Quantification of Creatine and Guanidinoacetate Using GC‐MS and LC‐MS/MS for the Detection of Cerebral Creatine Deficiency Syndromes

Sarah Young1, Eduard Struys2, Tim Wood3

1 Duke University Medical Center, Research Triangle Park, North Carolina, 2 VU University Medical Center, Amsterdam, The Netherlands, 3 Greenwood Genetic Center, Greenwood, South Carolina
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 17.3
DOI:  10.1002/0471142905.hg1703s54
Online Posting Date:  July, 2007
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Abstract

Inherited defects in creatine biosynthesis and cellular uptake are neurometabolic disorders characterized by seizures, developmental delay, mental retardation, autistic‐like behavior, and creatine deficiency in the brain. Metabolic screening of these disorders is possible using analytical techniques that quantify creatine and its precursor guanidinoacetate in urine, plasma, or cerebrospinal fluid (CSF). Elevated creatine in urine is suggestive of a deficiency of the X‐linked creatine transporter, SLC6A8. Decreased or elevated levels of guanidinoacetate in urine, plasma, or CSF suggest deficiencies of the creatine biosynthetic enzymes, arginine:glycine amidinotransferase (AGAT) or guanidinoacetate methyltransferase (GAMT), respectively. This unit describes three stable isotope dilution–mass spectrometric methods for analyzing creatine and guanidinoacetate. Gas chromatography/mass spectrometry with negative‐ion chemical ionization is a highly sensitive technique, suitable for detection of low analyte levels resulting from AGAT deficiency and in CSF. The two liquid chromatography–tandem mass spectrometric approaches are amenable to high‐throughput screening and have simple sample preparation requirements. Curr. Protoc. Hum. Genet. 54:17.3.1‐17.3.18. © 2007 by John Wiley & Sons, Inc.

Keywords: creatine; guanidinoacetate; mass spectrometry; GAMT deficiency; AGAT deficiency; creatine transporter

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Quantitative Determination of Creatine and Guanidinoacetate in Urine, Plasma, and Cerebrospinal Fluid by GC‐MS
  • Basic Protocol 2: LC‐MS/MS Quantification of Underivatized Creatine, Creatinine, and Guanidinoacetate in Urine and Plasma or Serum
  • Basic Protocol 3: Quantification of Creatine and Guanidinoacetate in Plasma/Serum by LC‐MS/MS, Using Butylated Derivatives
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Quantitative Determination of Creatine and Guanidinoacetate in Urine, Plasma, and Cerebrospinal Fluid by GC‐MS

  Materials
  • Patient specimen: random urine (50 µl), plasma (100 µl), or cerebrospinal fluid (CSF); 100 µl)
  • Creatinine reagent kit (e.g., Roche Diagnostics) or enzyme kit (e.g., Cobas Integra system, Roche Diagnostics)
  • Internal standards for urine assay
    • 500 µM [2H 3]creatine (CDN Isotopes) in deionized (d)H 2O
    • 250 µM [13C 2]guanidinoacetate (contact Dr. Herman J. ten Brink at hj.tenbrink@VUmc.nl or see Internet Resources) in dH 2O
  • Internal standards for plasma and CSF assay
    • 0.05 mM [2H 3]creatine (CDN Isotopes) in dH 2O (for both plasma and CSF)
    • 5 µM [13C 2]guanidinoacetate (contact Dr. Herman J. ten Brink at hj.tenbrink@VUmc.nl or see Internet Resources) in dH 2O (for plasma)
    • 0.5 µM [13C 2]guanidinoacetate (contact Dr. Herman J. ten Brink at hj.tenbrink@VUmc.nl or see Internet Resources) in dH 2O (for CSF)
  • Saturated aqueous sodium bicarbonate solution: freshly prepared by adding sodium bicarbonate to 5 ml dH 2O while stirring until crystals remain clearly visible in the solution
  • Toluene (highest purity available)
  • Hexafluoroacetylacetone (Fluka, Sigma Aldrich)
  • Nitrogen supply
  • 7% (v/v) pentafluorobenzoylbromide (PFB‐Br) in acetonitrile (Pierce)
  • Triethylamine (highest purity available)
  • 0.5 mol/liter HCl
  • Hexane (highest purity available)
  • Creatine, anhydrous (Sigma‐Aldrich)
  • Guanidinoacetic acid (guanidineacetic acid; glycocyamine; N‐amidinoglycine; Sigma‐Aldrich)
  • 1‐ to 2‐ml glass gas chromatograph (GC) vials and dedicated inserts for small volumes
  • 10‐ml conical borosilicate tubes with Teflon‐lined screw caps
  • Small magnetic stirrer
  • Heated magnetic stirring plate
  • Crimp capper and decapper
  • Heating block set to 40°C
  • Nitrogen evaporator suitable for gas chromatograph vials (e.g., Liebisch)
  • Tabletop centrifuge with rotor appropriate for conical borosilicate tubes (e.g., Hettich)
  • Gas chromatograph with a mass spectrometer detector with NCI capability (e.g., Agilent 6890N gas chromatograph equipped with an Agilent 5973 mass detector), using 5% ammonia in methane as moderating gas for NCI
  • SGE BPX‐70 column: 25 m, 0.32‐mm i.d., and 0.25‐µm film thickness (SIS)

