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Capillary Electrophoresis of Free Amino Acids in Physiological Fluids Without Derivatization Employing Direct or Indirect Absorbance Detection

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Amino Acid Analysis

Part of the book series: Methods in Molecular Biology ((MIMB,volume 828))

Abstract

Whole blood and/or plasma amino acids are useful for monitoring whole body protein and amino acid metabolism in an organism under various physiological and pathophysiological conditions. Various methodological procedures are in use for their measurement in biological fluids. From the time when capillary electrophoresis was introduced as a technology offering rapid separation of various ionic and/or ionizable compounds with low sample and solvent consumption, there were many attempts to use it for the measurement of amino acids present in physiological fluids. As a rule, these methods require derivatization procedures for detection purposes.

Here, we present two protocols for the analysis of free amino acids employing free zone capillary electrophoresis. Main advantage of both methods is an absence of any derivatization procedures that permits the analysis of free amino acid in physiological fluids. The method using direct detection and carrier electrolyte consisting of disodium monophosphate (10 mM at pH 2.90) permits determination of compounds that absorb in UV region (aromatic and sulfur containing amino acids, as well as some peptides, such as carnosine, reduced and oxidized glutathione). The other method uses indirect absorbance detection, employing 8 mM p-amino salicylic acid buffered with sodium carbonate at pH 10.2 as running electrolyte. It permits quantification of 30 underivatized physiological amino acids and peptides. In our experience, factorial design represents a useful tool for final optimization of the electrophoretic conditions if it is necessary.

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References

  1. Žunić G et al (2009) Increased nitric oxide formation followed by increased arginase activity induces relative lack of arginine at the wound site and alters whole nutritional status in rats almost within the early healing period. Nitric Oxide 20: 253–258

    Article  PubMed  Google Scholar 

  2. Žunić G et al (2000) Pulmonary blast injury increases nitric oxide production, disturbs arginine metabolism, and alters the plasma free amino acid pool in rabbits during the early posttraumatic period. Nitric Oxide 4: 123–128

    Article  PubMed  Google Scholar 

  3. Žunić G et al (1996) Early plasma amino acid pool alterations in patients with military gunshot/missile wound. J Trauma 40: S152–S156

    Article  PubMed  Google Scholar 

  4. Žunić G (1997) Free amino acids in the evaluation of protein metabolism. Vojnosanit Pregl 54: 615–620 (In Serbian)

    PubMed  Google Scholar 

  5. Pinna A et al (2010) Plasma thiols and taurine levels in central retinal vein occlusion. Curr Eye Res 35: 644–650

    Article  PubMed  CAS  Google Scholar 

  6. Guzman-Ramos K et al (2010) Off-line concomitant release of dopamine and glutamate involvement in taste memory consolidation. J Neurochem 144(1): 226–236

    Google Scholar 

  7. Cherla G, Jaimes EA (2004) Role of L-arginine in the Pathogenesis and treatment of renal disease. J Nutr 134: 2801S–2908S

    PubMed  CAS  Google Scholar 

  8. Sevaljević Lj, Zunic G (1996) Acute-phase protein synthesis in rats is influenced by alterations in plasma and muscle free amino acid pools related to lower plasma volume following trauma. J Nutr 126: 3136–3142

    Google Scholar 

  9. Cernak I, Savić J, Zunic G et al (1999). Recognizing, scoring and predicting blast injuries. World J Surg 23: 44–53

    Article  PubMed  CAS  Google Scholar 

  10. Ikai I et al (1989) Significance of hepatic mitochondrial redox potential on the concentration of plasma amino acids following hemorrhagic shock in rats. Circ Shock 27: 63–72

    PubMed  CAS  Google Scholar 

  11. Zinellu A et al (2010) Quantification of neurotransmitter amino acids by capillary electrophoresis laser-induced fluorescence detection in biological fluids. Anal Bioanal Chem 398: 1973–1978

    Article  PubMed  CAS  Google Scholar 

  12. Oberholzer VG, Briddon A (1990) A novel use of amino acid ratios as an indicator of nutritional status. In: Lubec G, Rosenthal, GA (eds) Amino Acids, Chemistry, Biology and Medicine, Escom Science Publishers, Leiden

    Google Scholar 

  13. Rafii M et al (2008) In vivo regulation of phenylalanine hydroxylation to tyrosine, studied using enrichment in apoB-100. Am J Physiol Endocrinol Metab 294: E475–E479

    Article  PubMed  CAS  Google Scholar 

  14. Wannemacher RW et al (1976) The significance and mechanism of an increased serum phenylalanine-tyrosine ratio during infection. Am J Clin Nutr 29: 997–1006

    PubMed  CAS  Google Scholar 

  15. Kawaguchi T et al (2007) Branched-chain amino acids improve insulin resistance in patients with hepatitis C virus-related liver disease: report of two cases. Liver Int 27: 1287–1292

    PubMed  CAS  Google Scholar 

  16. Bolander-Gouaille C (2001) Focus on homocysteine and the vitamins involved in its metabolism. Springer, Verlag, France

    Google Scholar 

  17. Ruderman NB, Berger M (1974) The formation of glutamine and alanine in skeletal muscle. J Biol Chem 249:5500–5506

    PubMed  CAS  Google Scholar 

  18. Žunić G, Stanimirović S, Savić J (1984) Rapid ion-exchange method for the determination of 3-methylhistidine in rat urine and skeletal muscle. J Chromatog 311:69–77

