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Asymmetric dimethylarginine (ADMA) in human blood: effects of extended haemodialysis in the critically ill patient with acute kidney injury, protein binding to human serum albumin and proteolysis by thermolysin

Amino Acids volume 47, pages 1983–1993 (2015)Cite this article

Abstract

Free, non-protein bound asymmetrically guanidine-dimethylated arginine (ADMA) is an endogenous inhibitor of nitric oxide (NO) synthesis. Human erythrocytic membrane comprises considerable amounts of large (>50 kDa) ADMA-containing proteins. Location in the erythrocyte membrane and identity and physiological functions of ADMA-containing proteins are unrevealed. In healthy subjects, the concentration of free ADMA in heparinised plasma is almost identical to that of serum. We hypothesised that the robustness of free ADMA concentration in human blood is due to a remarkable resistance of erythrocytic ADMA-containing proteins against proteases. In vivo, we investigated the course of the concentration of ADMA in serum and EDTA plasma of a critically ill patient with acute kidney injury during extended haemodialysis. In vitro, we studied the effects of thermolysin, a useful experimental proteolytic enzyme of erythrocyte membrane proteins, on erythrocytic ADMA. The protein binding (PB) of ADMA to human serum albumin (HSA) was also determined. In these studies, ADMA was measured by a previously reported, fully validated GC–MS/MS method. We measured almost identical ADMA concentrations in plasma and serum samples of the patient. During dialysis, the circulating ADMA concentration decreased slowly and moderately indicating removal of this substance, which was however much less than expected from its low molecular weight (202 Da) and high water solubility. After dialysis, circulating ADMA concentration increased again, a phenomenon called rebound, and ADMA reached higher levels compared to the baseline. The PB value of ADMA to HSA was about 30 %. This surprisingly high PB value of ADMA to HSA may be an explanation for the rather poor dialysance of ADMA. Washed human erythrocytes suspended in phosphate-buffered physiological saline were found not to release appreciable amounts of free and ADMA-containing proteins. The lack of effect of coagulation or anticoagulation on the concentration of circulating free ADMA in humans is likely to be due to a remarkable resistance of ADMA-containing proteins in the erythrocyte membrane against proteases in vivo in humans. Our study suggests that free ADMA is released in the circulating blood at relatively high rates. The considerable PB of ADMA to HSA is likely to add to the apparently poor dialysability of ADMA. Other contributing factors could be redistribution of free ADMA between plasma and erythrocytes in favour of plasma ADMA and parallel formation of free ADMA from erythrocytic ADMA-containing proteins during haemodialysis.

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Abbreviations

ADMA:

Asymmetric dimethylarginine (N G,N G-dimethyl-l-arginine)

CID:

Collision-induced dissociation

DDAH:

Dimethylarginine dimethylaminohydrolase

DMA:

Dimethylamine

ECNICI:

Electron-capture negative-ion chemical ionization

GC–MS/MS:

Gas chromatography-tandem mass spectrometry

HSA:

Human serum albumin

NO:

Nitric oxide

NOS:

Nitric oxide synthase

PB:

Protein binding

PBS:

Phosphate buffer saline

SDMA:

Symmetric dimethylarginine (N G,N′ G-dimethyl-l-arginine)

References

  • Adekoya OA, Sylte I (2009) The thermolysin family (M4) of enzymes: therapeutic and biotechnological potential. Chem Biol Drug Des 73:7–16

    CAS  Article  PubMed  Google Scholar 

  • Araujo P (2009) Key aspects of analytical method validation and linearity evaluation. J Chromatogr B 877:2224–2234

    CAS  Article  Google Scholar 

  • Billecke SS, Kitzmiller LA, Northrup JJ, Whitesall SE, Kimoto M, Hinz AV, D´Alecy LG (2006) Contribution of whole blood to the control of plasma asymmetrical dimethylarginine. Am J Physiol Heart Circ Physiol 291:H1788–H1796

    CAS  Article  PubMed  Google Scholar 

  • Billecke SS, D`Alecy LG, Platel R, Whitesall SE, Jamerson KA, Perlman RL, Gadegbeku CA (2009) Blood content of asymmetric dimethylarginine: new insights into its dysregulation in renal disease. Nephrol Dial Transplant 24:489–496

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  • Bland JM, Altman DG (1986) Statistical methods for assessing agreement between two methods of clinical measurement. Lancet 1:307–310

