUS from them in the same manner. A fixed amount of d3-creatinine was added to a 2 cm diameter disk excised from a DUS, then the creatinine and GAGs were co-extracted. An aliquot of the extract was analyzed for creatinine using LC-MS/MS and the remainder was dried, then methanolysed to produce dimeric subunits derived from the principal GAGs. These dimers primarily consist of uronic acid coupled with an N-acetylhexosamine moiety. A mixture of stable isotope-labeled dimers, prepared from standard GAGs by deuteriomethanolysis, enabled their quantification by pseudo-isotope-dilution using UPLC-ESI-MSMS. A Xevo-TQ MS tandem quadrupole mass spectrometer coupled with an Acquity UPLC system plus autosampler equipped with a BEH Amide 1.7 μm 2.1x 50 mm column (Waters Corporation, Milford, MA) was utilized. Full details of the method have been published [1].
Results. The mean inaccuracy of CS, DS and HS measurements was within acceptable limits (≤ ±9%) according to the back-calculated concentrations determined by replicate analysis of the calibrators. Concentrations were corrected for the endogenous contribution of these analytes in the pooled urine used to prepare the calibrators. Creatinine inaccuracy was 6.8% or less. According to the replicate analysis of quality control samples, intra-day imprecision was 8.9% or below for CS, DS and HS concentrations relative to creatinine. Inter-day imprecision was <15% except for low QC DS and HS, and medium QC HS measurements, which had an imprecision of <25%. For the ERNDIM PT samples, mean recovery of CS was 60 % and linear over the range of 0.7 to 23 g/mol creatinine ; recovery of DS was 63% and linear over the range of 2.0 to 56.8 g/mol creatinine and recovery of HS was 83% and linear over the range of 0.6 to 332 g/mol creatinine. Reference ranges for CS, DS and HS were determined for individuals in five different age ranges and compared with those determined previously in liquid urine samples.
Conclusion. We conclude that the use of dried urine spots for determining urinary GAG concentrations is a useful alternative to liquid samples. Despite lower recoveries relative to liquid urine GAG measurements, the DUS assay has sufficient sensitivity, precision, and accuracy to reliably measure an elevation of GAGs in samples from patients with suspected or confirmed MPS. Dried urine spots provide a convenient option for sample shipment thus facilitating long-term monitoring of urine GAGs in treated patients.
References & Acknowledgements:
1. Zhang H, Young SP, Millington DS. Quantification of glycosaminoglycans in urine by isotope-dilution liquid chromatography-electrospray ionization tandem mass spectrometry. Curr Protoc Hum Genet. 2013; Chapter 17:Unit 17.12. doi: 10.1002/0471142905.hg1712s76.
2. Zhang H, Wood T, Young SP, Millington DS. A straightforward, quantitative ultra-performance liquid chromatography-tandem mass spectrometric method for heparan sulfate, dermatan sulfate and chondroitin sulfate in urine: An improved clinical screening test for the mucopolysaccharidoses. Mol Genet Metab. 2015;114:123-8.
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