Skip to main content
SpringerLink
Log in

An on-column derivatization method for the determination of homocysteine-thiolactone and protein N-linked homocysteine

  • Original Article
  • Published:
Amino Acids Aims and scope Submit manuscript

Abstract

Homocysteine (Hcy) is incorporated into protein via a reaction of the thioester Hcy-thiolactone with ε-amino group of a protein lysine residue generating N-Hcy-protein. This reaction impairs and alters protein’s function and has been implicated in atherothrombotic disease. Here, we describe new high-performance liquid chromatography assays for the determination of Hcy-thiolactone, protein N-linked Hcy, and Hcy based on an on-column derivatization with o-phthaldialdehyde and fluorescence detection. The on-column derivatization generates narrow peaks, which allows fast run times (3–5 min) and facilitates determination of N-linked Hcy directly from acid hydrolysates of plasma protein. Utility of these assays was demonstrated with human urine and plasma samples.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

Abbreviations

Hcy:

Homocysteine

tHcy:

Total Hcy, i.e., free Hcy present in a sample after reductive cleavage of disulfide bonds

(Hcy)2 :

Homocystine

N-Hcy-protein:

N-homocysteinylated protein

protein N-linked Hcy:

Hcy bound to protein by an amide linkage to ε-amino group of lysine residue(s)

OPA:

o-phthaldialdehyde

SPE:

Solid phase extraction

CMQT:

2-chloro-1-methylquinolinium tetrafluoroborate

TCEP:

Tris(2-carboxyethyl)phosphine

CBS:

Cystathionine β-synthase

LOQ:

Lower limit of quantification

References

  • Bald E, Głowacki R (2001) 2-Chloro-1-methylquinolinium tetrafluoroborate as effective and thiol specific UV-tagging reagent for liquid chromatography. J Liq Chromatogr Rel Techn 24:1323–1339

    Article  CAS  Google Scholar 

  • Bald E, Głowacki R (2005) Analysis of saliva for glutathione and metabolically related thiols by liquid chromatography with ultraviolet detection. Amino Acids 28:431–433

    Article  PubMed  CAS  Google Scholar 

  • Bald E, Chwatko G, Głowacki R, Kusmierek K (2004) Analysis of plasma thiols by high-performance liquid chromatography with ultraviolet detection. J Chromatogr A 1032:109–115

    Article  PubMed  CAS  Google Scholar 

  • Chwatko G, Jakubowski H (2005a) Urinary excretion of homocysteine-thiolactone in humans. Clin Chem 51:408–415

    Article  PubMed  CAS  Google Scholar 

  • Chwatko G, Jakubowski H (2005b) The determination of homocysteine-thiolactone in human plasma. Anal Biochem 337:271–277

    Article  PubMed  CAS  Google Scholar 

  • Chwatko G, Boers GH, Strauss KA, Shih DM, Jakubowski H (2007) Mutations in methylenetetrahydrofolate reductase or cystathionine beta-synthase gene, or a high-methionine diet, increase homocysteine thiolactone levels in humans and mice. Faseb J 21:1707–1713

    Article  PubMed  CAS  Google Scholar 

  • Daneshvar P, Yazdanpanah M, Cuthbert C, Cole DE (2003) Quantitative assay of plasma homocysteine thiolactone by gas chromatography/mass spectrometry. Rapid Commun Mass Spectrom 17:358–362

    Article  PubMed  CAS  Google Scholar 

  • Głowacki R, Bald E (2009) Determination of N-acetylcysteine and main endogenous thiols in human plasma by HPLC with ultraviolet detection in the form of their S-quinolinium derivatives. J Liq Chromatogr Rel Techn 32:2530–2544

    Article  Google Scholar 

  • Głowacki R, Jakubowski H (2004) Cross-talk between Cys34 and lysine residues in human serum albumin revealed by N-homocysteinylation. J Biol Chem 279:10864–10871

    Article  PubMed  Google Scholar 

  • Głowacki R, Borowczyk K, Bald E, Jakubowski H (2010) On-column derivatization with o-phthaldialdehyde for fast determination of homocysteine in human urine. Anal Bioanal Chem (in press). doi:10.1007/s00216-010-3456-7

  • Gu W, Lu J, Yang G, Dou J, Mu Y, Meng J, Pan C (2008) Plasma homocysteine thiolactone associated with risk of macrovasculopathy in Chinese patients with type 2 diabetes mellitus. Adv Ther 25:914–924

    Article  PubMed  CAS  Google Scholar 

  • Jakubowski H (1990) Proofreading in vivo: editing of homocysteine by methionyl-tRNA synthetase in Escherichia coli. Proc Natl Acad Sci USA 87:4504–4508

    Article  PubMed  CAS  Google Scholar 

  • Jakubowski H (1997) Metabolism of homocysteine thiolactone in human cell cultures: possible mechanism for pathological consequences of elevated homocysteine levels. J Biol Chem 272:1935–1942

    Google Scholar 

  • Jakubowski H (1999) Protein homocysteinylation: possible mechanism underlying pathological consequences of elevated homocysteine levels. Faseb J 13:2277–2283

    PubMed  CAS  Google Scholar 

  • Jakubowski H (2000) Homocysteine thiolactone: metabolic origin and protein homocysteinylation in humans. J Nutr 130:377S–381S

    PubMed  CAS  Google Scholar 

  • Jakubowski H (2001a) Translational accuracy of aminoacyl-tRNA synthetases: implications for atherosclerosis. J Nutr 131:2983S–2987S

    PubMed  CAS  Google Scholar 

  • Jakubowski H (2001b) Protein N-homocysteinylation: implications for atherosclerosis. Biomed Pharmacother 55:443–447

