Quantification of Glycosaminoglycans in Urine by Isotope‐Dilution Liquid Chromatography‐Electrospray Ionization Tandem Mass Spectrometry

Haoyue Zhang1, Sarah P. Young1, David S. Millington1

1 Division of Medical Genetics, Department of Pediatrics, Duke University Medical Center, Durham, North Carolina
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 17.12
DOI:  10.1002/0471142905.hg1712s76
Online Posting Date:  January, 2013
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Abstract

Mucopolysaccharidoses (MPSs) are complex lysosomal storage disorders that result in the accumulation of glycosaminoglycans (GAGs) in urine, blood, and tissues. Lysosomal enzymes responsible for GAG degradation are defective in MPSs. GAGs including chondroitin sulfate (CS), dermatan sulfate (DS), heparan sulfate (HS), and keratan sulfate (KS) are disease‐specific biomarkers for MPSs. This unit describes a stable isotope dilution‐tandem mass spectrometric method for quantifying CS, DS, and HS in urine samples. The GAGs are methanolyzed to uronic or iduronic acid‐N‐acetylhexosamine or iduronic acid‐N‐sulfo‐glucosamine dimers and mixed with internal standards derived from deuteriomethanolysis of GAG standards. Specific dimers derived from HS, DS, and CS are separated by ultra‐performance liquid chromatography (UPLC) and analyzed by electrospray ionization tandem mass spectrometry (MS/MS) using selected reaction monitoring for each targeted GAG product and its corresponding internal standard. This new GAG assay is useful for identifying patients with MPS types I, II, III, VI, and VII. Curr. Protoc. Hum. Genet. 76:17.12.1‐17.12.14. © 2013 by John Wiley & Sons, Inc.

Keywords: glycosaminoglycans; dermatan sulfate; heparan sulfate; mucopolysaccharidosis; LC‐ESI‐MS/MS

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Urinary GAG Analysis by ESI‐MS/MS
  • Support Protocol 1: Prepare Calibration Samples
  • Support Protocol 2: Preparation of Stable Isotope Labeled Internal Standards
  • Support Protocol 3: Preparation of Quality Controls for GAG Analysis in Urine
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Urinary GAG Analysis by ESI‐MS/MS

  Materials
  • Urine sample for analysis
  • Nitrogen source
  • 3 N HCl in methanol (Sigma‐Aldrich)
  • Quality control samples (see protocol 4)
  • Calibration samples (see protocol 2)
  • Internal standards (IS; see protocol 3)
  • Ammonium acetate (Fluka Analytical, cat. no. 73594)
  • Acetonitrile (EMD Chemicals, cat. no. AX0145P‐1)
  • 1.8‐ml borosilicate E‐Z vials (Wheaton, cat. no. 225179SP) for derivatization with 11‐mm aluminum‐crimp vial caps with rubber/clear PTFE liner (Wheaton, cat. no W224211‐01SP); these vials require a crimping tool to seal the caps on the vials
  • 2‐ml 12 × 32–mm glass Maximum Recovery vials (Waters, cat. no. 18600327C) with cap and preslit PTFE/Silicone septa for autosampler (or similar vial for model of autosampler used)
  • Heated nitrogen drier
  • Incubation oven set at 65°C
  • Xevo‐TQ MS tandem quadrupole mass spectrometer equipped with an Acquity UPLC system with autosampler (Waters Corporation)
  • Acquity UPLC BEH Amide 1.7 µm, 2.1 × 50–mm column (Waters cat. no. 186004800)
  • VanGuard BEH Amide 1.7‐µm guard column (Waters cat. no. 186004799)
  • TargetLynx software (Waters Corp.)
NOTE: The creatinine value for urine samples is required for the calculation of GAG concentration. It is expected the laboratories will use standard procedures to measure creatinine, and the methodology for that determination is not described in this unit.

Support Protocol 1: Prepare Calibration Samples

  Materials
  • Chondroitin sulfate A (Sigma‐Aldrich C‐8529)
  • Chondroitin sulfate B (dermatan sulfate; Sigma‐Aldrich C‐3788)
  • Heparan sulfate (Sigma‐Aldrich C‐7640)
  • Control urine with low endogenous concentrations of GAG species: a pooled sample from normal volunteers; our criteria for the control urine is that the concentration CS, DS, and HS should each be less than 4 µg/ml and the expected pattern of GAG species should be consistent with other samples in the control group
  • Nitrogen Source
  • 10‐ml volumetric flask
  • 1.8‐ml borosilicate E‐Z vials (Wheaton, cat. no. 225179SP) with 11‐mm aluminum‐crimp vial caps with rubber/clear PTFE liner (Wheaton, cat no. W224211‐01SP); these vials require a crimping tool to seal the caps on the vials

