Investigational Methods for Peroxisomal Disorders

Steven Steinberg1, Richard Jones1, Carol Tiffany1, Ann Moser1

1 Department of Neurology and Neurosurgery, Johns Hopkins University School of Medicine, Baltimore, Maryland
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 17.6
DOI:  10.1002/0471142905.hg1706s58
Online Posting Date:  July, 2008
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Abstract

Peroxisomes play an important role in cellular metabolism. Defects in peroxisome assembly or of a single peroxisomal pathway are associated with a wide variety of inherited disorders, including X‐linked adrenoleukodystrophy, Zellweger spectrum disorders, rhizomelic chondrodysplasia punctata, and Refsum disease. A group of peroxisome‐specific biomarkers has been shown to be characteristic of specific defects. Patients with defects in peroxisome fatty acid β‐oxidation accumulate very long–chain fatty acids (VLCFA), patients with defects in plasmalogen synthesis are deficient in erythrocyte membrane plasmalogens, and patients with mislocalized pipecolic acid oxidase accumulate pipecolic acid in body fluids. This unit describes three protocols that can be used to measure plasma VLCFA, erythrocyte plasmalogens, and plasma or urine pipecolic acid by capillary gas chromatography (GC) or GC‐mass spectrometry. These techniques can be used to identify the majority of patients with known neurogenetic peroxisome disorders. Curr. Protoc. Hum. Genet. 58:17.6.1‐17.6.23. © 2008 by John Wiley & Sons, Inc.

Keywords: very long–chain fatty acids (VLCFA); pipecolic acid; plasmalogens; gas chromatograph‐mass spectrometry (GC‐MS); X‐linked adrenoleukodystrophy (X‐ALD); peroxisome biogenesis disorder (PBD)

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Analysis of Plasma Branched‐Chain Fatty Acids (BCFA) and Very Long–Chain Fatty Acids (VLCFA) by GC‐MS
  • Basic Protocol 2: Analysis of Erythrocyte Plasmalogens
  • Basic Protocol 3: Analysis of Plasma or Urine Pipecolic Acid
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Analysis of Plasma Branched‐Chain Fatty Acids (BCFA) and Very Long–Chain Fatty Acids (VLCFA) by GC‐MS

  Materials
  • 2:1 (v/v) chloroform (CHCl 3; glass distilled with alcohol as a preservative, recommended; e.g., Jackson and Burdick)/methanol (MeOH) containing 50 mg/liter butylated hydroxytoluene (2,6‐di‐tert‐butyl‐p‐cresol or BHT; e.g., Sigma)
  • Nitrogen (N 2) source with multisample evaporator apparatus
  • Phytanic/pristanic mix (see recipe)
  • D mix fatty acids (see recipe)
  • Free mix 1 and 2 (see recipes)
  • Normal control fasting plasma samples in tubes containing an anticoagulant (e.g., EDTA)
  • Abnormal control fasting plasma samples: e.g., pooled from X‐linked adrenoleukodystrophy (X‐ALD) patients in tubes containing an anticoagulant (e.g., EDTA)
  • Patient samples: overnight fasting or preprandial plasma in tubes containing an anticoagulant (e.g., EDTA)
  • 0.6 N hydrochloric acid (HCl) in acetonitrile: prepare fresh on day of use by diluting 6 N HCl in acetonitrile
  • 1.0 N methanolic sodium hydroxide (NaOH): prepare fresh on day of use by diluting 10 N NaOH in methanol
  • 6 N HCl
  • Hexane
  • Triethylamine (e.g., Pierce)
  • 10% (v/v) pentafluorobenzyl bromide (PFB‐Br, e.g., Sigma) in acetonitrile: prepare fresh on day of use
  • 13 × 100–mm glass screw‐cap tubes with standard Teflon‐lined caps and open caps with standard Teflon‐lined 12‐mm silicon disc inserts
  • Glass capillary micropipets (e.g., Wiretrol, Drummond Scientific)
  • 104°C oven (e.g., Precision)
  • Centrifuge, refrigerated tabletop (e.g., Beckman)
  • GC sample vials with Teflon‐lined caps (e.g., Agilent)
  • Gas chromatograph (GC) or GC‐mass spectrometer (GC‐MS; e.g., Agilent) with capillary GC column (e.g., 50 m × 0.25 mm × 0.1 µm, Supelco) and analysis software

