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GC–MS and GC–MS/MS measurement of the cardiovascular risk factor homoarginine in biological samples

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Abstract

l-Homoarginine (hArg) has recently emerged as a novel cardiovascular risk factor and to herald a poor prognosis in heart failure patients. Here, we report on the development and thorough validation of gas chromatography–mass spectrometry (GC–MS) and gas chromatography–tandem mass spectrometry (GC–MS/MS) methods for the quantitative determination of hArg in biological samples, including human plasma, urine and sputum. For plasma and serum samples, ultrafiltrate (10 µL; cutoff, 10 kDa) was used. For urine samples, native urine (10 µL) was used. For sputum, protein precipitation by acetone was performed. hArg is derivatized to its methyl ester tri(N-pentafluoropropionyl) derivative; de novo synthesized trideutero-methyl ester hArg is used as the internal standard (IS). Alternatively, [guanidino-15N2]-arginine can be used as an IS. Quantitative analyses were performed after electron-capture negative-ion chemical ionization by selected-ion monitoring in GC–MS and selected-reaction monitoring in GC–MS/MS. We obtained very similar hArg concentrations by GC–MS and GC–MS/MS, suggesting that GC–MS suffices for accurate and precise quantification of hArg in biological samples. In plasma and serum samples of the same subjects very close hArg concentrations were measured. The plasma-to-serum hArg concentration ratio was determined to be 1.12 ± 0.21 (RSD, 19 %), suggesting that blood anticoagulation is not a major preanalytical concern in hArg analysis. In healthy subjects, the creatinine-corrected urinary excretion of hArg varies considerably (0.18 ± 0.22 µmol/mmol, mean ± SD, n = 19) unlike asymmetric dimethylarginine (ADMA, 2.89 ± 0.89 µmol/mmol). In urine, hArg correlated with ADMA (r = 0.475, P = 0.040); in average, subjects excreted in the urine about 17.5 times more ADMA than hArg. In plasma of healthy humans, the concentration of hArg is of the order of 2 µM. hArg may be a low-abundance constituent of human plasma proteins. The GC–MS and GC-MS/MS methods we report in this article are useful to study the physiology and pathology of hArg in experimental and clinical settings.

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Abbreviations

ADMA:

N G ,N G-Dimethyl-l-arginine

AGAT:

Arginine:glycine amidinotransferase

CID:

Collision-induced dissociation

DMA:

Dimethyl amine

DSQ:

Double-stage quadrupole

ECNICI:

Electron-capture negative-ion chemical ionization

GC–MS:

Gas chromatography–mass spectrometry

GC–MS/MS:

Gas chromatography–tandem mass spectrometry

hArg:

Homoarginine

IS:

Internal standard

LC–MS/MS:

Liquid chromatography–tandem mass spectrometry

NO:

Nitric oxide

NOS:

Nitric oxide synthase

PFP:

Pentafluoropropionyl

PFPA:

Pentafluoropropionic anhydride

SDMA:

Symmetric dimethylarginine

SIM:

Selected-ion monitoring

SRM:

Selected-reaction monitoring

TSQ:

Triple-stage quadrupole

References

  • Albsmeier J, Schwedhelm E, Schulze F, Kastner M, Böger RH (2004) Determination of NG, NG-dimethyl-l-arginine, an endogenous NO synthase inhibitor, by gas chromatography-mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci 809(1):59–65

  • Atzler D, Mieth M, Maas R, Böger RH, Schwedhelm E (2011) Stable isotope dilution assay for liquid chromatography-tandem mass spectrometric determination of l-homoarginine in human plasma. J Chromatogr B 879:2294–2298

    Article  CAS  Google Scholar 

  • Bretscher LE, Li H, Poulos TL, Griffith OW (2003) Structural characterization and kinetics of nitric-oxide synthase inhibition by novel N5-(iminoalkyl)- and N5- (iminoalkenyl)-ornithines. J Biol Chem 278:46789–46797

    Article  CAS  PubMed  Google Scholar 

  • Choe CU, Atzler D, Wild PS, Carter AM, Böger RH, Ojeda F, Simova O, Stockebrand M, Lackner K, Nabuurs C, Marescau B, Streichert T, Muller C, Luneburg N, De Deyn PP, Benndorf RA, Baldus S, Gerloff C, Blankenberg S, Heerschap A, Grant PJ, Magnus T, Zeller T, Isbrandt D, Schwedhelm E (2013) Homoarginine levels are regulated by l-arginine:glycine amidinotransferase and affect stroke outcome: results from human and murine studies. Circulation 128:1451–1461

    Article  CAS  PubMed  Google Scholar 

  • Dai Z, Wu Z, Jia S, Wu G (2014) Analysis of amino acid composition in proteins of animal tissues and foods as pre-column o-phthaldialdehyde derivatives by HPLC with fluorescence detection. J Chromatogr B. doi:10.1016/j.jchromb.2014.03.025