    CAS  Article  PubMed  Google Scholar 

  • Böhmer A, Großkopf H, Jordan J, Tsikas D (2012a) Human hemoglobin does not contain asymmetric dimethylarginine (ADMA). Nitric Oxide 27:72–74

    Article  PubMed  Google Scholar 

  • Böhmer A, Beckmann B, Sandmann J, Tsikas D (2012b) Doubts concerning functional endothelial nitric oxide synthase in human erythrocytes. Blood 119:1322–1323

    Article  PubMed  Google Scholar 

  • Bouteldja N, Woodman RJ, Hewitson CL, Domingo E, Barbara JA, Mangoni AA (2013) Methylated arginines and nitric oxide in end-stage renal disease: impact of inflammation, oxidative stress and haemodialysis. Biomarkers 18:357–364

    CAS  Article  PubMed  Google Scholar 

  • Burkhardt O, Hafer C, Langhoff A, Kaever V, Kumar V, Welte T, Haller H, Fliser D, Kielstein JT (2009) Pharmacokinetics of ertapenem in critically ill patients with acute renal failure undergoing extended daily dialysis. Nephrol Dial Transplant 24:267–271

    CAS  Article  PubMed  Google Scholar 

  • D`Alecy LG, Billecke SS (2010) Massive quantities of asymmetric dimethylarginine (ADMA) are incorporated in red blood cell proteins and may be released by proteolysis following hemolytic stress. Blood Cells Mol Dis 45:40

    CAS  Article  Google Scholar 

  • Damoglou AP, Lindley H, Stapleton IW (1971) The hydrolysis by thermolysin of dipeptide derivatives that contain substituted cysteine. Biochem J 123:379–384

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  • Davids M, van Hell AJ, Visser M, Nijveldt RJ, van Leeuwen PAM, Teerlink T (2012) Role of the human erythrocyte in generation and storage of asymmetric dimethylarginine. Am J Physiol Heart Circ Physiol 302:H1762–H1770

    CAS  Article  PubMed  Google Scholar 

  • Davids M, Peters JHC, de Jong S, Teerlink T (2013) Measurement of nitric oxide-related amino acids in serum and plasma: effects of blood clotting and type of anticoagulant. Clin Chim Acta 421C:164–167

    Article  Google Scholar 

  • Eksborg S (1981) Evaluation of method-comparison data. Clin Chem 27:1311–1312

    CAS  PubMed  Google Scholar 

  • Ellmerer M, Schaupp L, Brunner GA, Sendlhofer G, Wutte A, Wach P, Pieber TR (2000) Measurement of interstitial albumin in human skeletal muscle and adipose tissue by open-flow microperfusion. Am J Physiol Endocrinol Metab 278:E352–E356

    CAS  PubMed  Google Scholar 

  • Feder J, Garrett LR, Wildi BS (1971) Studies on the role of calcium in thermolysin. Biochemistry 10:4552–4556

    CAS  Article  PubMed  Google Scholar 

  • Girg R, Rudolph R, Jaenicke R (1981) Limited proteolysis of porcine-muscle lactic dehydrogenase by thermolysin during reconstitution yields dimers. Eur J Biochem 119:301–305

    CAS  Article  PubMed  Google Scholar 

  • Großkopf H, Böhmer A, Tsikas D (2012) Letter to the editor: “Role of the human erythrocyte in generation and storage of asymmetric dimethylarginine”. Am J Physiol Heart Circ Physiol 303:751–752

    Article  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

    CAS  Article  Google Scholar 

  • Ikizler TA (2005) Effects of haemodialysis on protein metabolism. J Ren Nutr 15:39–43

    Article  PubMed  Google Scholar 

  • Jenkins RE, Tanner JA (1977) The structure of the major protein of the human erythrocyte membrane. Characterization of the intact protein and major fragments. Biochem J 161:139–147

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  • Kang ES, Cates TB, Harper DN, Chiang TM, Myers LK, Acchiardo SR, Kimoto M (2011) An enzyme hydrolyzing methylated inhibitors of nitric oxide synthase is present in circulating human red blood cells. Free Rad Res 35:693–707

    Article  Google Scholar 

  • Karim AR, Rees WD, Holman GD (1987) Binding of cytochalasin B to trypsin and thermolysin fragments of the human erythrocyte hexose transporter. Biochim Biophys Acta 902:402–405