Support Protocol 2: Preparation of Stable Isotope Labeled Internal Standards

  Materials
  • [2H] 4methanol (store at 4°C)
  • Acetyl chloride (store at 4°C)
  • 1.8‐ml borosilicate vials each containing 300 µg dried DS or HS standard prepared by pipetting 300 µl each of DS or HS working standard solution 1 (1 mg/ml) into separate vials and removal of the solvent using nitrogen at 30°C
  • Sep‐Pak Vac C18 solid phase extraction cartridges (100 mg) (Waters Corporation, cat. no. WAT023590) (for internal standard preparation)
  • Nitrogen source
  • Acetonitrile (EMD Chemicals, cat. no. AX0145P‐1)
  • 65°C water bath
  • Heated nitrogen drier
  • Sep‐Pak Vac C18 solid‐phase extraction cartridges (100 mg; Waters Corporation, cat. no. WAT023590)

Support Protocol 3: Preparation of Quality Controls for GAG Analysis in Urine

  Materials
  • GAG stock solution (5.0 mg/ml; see protocol 2, step 1a)
  • GAG working standard solution 2 (200 µg/ml; see protocol 2, step 1c)
  • Pooled normal control urine (40 ml needed): a pooled sample from normal volunteers; our criteria for the control urine is that the concentration CS, DS, and HS should each be less than 4 µg/ml and the expected pattern of GAG species should be consistent with other samples in the control group
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Figures