    Google Scholar 

  • Davids M, Ndika JD, Salomons GS, Blom HJ, Teerlink T (2012a) Promiscuous activity of arginine:glycine amidinotransferase is responsible for the synthesis of the novel cardiovascular risk factor homoarginine. FEBS Lett 586:3653–3657

    Article  CAS  PubMed  Google Scholar 

  • Davids M, Swieringa E, Palm F, Smith DE, Smulders YM, Scheffer PG, Blom HJ, Teerlink T (2012b) Simultaneous determination of asymmetric and symmetric dimethylarginine, L-monomethylarginine, l-arginine, and L-homoarginine in biological samples using stable isotope dilution liquid chromatography tandem mass spectrometry. J Chromatogr B 900:38–47

    Article  CAS  Google Scholar 

  • Drechsler C, Meinitzer A, Pilz S, Krane V, Tomaschitz A, Ritz E, März W, Wanner C (2011) Homoarginine, heart failure, and sudden cardiac death in haemodialysis patients. Eur J Heart Fail 13:852–859

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Engeli S, Tsikas D, Lehmann AC, Böhnke J, Haas V, Strauß A, Janke J, Gorzelniak K, Luft FC, Jordan J (2012) Influence of dietary fat ingestion on asymmetrical dimethylarginine in lean and obese human subjects. Nutr Metab Cardiovasc Dis 22:720–726

    Article  CAS  PubMed  Google Scholar 

  • Horowitz JD, Heresztyn T (2007) An overview of plasma concentrations of asymmetric dimethylarginine (ADMA) in health and disease and in clinical studies: methodological considerations. J Chromatogr B 851:42–50

    Article  CAS  Google Scholar 

  • Kayacelebi AA, Nguyen TH, Neil C, Horowitz JD, Jordan J, Tsikas D (2014) Homoarginine and 3-nitrotyrosine in patients with takotsubo cardiomyopathy. Int J Cardiol. doi:10.1016/j.ijcard.2014.03.080

    PubMed  Google Scholar 

  • Khalil AA, Tsikas D, Akolekar R, Jordan J, Nicolaides KH (2013) Asymmetric dimethylarginine, arginine and homoarginine at 11–13 weeks gestation and preeclampsia: a case-control study. J Hum Hypertens 27:38–43

    Article  CAS  PubMed  Google Scholar 

  • Leiper J, Vallance P (1999) Biological significance of endogenous methylarginines that inhibit nitric oxide synthases. Cardiovasc Res 43:542–548

    Article  CAS  PubMed  Google Scholar 

  • Martens-Lobenhoffer J, Bode-Böger SM (2014) Mass spectrometric quantification of l-arginine and its pathway related substances in biofluids: the road to maturity. J Chromatogr B. doi:10.1016/j.jchromb.2013.10.030

    Google Scholar 

  • Martens-Lobenhoffer J, Schwedhelm E, Tsikas D (2009) Quantification of arginine and its mono- and dimethylated analogs NMMA, ADMA and SDMA in biological fluids by LC-MS/MS: is LC superfluous? J Chromatogr B 877:3261–3266

    Article  CAS  Google Scholar 

  • März W, Meinitzer A, Drechsler C, Pilz S, Krane V, Kleber ME, Fischer J, Winkelmann BR, Böhm BO, Ritz E, Wanner C (2010) Homoarginine, cardiovascular risk, and mortality. Circulation 122:967–975

    Article  PubMed  Google Scholar 

  • Moali C, Boucher JL, Sari MA, Stuehr DJ, Mansuy D (1998) Substrate specificity of NO synthases: detailed comparison of l-arginine, homo-l-arginine, their N omega-hydroxy derivatives, and N omega-hydroxynor-l-arginine. Biochemistry 37:10453–10460

    Article  CAS  PubMed  Google Scholar 

  • Moali C, Brollo M, Custot J, Sari MA, Boucher JL, Stuehr DJ, Mansuy D (2000) Recognition of alpha-amino acids bearing various C = NOH functions by nitric oxide synthase and arginase involves very different structural determinants. Biochemistry 39:8208–8218

    Article  CAS  PubMed  Google Scholar 

  • Moncada S, Higgs A (1993) The l-arginine-nitric oxide pathway. New Engl J Med 329:2002–2012

    Article  CAS  PubMed  Google Scholar 

  • Pilz S, Tomaschitz A, Meinitzer A, Drechsler C, Ritz E, Krane V, Wanner C, Bohm BO, März W (2011) Low serum homoarginine is a novel risk factor for fatal strokes in patients undergoing coronary angiography. Stroke 42:1132–1134

    Article  CAS  PubMed  Google Scholar 

  • Ryan WL, Wells IC (1964) Homocitrulline and homoarginine synthesis from lysine. Science 144:1122–1127