Basic Protocol 2: LC‐MS/MS Quantification of Underivatized Creatine, Creatinine, and Guanidinoacetate in Urine and Plasma or Serum

  Materials
  • Patient specimen: 200 µl random urine, plasma, or serum
  • Internal standards for urine assay
    • 25 µg/ml (210 µM) [2H 2]guanidinoacetic acid (guanidineacetic‐2,2‐d2 acid) (Isotech, Sigma‐Aldrich) prepared in deionized (d)H 2O or [13C,15N]guanidinoacetate synthesized from glycine‐2‐13C,15N (MSD), cyanamide (Sigma‐Aldrich), and concentrated NH 3, using the method of Struys et al. ( )
    • 30 µg/ml (224 µM) [2H 3]creatine (CDN Isotopes) prepared in dH 2O
    • 370 µg/ml (3.2 mM) [2H 3]creatinine (CDN Isotopes) prepared in dH 2O
  • Matrix and mobile phase (urine and plasma assays): 10 mM ammonium acetate in 65:35 (v/v) acetonitrile/water
  • Internal standards for plasma or serum assay
    • 4 µg/ml (17 µM) [2H 2]guanidinoacetic acid ([2H 2]guanidineacetic acid; Isotec, Sigma) prepared in dH 2O
    • 16 µg/ml (122 µM) [2H 3]creatine (CDN Isotopes) prepared in dH 2O
  • 100% acetonitrile, HPLC grade (plasma assay)
  • Nitrogen tank
  • Creatine, anhydrous (Sigma‐Aldrich)
  • Guanidinoacetic acid (guanidineacetic acid; glycocyamine; N‐amidinoglycine; Sigma‐Aldrich)
  • Creatinine calibration standards in 0.02 M HCl (Sigma‐Aldrich), including 1 mg/dl, 3 mg/dl, and 10 mg/dl
  • 1.5‐ml (for urine and plasma assays) and 2‐ml (for plasma assay only) polypropylene microcentrifuge tubes
  • Microcentrifuge
  • Vortex mixer
  • 96‐well microtiter plates (e.g., sterile, round, U‐bottom untreated polystyrene; Evergreen Scientific) or sample vials for LC system
  • 0.2‐µm nylon micro‐spin filter tubes, 1.5‐ml (for plasma assay only)
  • Nitrogen evaporator
  • Liquid chromatograph and electrospray tandem mass spectrometer: e.g., Waters Quattro‐Micro with HP1100 LC pump (Agilent) and CTC HTS PAL liquid sample handler (Leap Technologies)
  • LC column: 5‐µm, 2.0 × 100–mm TSK‐GEL amide‐80 LC column with a 5‐µm 2.0 × 10–mm TSK‐GEL amide‐80 guard cartridge (Tosoh Bioscience)