Videos

Literature Cited

Literature Cited
   Auclair, D., Finnie, J., White, J., Nielsen, T., Fuller, M., Kakkis, E., Cheng, A., O'Neill, C.A., and Hopwood, J.J. 2010. Repeated intrathecal injections of recombinant human 4‐sulphatase remove dural storage in mature mucopolysaccharidosis VI cats primed with a short‐course tolerisation regimen. Mol. Genet. Metab. 99:132‐141.
   Auray‐Blais, C., Bherer, P., Gagnon, R., Young, S.P., Zhang, H.H., An, Y., Clarke, J.T., and Millington, D.S. 2011. Efficient analysis of urinary glycosaminoglycans by LC‐MS/MS in mucopolysaccharidoses type I, II and VI. Mol. Genet. Metab. 102:49‐56.
   Cox‐Brinkman, J., Boelens, J.J., Wraith, J.E., O'Meara, A., Veys, P., Wijburg, F.A., Wulffraat, N., and Wynn, R.F. 2006. Haematopoietic cell transplantation (HCT) in combination with enzyme replacement therapy (ERT) in patients with Hurler syndrome. Bone Marrow Transplant. 38:17‐21.
   Cox‐Brinkman, J., Timmermans, R.G., Wijburg, F.A., Donker, W.E., van de Ploeg, A.T., Aerts, J.M., and Hollak, C.E. 2007. Home treatment with enzyme replacement therapy for mucopolysaccharidosis type I is feasible and safe. J. Inherit. Metab. Dis. 30:984.
   de Jong, J.G., Wevers, R.A., Laarakkers, C., and Poorthuis, B.J. 1989. Dimethylmethylene blue‐based spectrophotometry of glycosaminoglycans in untreated urine: A rapid screening procedure for mucopolysaccharidoses. Clin. Chem. 35:1472‐1477.
   de Jong, J.G., Wevers, R.A., and Liebrand‐van Sambeek, R. 1992. Measuring urinary glycosaminoglycans in the presence of protein: An improved screening procedure for mucopolysaccharidoses based on dimethylmethylene blue. Clin. Chem. 38:803‐807.
   Dembure, P.P., Drumheller, J.E., Barr, S.M., and Elsas, L.J. 1990. Selective urinary screening for mucopolysaccharidoses. Clin. Biochem. 23:91‐96.
   Dickson, P., McEntee, M., Vogler, C., Le, S., Levy, B., Peinovich, M., Hanson, S., Passage, M., and Kakkis, E. 2007. Intrathecal enzyme replacement therapy: Successful treatment of brain disease via the cerebrospinal fluid. Mol. Genet. Metab. 91:61‐68.
   Dickson, P., Peinovich, M., McEntee, M., Lester, T., Le, S., Krieger, A., Manuel, H., Jabagat, C., Passage, M., and Kakkis, E.D. 2008. Immune tolerance improves the efficacy of enzyme replacement therapy in canine mucopolysaccharidosis I. J. Clin. Invest. 118:2868‐2876.
   Dickson, P.I., Hanson, S., McEntee, M.F., Vite, C.H., Vogler, C.A., Mlikotic, A., Chen, A.H., Ponder, K.P., Haskins, M.E., Tippin, B.L., Le, S.Q., Passage, M.B., Guerra, C., Dierenfeld, A., Jens, J., Snella, E., Kan, S.H., and Ellinwood, N.M. 2010. Early versus late treatment of spinal cord compression with long‐term intrathecal enzyme replacement therapy in canine mucopolysaccharidosis type I. Mol. Genet. Metab. 101:115‐122.
   Farndale, R.W., Sayers, C.A., and Barrett, A.J. 1982. A direct spectrophotometric microassay for sulfated glycosaminoglycans in cartilage cultures. Connect. Tissue Res. 9:247‐248.
   Kakkis, E., McEntee, M., Vogler, C., Le, S., Levy, B., Belichenko, P., Mobley, W., Dickson, P., Hanson, S., and Passage, M. 2004. Intrathecal enzyme replacement therapy reduces lysosomal storage in the brain and meninges of the canine model of MPS I. Mol. Genet. Metab. 83:163‐174.
   Mason, K.E., Meikle, P.J., Hopwood, J.J., and Fuller, M. 2006. Characterization of sulfated oligosaccharides in mucopolysaccharidosis type IIIA by electrospray ionization mass spectrometry. Anal. Chem. 78:4534‐4542.
   Muenzer, J. 2004. The mucopolysaccharidoses: A heterogeneous group of disorders with variable pediatric presentations. J. Pediatr. 144:S27‐S34.
   Muenzer, J. 2011. Overview of the mucopolysaccharidoses. Rheumatology (Oxford) 50:v4‐v12.
   Munoz‐Rojas, M.V., Vieira, T., Costa, R., Fagondes, S., John, A., Jardim, L.B., Vedolin, L.M., Raymundo, M., Dickson, P.I., Kakkis, E., and Giugliani, R. 2008. Intrathecal enzyme replacement therapy in a patient with mucopolysaccharidosis type I and symptomatic spinal cord compression. Am. J. Med. Genet. A 146A:2538‐2544.
   Munoz‐Rojas, M.V., Horovitz, D.D., Jardim, L.B., Raymundo, M., Llerena, J.C. Jr., de Magalhaes Tde, S., Vieira, T.A., Costa, R., Kakkis, E., and Giugliani, R. 2010. Intrathecal administration of recombinant human N‐acetylgalactosamine 4‐sulfatase to a MPS VI patient with pachymeningitis cervicalis. Mol. Genet. Metab. 99:346‐350.
   Nielsen, T.C., Rozek, T., Hopwood, J.J., and Fuller, M. 2010. Determination of urinary oligosaccharides by high‐performance liquid chromatography/electrospray ionization‐tandem mass spectrometry: Application to Hunter syndrome. Anal. Biochem. 402:113‐120.
   Oguma, T., Tomatsu, S., Montano, A.M., and Okazaki, O. 2007. Analytical method for the determination of disaccharides derived from keratan, heparan, and dermatan sulfates in human serum and plasma by high‐performance liquid chromatography/turbo ionspray ionization tandem mass spectrometry. Anal. Biochem. 368:79‐86.
   Ramsay, S.L., Meikle, P.J., and Hopwood, J.J. 2003. Determination of monosaccharides and disaccharides in mucopolysaccharidoses patients by electrospray ionisation mass spectrometry. Mol. Genet. Metab. 78:193‐204.
   Stone, J.E. 1998. Urine analysis in the diagnosis of mucopolysaccharide disorders. Ann. Clin. Biochem. 35:207‐225.
   Tomatsu, S., Montano, A.M., Oguma, T., Dung, V.C., Oikawa, H., de Carvalho, T.G., Gutierrez, M.L., Yamaguchi, S., Suzuki, Y., Fukushi, M., Kida, K., Kubota, M., Barrera, L., and Orii, T. 2010a. Validation of keratan sulfate level in mucopolysaccharidosis type IVA by liquid chromatography‐tandem mass spectrometry. J. Inherit. Metab. Dis.Epub ahead of print.
   Tomatsu, S., Montano, A.M., Oguma, T., Dung, V.C., Oikawa, H., de Carvalho, T.G., Gutierrez, M.L., Yamaguchi, S., Suzuki, Y., Fukushi, M., Sakura, N., Barrera, L., Kida, K., Kubota, M., and Orii, T. 2010b. Dermatan sulfate and heparan sulfate as a biomarker for mucopolysaccharidosis I. J. Inherit. Metab. Dis. 33:141‐150.
   Valayannopoulos, V. and Wijburg, F.A. 2011. Therapy for the mucopolysaccharidoses. Rheumatology (Oxford) 50:v49‐v59.
   Zhang, H., Young, S.P., Auray‐Blais, C., Orchard, P.J., Tolar, J., and Millington, D.S. 2011. Analysis of glycosaminoglycans in cerebrospinal fluid from patients with mucopolysaccharidoses by isotope‐dilution ultra‐performance liquid chromatography‐tandem mass spectrometry. Clin. Chem. 57:1005‐1012.
   Zhou, H., Fernhoff, P., and Vogt, R.F. 2011. Newborn bloodspot screening for lysosomal storage disorders. J. Pediatr. 159:7‐13.
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