    Article  Google Scholar 

  19. Peranzoni E et al (2007) Role of arginine in the metabolism, immunity and immunopathology. Immunobiology 212:795–812

    Article  PubMed  CAS  Google Scholar 

  20. Wischemeyer PE (2008) Glutamine: role in critical illness and ongoing clinical trials. Curr Opin Gastroenterol 24:190–197

    Article  Google Scholar 

  21. Shi HP et al (2007) Supplemental L-arginine enhances wound healing following trauma/hemorrhagic shock. Wound Repair Regen 15: 66–70

    Article  PubMed  CAS  Google Scholar 

  22. Zinellu A et al (2005) Quantification of thiol-containing amino acids linked by disulfides to LDL. Clin Chem 51:658–660

    Article  PubMed  CAS  Google Scholar 

  23. Ziegler F et al (1992) Plasma amino acid determinations by reversed-phase HPLC: improvement of the orthophthalaldehyde method and comparison with ion-exchange chromatography. J Automat Chem 14:145–149

    Article  PubMed  CAS  Google Scholar 

  24. Moore S, Spackman DH, Stein WH (1958) Chromatography of amino acids on sulfonated polystyrene resins. Anal Chem 30:1185–1190

    Article  CAS  Google Scholar 

  25. Cynober L et al (1985) High performance ion-exchange chromatography of amino acids in biological fluids using Chromakon 500—performance of the apparatus. J Automat Chem 7:201–203

    Article  PubMed  CAS  Google Scholar 

  26. Kuhr WG, Yeung ES (1988) Indirect fluorescence detection of native amino acids in capillary zone electrophoresis. Anal Chem 60:1832–1834

    Article  PubMed  CAS  Google Scholar 

  27. Liu J et al (1991) Design of 3-(4-carboxybenzoyl)-2-quinolinecarboxaldehyde as a reagent for ultrasensitive determination of primary amines by capillary electrophoresis using laser fluorescence detection. Anal Chem 63:408–412

    Article  PubMed  CAS  Google Scholar 

  28. Altria KD (1996) Additional application areas of capillary electrophoresis. In: Altria KD (ed) Capillary Electrophoresis Guide Book – Principles, Operations and Applications, Humana Press, Totowa, New Jersey

    Google Scholar 

  29. Žunić G, Spasić S (2008) Capillary electrophoresis method optimized with a factorial design for the determination of glutathione and amino acid status using human capillary blood. J Chromatogr B 873:70–76

    Article  Google Scholar 

  30. Poinsot V et al (2010) Recent advances in amino acid analysis by CE. Electrophoresis 31:105–121

    Article  PubMed  CAS  Google Scholar 

  31. Zhai C et al (2010) Ultraviolet detection of amino acids based on their on-column conjugation with cupric cation using a disposable electrophoresis microdevice. Talanta 82:67–71

    Article  PubMed  CAS  Google Scholar 

  32. Lee Y-H, Lin T-I (1994) Capillary electrophoretic determination of amino acids with indirect absorbance detection. J Chromatogr A 680:287–297

    Article  CAS  Google Scholar 

  33. Martin-Girardeu A, Renou-Gonnord MF (2000) Optimization of a capillary electrophoresis – electrospray mass spectrometry for the quantification of the 20 natural amino acids in children blood. J Chromatogr B 742:163–171

    Article  Google Scholar 

  34. Deming SN, Morgan SL (1993) Experimental design: a chemometric approach. Elsevier, Amsterdam – London – New York – Tokyo, Netherlands

    Google Scholar 

  35. Žunić G et al (2002) Optimization of a free separation of 30 free amino acids and peptides by capillary zone electrophoresis with indirect absorbance detection: a potential for quantification in physiological fluids. J Chromatogr B 772:19–33

    Article  Google Scholar 

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Acknowledgement

This work was supported by a grant of MMA (Project No. 06-10/B.5.)

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Authors and Affiliations

  1. Institute for Medical Research, Military Medical Academy, Belgrade, Serbia

    Gordana D. Žunić

  2. Department of Medical Biochemistry, Faculty of Pharmacy, University of Belgrade, Belgrade, Serbia

    Slavica Spasić & Zorana Jelić-Ivanović

Authors
  1. Gordana D. Žunić
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  2. Slavica Spasić
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  3. Zorana Jelić-Ivanović
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Corresponding author

Correspondence to Gordana D. Žunić .

Editor information

Editors and Affiliations

  1. Ctr. Biologics Evaluation & Research, Div. Cellular & Gene Therapies, U.S. Food and Drug Administration, Rockville Pike 8800, Bethesda, 20892, Maryland, USA

    Michail A. Alterman

  2. Functional Genomics Center Zürich, Protein Analysis Group, Universität Zürich, Winterthurerstr. 190, Zürich, 8057, Switzerland

    Peter Hunziker

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Žunić, G.D., Spasić, S., Jelić-Ivanović, Z. (2012). Capillary Electrophoresis of Free Amino Acids in Physiological Fluids Without Derivatization Employing Direct or Indirect Absorbance Detection. In: Alterman, M., Hunziker, P. (eds) Amino Acid Analysis. Methods in Molecular Biology, vol 828. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-445-2_19

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  • DOI: https://doi.org/10.1007/978-1-61779-445-2_19

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-61779-444-5

  • Online ISBN: 978-1-61779-445-2

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