Basic Protocol 3: Quantification of Creatine and Guanidinoacetate in Plasma/Serum by LC‐MS/MS, Using Butylated Derivatives

  Materials
  • Patient specimen: 100 µl plasma or serum
  • Internal standards
    • 100 µM [2H 2]guanidinoacetic acid (guanidineacetic‐2,2‐d2 acid) (Isotec, Sigma‐Aldrich) prepared in deionized (d)H 2O or synthesize from glycine‐2‐13C; 15N (MSD), cyanamide (Sigma‐Aldrich), and concentrated NH 3 using the method of Struys et al. ( )
    • 100 µM [2H 3]creatine (CDN Isotopes) prepared in H 2O
  • 100% acetonitrile, HPLC grade
  • Nitrogen supply
  • Butanolic HCl (Regis Technologies)
  • 80:20 (v/v) acetonitrile/dH 2O
  • Creatine, anhydrous (Sigma‐Aldrich)
  • Guanidinoacetic acid (guanidineacetic acid; glycocyamine; N‐amidinoglycine; Sigma‐Aldrich)
  • 1.5‐ml polypropylene microcentrifuge tubes
  • Vortex mixer
  • Microcentrifuge
  • Nitrogen tank
  • Heating block set to 60°C
  • 96‐well microtiter plates (e.g., sterile, round, U‐bottom untreated polystyrene; Evergreen Scientific) or sample vials for LC system
  • Liquid chromatograph and electrospray tandem mass spectrometer: e.g., Waters Quattro‐Micro with HP1100 LC pump (Agilent) and CTC HTS PAL liquid sample handler (Leap Technologies)
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Figures