Basic Protocol 2: Analysis of Erythrocyte Plasmalogens

  Materials
  • Patient sample: 1 to 5 ml of whole blood in tubes containing an anticoagulant (e.g., EDTA), transported overnight and protected from temperature extremes
  • Normal control samples: blood from healthy individuals
  • Abnormal control samples: whole blood from rhizomelic chondrodysplasia punctata (RCDP) or peroxisome biogenesis disorder (PBD) patients with low red blood cell (RBC) plasmalogens, whole blood from a normal individual left at room temperature for 2 to 3 weeks, or 50 µg of brain sphingomyelin
  • Phosphate‐buffered saline (PBS), pH 7.4 (e.g., Sigma), 4°C
  • Nitrogen (N 2) source with multisample evaporator apparatus
  • Milli‐Q‐purified water (or equivalent)
  • 2:3 (v/v) isopropanol/hexane
  • Internal standards working solution (ISWS, see recipe)
  • 1 N methanolic HCl (see recipe)
  • Hexane
  • External standards working solution (ESWS, see recipe)
  • FAME 37 working solution (see recipe)
  • Refrigerated tabletop centrifuge (e.g., Beckman), 4°C
  • Plastic bulb transfer pipets (e.g., Samio Scientific)
  • 2‐ml plastic, screw‐cap tubes with low phthalate content (e.g., Nunc freezer tubes)
  • 13 × 100–mm glass screw‐cap tubes (prerinsed with 2:1 CHCl 3/MeOH), with open‐top caps having Teflon‐lined 12‐mm silicon disc inserts
  • Rocking mixer (e.g., Speci‐Mix, Thermolyne)
  • Glass capillary micropipets (e.g., Wiretrol, Drummond Scientific)
  • Oven, capable of 75°C ± 1°C (e.g., Precision)
  • Glass Pasteur pipets
  • Capillary gas chromatograph (e.g., Agilent) with polar capillary column (e.g., 100 m × 0.25 mm × 0.2 µm, Supelco) and nonpolar capillary column (e.g., 60 m × 0.25 mm × 0.1 µm, Agilent)
  • 100‐µl GC vial inserts
  • GC sample vials with Teflon‐lined caps (e.g., Agilent)

Basic Protocol 3: Analysis of Plasma or Urine Pipecolic Acid

  Materials
  • Pipecolic acid external standard (PIP‐ES): 50 µM l‐pipecolic acid (e.g., Sigma) in deionized water
  • Normal control plasma: from healthy individuals
  • Abnormal control plasma: from a Zellweger spectrum disorders (ZSD) patient
  • Patient plasma specimen: fasting plasma or serum or random urine
  • Pipecolic acid internal standard (PIP‐IS): 50 µM pipecolic acid‐d 11 in deionized water (e.g., Medical Isotopes)
  • 1 M phosphate‐buffered saline (PBS), pH 11.5: prepare by mixing 1 M Na 2CO 3 and 1 M K 3PO 4 until pH 11.5 is obtained
  • Methylchloroformate (e.g., Sigma)
  • 6 N and 0.1 N HCl
  • Ethyl acetate (e.g., Sigma)
  • Nitrogen (N 2) source with multi‐sample drying apparatus
  • Triethylamine (e.g., Burdick and Jackson)
  • 10% (v/v) pentafluorobenzyl bromide (PFB‐Br) in acetonitrile
  • Hexane
  • Silicic acid (100 to 200 mesh; e.g., Sigma) slurry in 3% ethyl acetate in hexane
  • 13 × 100–mm and 16 × 125–mm glass screw‐top tubes with Teflon‐lined caps
  • Creatinine assay instrument (e.g., Beckman Coulter Creatinine 2 Analyzer)
  • Rocking mixer (e.g., Speci‐Mix, Thermolyne)
  • Centrifuge, refrigerated tabletop centrifuge (e.g., Beckman GS‐6R)
  • 9‐in. glass Pasteur pipets and ring stand with holder apparatus (see Fig. )
  • Glass wool
  • 1.5‐ml GC vials
  • 200‐µl flat‐bottom GC vial inserts for 1.5‐ml GC vials
  • Gas chromatograph‐mass spectrometer (GC‐MS, e.g., Agilent)
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Figures