    Article  PubMed  CAS  Google Scholar 

  • Jakubowski H (2002a) Homocysteine is a protein amino acid in humans. Implications for homocysteine-linked disease. J Biol Chem 277:30425–30428

    Article  PubMed  CAS  Google Scholar 

  • Jakubowski H (2002b) The determination of homocysteine-thiolactone in biological samples. Anal Biochem 308:112–119

    Article  PubMed  CAS  Google Scholar 

  • Jakubowski H (2004) Molecular basis of homocysteine toxicity in humans. Cell Mol Life Sci 61:470–487

    Article  PubMed  CAS  Google Scholar 

  • Jakubowski H (2005) Anti-N-homocysteinylated protein autoantibodies and cardiovascular disease. Clin Chem Lab Med 43:1011–1014

    Article  PubMed  CAS  Google Scholar 

  • Jakubowski H (2006a) Pathophysiological consequences of homocysteine excess. J Nutr 136:1741S–1749S

    PubMed  CAS  Google Scholar 

  • Jakubowski H (2006b) Mechanism of the condensation of homocysteine thiolactone with aldehydes. Chem Eur J 12:8039–8043

    Article  CAS  Google Scholar 

  • Jakubowski H (2007) The molecular basis of homocysteine thiolactone-mediated vascular disease. Clin Chem Lab Med 45:1704–1716

    Article  PubMed  CAS  Google Scholar 

  • Jakubowski H (2008a) The pathophysiological hypothesis of homocysteine thiolactone-mediated vascular disease. J Physiol Pharmacol 59(suppl 9):155–167

    Google Scholar 

  • Jakubowski H (2008b) New method for the determination of protein N-linked homocysteine. Anal Biochem 380:257–261

    Article  PubMed  CAS  Google Scholar 

  • Jakubowski H, Goldman E (1993) Synthesis of homocysteine thiolactone by methionyl-tRNA synthetase in cultured mammalian cells. FEBS Lett 317:237–240

    Article  PubMed  CAS  Google Scholar 

  • Jakubowski H, Boers GH, Strauss KA (2008) Mutations in cystathionine {beta}-synthase or methylenetetrahydrofolate reductase gene increase N-homocysteinylated protein levels in humans. FASEB J 22:4071–4076

    Article  PubMed  CAS  Google Scholar 

  • Jakubowski H, Perla-Kajan J, Finnell RH, Cabrera RM, Wang H, Gupta S, Kruger WD, Kraus JP, Shih DM (2009) Genetic or nutritional disorders in homocysteine or folate metabolism increase protein N-homocysteinylation in mice. FASEB J 23:1721–1727

    Article  PubMed  CAS  Google Scholar 

  • Lentz SR (2005) Mechanisms of homocysteine-induced atherothrombosis. J Thromb Haemost 3:1646–1654

    Article  PubMed  CAS  Google Scholar 

  • Mukai Y, Togawa T, Suzuki T, Ohata K, Tanabe S (2002) Determination of homocysteine thiolactone and homocysteine in cell cultures using high-performance liquid chromatography with fluorescence detection. J Chromatogr B Analyt Technol Biomed Life Sci 767:263–268

    Article  PubMed  CAS  Google Scholar 

  • Perla-Kajan J, Twardowski T, Jakubowski H (2007) Mechanisms of homocysteine toxicity in humans. Amino Acids 32:561–572

    Article  PubMed  CAS  Google Scholar 

  • Perla-Kajan J, Stanger O, Luczak M, Ziolkowska A, Malendowicz LM, Twardowski T, Lhotak S, Austin RC, Jakubowski H (2008) Immunohistochemical detection of N-homocysteinylated proteins in humans and mice. Biomed Pharmacother 62:473–479

    Article  PubMed  CAS  Google Scholar 

  • Perna AF, Satta E, Acanfora F, Lombardi C, Ingrosso D, De Santo NG (2006) Increased plasma protein homocysteinylation in hemodialysis patients. Kidney Int 69:869–876

    Article  PubMed  CAS  Google Scholar 

  • Uji Y, Motomiya Y, Hanyu N, Ukaji F, Okabe H (2002) Protein-bound homocystamide measured in human plasma by HPLC. Clin Chem 48:941–944

    PubMed  CAS  Google Scholar 

  • Yang X, Gao Y, Zhou J, Zhen Y, Yang Y, Wang J, Song L, Liu Y, Xu H, Chen Z, Hui R (2006) Plasma homocysteine thiolactone adducts associated with risk of coronary heart disease. Clin Chim Acta 364:230–234

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported in part by grants from the American Heart Association, the University of Łódź, and the Ministry of Science and Higher Education, Poland (NN 401 230634, N401 065 32/1504, POIG.01.03.01-00-097/08).

Author information

Authors and Affiliations

  1. Department of Environmental Chemistry, University of Łódź, Lodz, Poland

    Rafał Głowacki & Edward Bald

  2. Department of Microbiology and Molecular Genetics, UMDNJ-New Jersey Medical School, International Center for Public Health, Newark, NJ, 07101, USA

    Hieronim Jakubowski

  3. Institute of Bioorganic Chemistry, Polish Academy of Sciences, 61-704, Poznan, Poland

    Hieronim Jakubowski

  4. Department of Biochemistry and Biotechnology, University of Life Sciences, 60-637, Poznan, Poland

    Hieronim Jakubowski

Authors
  1. Rafał Głowacki
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Edward Bald
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Hieronim Jakubowski
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Edward Bald.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Głowacki, R., Bald, E. & Jakubowski, H. An on-column derivatization method for the determination of homocysteine-thiolactone and protein N-linked homocysteine. Amino Acids 41, 187–194 (2011). https://doi.org/10.1007/s00726-010-0521-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00726-010-0521-7

Keywords

Search

Navigation