Videos

Literature Cited

   Almeida, L.S., Verhoeven, N.M., Roos, B., Valongo, C., Cardoso, M.L., Vilarinho, L., Salomons, G.S., and Jakobs, C. 2004. Creatine and guanidinoacetate: Diagnostic markers for inborn errors in creatine biosynthesis and transport. Mol. Genet. Metab. 82:214‐219.
   Bodamer, O.A., Bloesch, S.M., Gregg, A.R., Stockler‐Ipsiroglu, S., and O'Brien, W.E. 2001. Analysis of guanidinoacetate and creatine by isotope dilution electrospray tandem mass spectrometry. Clin. Chim. Acta 308:173‐178.
   Cecil, K.M., Salomons, G.S., Ball, W.S., Wong, B., Chuck, G., Verhoeven, N.M., Jakobs, C., and DeGrauw, T.J. 2001. Irreversible brain creatine deficiency with elevated serum and urine creatine: A creatine transporter defect? Ann. Neurol. 49:401‐104.
   Clark, A.J., Rosenberg, E.H., Almeida, L.S., Wood, T.C., Jakobs, C., Stevenson, R.E., Schwartz, C.E., and Salomons, G.S. 2006. X‐linked creatine transporter (SLC6A8) mutations in about 1% of males with mental retardation of unknown etiology. Hum. Genet. 119:604‐610.
   Cognat, S., Cheillan, D., Piraud, M., Roos, B., Jakobs, C., and Vianey‐Saban, C. 2004. Determination of guanidinoacetate and creatine In Urine and Plasma by Liquid Chromatography–Tandem Mass Spectrometry. Clin. Chem. 50:1459‐1461.
   Item, C.B., Stockler‐Ipsiroglu, S., Stromberger, C., Muhl, A., Alessandri, M.G., Bianchi, M.C., Tosetti, M., Fornai, F., and Cioni, G. 2001. Arginine:glycine amidinotransferase deficiency: The third inborn error of creatine metabolism in humans. Am. J. Hum. Genet. 69:1127‐1133.
   Jansen, E.E., Verhoeven, N.M., Jakobs, C., Schulze, A., Senephansiri, H., Gupta, M., Snead, O.C., and Gibson, K.M. 2006. Increased guanidino species in murine and human succinate semialdehyde dehydrogenase (SSADH) deficiency. Biochim. Biophys. Acta 1762:494‐498.
   Lion‐Francois, L., Cheillan, D., Pitelet, G., Acquaviva‐boudain, C., Bussy, G., Cotton, F., Guibaud, L., Gerard, D., Rivier, C., Vianey‐Saban, C., Jakobs, C., Salomons, G.S., and des Portes, V. 2006. High frequency of creatine deficiency syndromes in patients with unexplained mental retardation. Neurology 67:1713‐1714.
   Mercimek‐Mahmutoglu, S., Stoeckler‐Ipsiroglu, S., Adami, A., Appleton, R., Araujo, H.C., Duran, M., Ensenauer, R., Fernandez‐Alvarez, E., Garcia, P., Grolik, C., Item, C.B., Leuzzi, V., Marquardt, I., Muhl, A., Saelke‐Kellermann, R.A., Salomons, G.S., Schulze, A., Surtees, R., van der Knaap, M.S., Vasconcelos, R., Verhoeven, N.M., Vilarinho, L., Wilichowski, E., and Jakobs, C. 2006. GAMT deficiency: Features, treatment, and outcome in an inborn error of creatine synthesis. Neurology 67:480‐484.
   Newmeyer, A., Cecil, K.M., Schapiro, M., Clark, J.F., and Degrauw, T.J. 2005. Incidence of brain creatine transporter deficiency in males with developmental delay referred for brain magnetic resonance imaging. J. Dev. Behav. Pediatr. 26:276‐282.
   Rosenberg, E.H., Almeida, L.S., Kleefstra, T., deGrauw, R.S., Yntema, H.G., Bahi, N., Moraine, C., Ropers, H.H., Fryns, J.P., deGrauw, T., Jakobs, C., and Salomons, G.S. 2004. High prevalence of SLC6A8 deficiency in X‐linked mental retardation. Am. J. Hum. Genet. 75:97‐105.
   Rosenberg, E.H., Almeida, L.S., Verhoeven, N.M., Jakobs, C., and Salomons, G.S. 2006. Are Cerebral creatine deficiency syndromes on the radar screen? Fut. Neurol. 1:647‐649.
   Salomons, G.S., van Dooren, S.J.M., Verhoeven, N.M., Cecil, K.M., Ball, W.S., Degrauw, T.J., and Jakobs, C. 2001. X‐linked creatine transporter gene (SLC6A8) defect: A new creatine‐deficiency syndrome. Am. J. Hum. Genet. 68:1497‐1500.
   Salomons, G.S., van Dooren, S.J.M, Verhoeven, N.M., Marsden, D., Schwartz, C., Cecil, K.M., DeGrauw, T.J., and Jakobs, C. 2003. X‐linked creatine transporter defect: An overview. J. Inherit. Metab. Dis. 26:309‐318.
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  Stockler, S., Marescau, B., De Deyn, P.P., Trijbels, J.M.F., and Hanefeld, F. 1997. Guanidino compounds in guanidinoacetate methyltransferase deficiency, a new inborn error of creatine synthesis. Metabolism 46:1189–1193.
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   Struys, E.A., Jansen, E.E.W., ten Brink, H.J., Verhoeven, N.M., van der Knaap, M.S., and Jakobs C. 1998. An accurate stable isotope dilution gas chromatographic‐mass spectrometric approach to the diagnosis of guanidinoacetate methyltransferase deficiency. J. Pharm. Biomed. Anal. 18:659‐665.
   Verhoeven, N.M., Guérand, W.S., Struys, E.A., Bouman, A.A., van der Knaap, M.S., and Jakobs, C. 2000. Plasma creatinine assessment in creatine deficiency: A diagnostic pitfall. J. Inherit. Metab. Dis. 23:835‐840.
   Verhoeven, N.M., Salomons, G.S., and Jakobs, C. 2005. Laboratory diagnosis of defects of creatine biosynthesis and transport Clin. Chim. Acta 361:1‐9. Review.
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Internet Resources
  http://www.vumc.nl/metabool/index.html?metabolic_research.html∼hoofd
  Web site with information about obtaining labeled internal standards for the assay in .
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