Videos

Literature Cited

   Baas, J.C.M., van de Laar, R., Doralnd, L., Duran, M., Berger, R., Poll‐The, B.T., and de Konig, T.J. 2002. Plasma pipecolic acid is frequently elevated in non‐peroxisomal disease. J. Inherit. Metab. Dis. 25:699‐701.
   Bjorkhem, I., Sisfontes, L., Bostrom, B., Kase, B.F., and Blomstrand, R. 1986. Simple diagnosis of the Zellweger syndrome by gas‐liquid chromatography of dimethylacetals. J. Lipid Res. 27:786‐791.
   Danks, D.M., Tippett, P., Adams, C., and Campbell, P. 1975. Cerebro‐hepato‐renal syndrome of Zellweger. A report of eight cases with comments upon the incidence, the liver lesion, and a fault in pipecolic acid metabolism. J. Pediatr. 86:382‐387.
   Ferdinandusse, S., Rusch, H., van Lint, A.E., Dacremont, G., Wanders, R.J., and Vreken, P. 2002. Stereochemistry of the peroxisomal branched‐chain fatty acid alpha‐ and beta‐oxidation systems in patients suffering from different peroxisomal disorders. J. Lipid Res. 43:438‐444.
   Hubbard, W.C., Moser, A.B., Tortorelli, S., Liu, A., Jones, D., and Moser, H. 2006. Combined liquid chromatography‐tandem mass spectrometry as an analytical method for high throughput screening for X‐linked adrenoleukodystrophy and other peroxisomal disorders. Mol. Genet. Metab. 89:185‐187.
   Kelley, R.I. 1991. Quantification of pipecolic acid in plasma and urine by isotope‐dilution gas chromatography/mass spectrometry. In Techniques in Diagnostic Human Biochemical Genetics (F.A. Hommes, ed.) pages 205‐218. Wiley‐Liss.
   Kok, R.M., deJung, A.P., Poll‐Thé, B. Saudubray, J.M., and Jakobs, C. 1987. Stable isotope dilution analysis of pipecolic acid in cerebrospinal fluid, plasma, urine, and amniotic fluid using electron capture negative ion mass fragmentrography. Clin. Chim. Acta 168:143‐152.
   Lagerstedt, S.A., Hinrichs, D.R., Batt, S.M., Magera, M.J., Rinaldo, P., and McConnell, J.P. 2001. Quantitative determination of plasma C8‐C26 total fatty acids for the biochemical diagnosis of nutritional and metabolic disorders. Mol. Genet. Metab. 73:38‐45.
   Mills, P.B., Stuys, E., Jakobs, C., Plecko, B., Baxter, P., Baumgartner, M., Willemsen, M.A.A.P., Omran, H., Tacke, U., Uhlenberg, B., Weschke, B, and Clayton, P.T. 2006. Mutations in antiquitin in individuals with pyridoxine‐dependent seizures. Nature Med. 12:307‐309.
   Moser, H.W. and Moser, A.B. 1991a. Measurement of phytanic acid levels. In Techniques in Diagnostic Human Biochemical Genetics, pages 194‐203. Wiley‐Liss, New York.
   Moser, H.W. and Moser, A.B. 1991b. Measurement of saturated very long chain fatty acids in plasma. In Techniques in Diagnostic Human Biochemical Genetics, pages 177‐191. Wiley‐Liss, New York.
   Moser, H.W., Raymond, G.V., Lu, S.E., Muenz, L.R., Moser, A.B., Xu, J., Jones, R.O., Loes, D.J., Melhem, E.R., Dubey, P., Bezman, L., Brereton, N.H., and Odone, A. 2005. Follow‐up of 89 asymptomatic patients with adrenoleukodystrophy treated with Lorenzo's oil. Arch Neurol. 62:1073‐1080.
   Steinberg, S.J., Dodt, G., Raymond, G.V., Braverman, N.E., Moser, A.B., and Moser, H.W. 2006. Peroxisome biogenesis disorders. Biochem. Biophys. Acta. 1763:1733‐1748.
   Theda, C., Woody, R.C., Naidu, S., Moser, A.B., and Moser, H.W. 1993. Increased very long chain fatty acids in patients on a ketogenic diet: a cause of diagnostic confusion. J. Pediatr. 122:724‐726.
   Van den Brink, D.M., Brites, P., Haasjes, J., Wierzbicki, A.S., Mitchell, J., Lambert‐Hamill, M., de Belleroche, J., Jansen, G.A., Waterham, H.R., and Wanders, R.J. 2003. Identification of PEX7 as the second gene involved in Refsum disease. Adv. Exp. Med. Biol. 544:69‐70.
   Wanders, R.J.A. 2004. Metabolic and molecular basis of peroxisomal disorders: A review. Am. J. Med. Genet. 126A:355‐375.
   Wanders, R.J., Purvis, Y.R., Heymans, H.S., Bakkeren, J.A., Parmentier, G.G., van Eldere, J., Eyssen, H., van den Bosch, H., Tager, J.M., and Schutgens, R.B. 1986. Age‐related differences in plasmalogen content of erythrocytes from patients with the cerebro‐hepato‐renal (Zellweger) syndrome: implications for postnatal detection of the disease. J. Inherit. Metab. Dis. 9:335‐342.
Key References
   Kelley, 1991. See above.
  Provides a good discussion of approaches to pipecolic acid measurement.
   Wanders, 2004. See above.
  Provides a good overview of the biochemicyal markers associated with the known peroxisomal disorders.
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