    CAS  Article  PubMed  Google Scholar 

  • Kielstein JT, Böger RH, Bode-Böger SM, Schäffer J, Barbey M, Koch KM, Frölich JC (1999) Asymmetric dimethylarginine plasma concentrations differ in patients with end-stage renal disease: relationship to treatment method and atherosclerotic disease. J Am Soc Nephrol 10:594–600

    CAS  PubMed  Google Scholar 

  • Kielstein JT, Böger RH, Bode-Böger SM, Martens-Lobenhoffer J, Lonnemann G, Frölich JC, Haller H, Fliser D (2004) Low dialysance of asymmetric dimethylarginine (ADMA)—in vivo and in vitro evidence of significant protein binding. Clin Nephrol 62:295–300

    CAS  Article  PubMed  Google Scholar 

  • Kielstein A, Tsikas D, Galloway GP, Mendelson JE (2007) Asymmetric dimethylarginine (ADMA)—a modulator of nociception in opiate tolerance and addiction? Nitric Oxide 17:55–59

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  • Kleinbongard P, Schulz R, Rassaf T, Lauer T, Dejam A, Jax T, Kumara I, Gharini P, Kabanova S, Ozüyaman B, Schnürch HG, Gödecke A, Weber AA, Robenek M, Robenek H, Bloch W, Rösen P, Kelm M (2006) Red blood cells express a functional endothelial nitric oxide synthase. Blood 107:2943–2951

    CAS  Article  PubMed  Google Scholar 

  • Komori Y, Nonogaki T, Nikai T (2001) Hemorrhagic activity and muscle damaging effect of Pseudomonas aeruginosa metalloproteinase (elastase). Toxicon 39:1327–1332

    CAS  Article  PubMed  Google Scholar 

  • Kramer E, Routh JI (1973) The binding of salicylic acid and acetylsalicylic acid to human serum albumin. Clin Biochem 6:98–105

    CAS  Article  PubMed  Google Scholar 

  • Kurz H, Trunk H, Weitz B (1977) Evaluation of methods to determine protein-binding of drugs: equilibrium dialysis, ultrafiltration, ultracentrifugation, gel filtration. Arzneimittelforschung 27:1373–1380

    CAS  PubMed  Google Scholar 

  • Leiper JM, Vallance P (2006) The synthesis and metabolism of asymmetric dimethylarginine (ADMA). Eur J Clin Pharmacol 26:33–38

    Article  Google Scholar 

  • Lenzen H, Tsikas T, Böger RH (2006) Asymmetric dimethylarginine (ADMA) and the risk for coronary heart disease. The multicenter CARDIAC study. Eur J Clin Pharmacol 62:45–49

    CAS  Article  Google Scholar 

  • May M, Batkai S, Zörner AA, Tsikas D, Jordan J, Engeli S (2014) Clinical evaluation of extracellular ADMA concentrations in human blood and adipose tissue. Int J Mol Sci 15:1189–1200

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  • May M, Kayacelebi AA, Batkai S, Jordan J, Tsikas D, Engeli S (2015) Plasma and tissue homoarginine concentrations in healthy and obese humans. Amino Acids. doi:10.1007/s00726-015-1922-4

    PubMed  Google Scholar 

  • Morris ME, Levy G (1984) Renal clearance and serum protein binding of acetaminophen and its major conjugates in humans. J Pharm Sci 73:1038–1041

    CAS  Article  PubMed  Google Scholar 

  • Oldham PD (1968) Measurement in medicine: the interpretation of numerical data. English Universities Press, London

    Google Scholar 

  • Rifai K, Bode-Boeger SM, Martens-Lobenhoffer J, Ernst T, Kretschmer U, Hafer C, Fliser D, Manns MP, Kielstein JT (2010) Removal of asymmetric dimethylarginine during artificial liver support using fractionated plasma separation and adsorption. Scand J Gastroenterol 45:1110–1115

    CAS  Article  PubMed  Google Scholar 

  • Schepers E, Speer T, Bode-Böger SM, Fliser D, Kielstein JT (2014) Dimethylarginines ADMA and SDMA: the real water-soluble small toxins? Semin Nephrol 34:97–105

    CAS  Article  PubMed  Google Scholar 

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

    CAS  Article  Google Scholar 

  • Trettin A, Modun D, Madunic S, Vukovic J, Radman M, Batkai S, Thum T, Jordan J, Tsikas D (2014) LC–MS/MS and GC–MS/MS measurement of plasma and urine di-paracetamol and 3-nitro-paracetamol: proof-of-concept studies on a novel human model of oxidative stress based on oral paracetamol administration. J Chromatogr B 959:71–81