    Article  CAS  PubMed  Google Scholar 

  • Ryan WL, Johnson RJ, Dimari S (1969) Homoarginine synthesis by rat kidney. Arch Biochem Biophys 131:521–526

    Article  CAS  PubMed  Google Scholar 

  • Schwedhelm E (2005) Quantification of ADMA: analytical approaches. Vascular Med (London, England) 10(Suppl 1):S89–S95

    Article  Google Scholar 

  • Schwedhelm E, Maas R, Tan-Andresen J, Schulze F, Riederer U, Böger RH (2007) High-throughput liquid chromatographic-tandem mass spectrometric determination of arginine and dimethylated arginine derivatives in human and mouse plasma. J Chromatogr B 851:211–219

    Article  CAS  Google Scholar 

  • Sobczak A, Prokopowicz A, Radek M, Szula M, Zaciera M, Kurek J, Goniewicz ML (2014) Tobacco smoking decreases plasma concentration of the emerging cardiovascular risk marker, l-homoarginine. Circ J 78:1254–1258

    Article  CAS  PubMed  Google Scholar 

  • Teerlink T (2007) HPLC analysis of ADMA and other methylated l-arginine analogs in biological fluids. J Chromatogr B 851:21–29

    Article  CAS  Google Scholar 

  • Tsikas D (2008) A critical review and discussion of analytical methods in the l-arginine/nitric oxide area of basic and clinical research. Anal Biochem 379:139–163

    Article  CAS  PubMed  Google Scholar 

  • Tsikas D, Beckmann B (2009) Albumin from human serum does not contain asymmetric dimethylarginine (ADMA). Clin Biochem 42:1739–1740

    Article  CAS  PubMed  Google Scholar 

  • Tsikas D, Kayacelebi AA (2014) Do homoarginine and asymmetric dimethylarginine act antagonistically in the cardiovascular system? Circ J 20

  • Tsikas D, Schubert B, Gutzki FM, Sandmann J, Frölich JC (2003) Quantitative determination of circulating and urinary asymmetric dimethylarginine (ADMA) in humans by gas chromatography-tandem mass spectrometry as methyl ester tri(N-pentafluoropropionyl) derivative. J Chromatogr B 798:87–99

    Article  CAS  Google Scholar 

  • Tsikas D, Engeli S, Beckmann B, Jordan J (2010) Asymmetric dimethylarginine (ADMA) is present in plasma proteins of healthy subjects at the low nmol-per-g-level. Nitric Oxide 22:316–317

    Article  CAS  PubMed  Google Scholar 

  • Tsikas D, Beckmann B, Gutzki FM, Jordan J (2011) Simultaneous gas chromatography-tandem mass spectrometry quantification of symmetric and asymmetric dimethylarginine in human urine. Anal Biochem 413:60–62

    Article  CAS  PubMed  Google Scholar 

  • Valtonen P, Laitinen T, Lyyra-Laitinen T, Raitakari OT, Juonala M, Viikari JS, Heiskanen N, Vanninen E, Punnonen K, Heinonen S (2008) Serum l-homoarginine concentration is elevated during normal pregnancy and is related to flow-mediated vasodilatation. Circ J 72:1879–1884

    Article  CAS  PubMed  Google Scholar 

  • Wu G, Bazer FW, Davis TA, Kim SW, Li P, Marc Rhoads J, Carey Satterfield M, Smith SB, Spencer TE, Yin Y (2009) Arginine metabolism and nutrition in growth, health and disease. Amino Acids 37:153–168

    Article  CAS  PubMed Central  PubMed  Google Scholar 

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Acknowledgments

We thank the staff of the Institute of Clinical Pharmacology, Hannover Medical School, for providing us with spot urine samples for the analysis of hArg and its relatives.

Conflict of interest

The authors declare that they have no conflict of interest.

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Authors and Affiliations

  1. Institute of Clinical Pharmacology, Hannover Medical School, Carl-Neuberg-Strasse 1, 30625, Hannover, Germany

    Arslan Arinc Kayacelebi, Bibiana Beckmann, Frank-Mathias Gutzki, Jens Jordan & Dimitrios Tsikas

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  1. Arslan Arinc Kayacelebi
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  2. Bibiana Beckmann
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  3. Frank-Mathias Gutzki
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  4. Jens Jordan
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  5. Dimitrios Tsikas
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Correspondence to Dimitrios Tsikas.

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Kayacelebi, A.A., Beckmann, B., Gutzki, FM. et al. GC–MS and GC–MS/MS measurement of the cardiovascular risk factor homoarginine in biological samples. Amino Acids 46, 2205–2217 (2014). https://doi.org/10.1007/s00726-014-1774-3

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  • DOI: https://doi.org/10.1007/s00726-014-1774-3

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