    CAS  Article  Google Scholar 

  • Tsikas D (2007) Analysis of nitrite and nitrate in biological fluids by assays based on the Griess reaction: appraisal of the Griess reaction in the l-arginine/nitric oxide area of research. J Chromatogr B 851:51–70

    CAS  Article  Google Scholar 

  • Tsikas D (2008) Determination of asymmetric dimethylarginine in biological fluids: a paradigm for a successful analytical story. Curr Opin Clin Nutr Metab Care 11:592–600

    CAS  Article  PubMed  Google Scholar 

  • Tsikas D (2009) De novo synthesis of trideuteromethyl esters of amino acids for use in GC–MS and GC–tandem MS exemplified for ADMA in in human plasma and urine: standardization, validation, comparison and proof of evidence for their aptitude as internal standards. J Chromatogr B 877:2308–2320

    CAS  Article  Google Scholar 

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

    CAS  Article  PubMed  Google Scholar 

  • Tsikas D, Böger RH, Sandmann J, Bode-Böger SM, Frölich JC (2000) Endogenous nitric oxide synthase inhibitors are responsible for the l-arginine paradox. FEBS Lett 478:1–3

    CAS  Article  PubMed  Google Scholar 

  • Tsikas D, Schubert B, Gutzki FM, Sandmann J, Schwedhelm E, 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

    CAS  Article  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

    CAS  Article  PubMed  Google Scholar 

  • Tsikas D, Böhmer A, Großkopf H, Beckmann B, Dreißigacker U, Jordan J, Maassen N (2012) Clinical-chemistry laboratory relevant hemolysis is unlikely to compromise human plasma concentration of free asymmetric dimethylarginine (ADMA). Clin Biochem 45:1536–1538

    CAS  Article  PubMed  Google Scholar 

  • Vallance P, Leone A, Calver A, Collier J, Moncada S (1992) Accumulation of an endogenous inhibitor of nitric oxide synthesis in chronic renal failure. Lancet 339(8793):572–575

    CAS  Article  PubMed  Google Scholar 

  • Zinellu A, Sotgia S, Scanu B, Formato M, Deiana L, Carru C (2007) Assessment of protein-incorporated arginine methylation in biological specimens by CZE UV detection. Electrophoresis 28:4452–4458

    CAS  Article  PubMed  Google Scholar 

  • Zinellu A, Sotgia S, Scanu B, Deiana L, Carru C (2008) Determination of proteinincorporated methylated arginine reference values in healthy subjects whole blood and evaluation of factors affecting protein methylation. Clin Biochem 41:1218–1223

    CAS  Article  PubMed  Google Scholar 

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Acknowledgments

This study was supported by the Deutsche Forschungsgemeinschaft (DFG; grant TS60/4-1). We thank F.-M. Gutzki for performing GC–MS/MS analyses.

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical standard

The studies involving human participants were approved by the Ethics Committee of the Hannover Medical School.

Author information

Affiliations

  1. Centre of Pharmacology and Toxicology, Hannover Medical School, 30623, Hannover, Germany

    Mustafa Erinc Sitar, Arslan Arinc Kayacelebi, Bibiana Beckmann & Dimitrios Tsikas

  2. Department of Nephrology and Hypertension, Hannover Medical School, 30623, Hannover, Germany

    Jan T. Kielstein

  3. Centre of Pharmacology and Toxicology, Hannover Medical School, Carl-Neuberg-Strasse 1, 30625, Hannover, Germany

    Dimitrios Tsikas

Authors
  1. Mustafa Erinc Sitar
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  2. Arslan Arinc Kayacelebi
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  3. Bibiana Beckmann
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  4. Jan T. Kielstein
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  5. Dimitrios Tsikas
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Correspondence to Dimitrios Tsikas.

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Sitar, M.E., Kayacelebi, A.A., Beckmann, B. et al. Asymmetric dimethylarginine (ADMA) in human blood: effects of extended haemodialysis in the critically ill patient with acute kidney injury, protein binding to human serum albumin and proteolysis by thermolysin. Amino Acids 47, 1983–1993 (2015). https://doi.org/10.1007/s00726-015-1991-4

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  • DOI: https://doi.org/10.1007/s00726-015-1991-4

Keywords

  • Anticoagulation
  • Coagulation
  • Erythrocytes
  • Haemodialysis
  • Mass spectrometry
  • Nitric oxide
  • Proteins