Nitric Oxide 27 (2012) 72–74
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Letter to the Editor Human hemoglobin does not contain asymmetric dimethylarginine (ADMA)
To the Editor, The guanidine (NG)-methylated arginines monomethylarginine (MMA), symmetric dimethylarginine (SDMA), and asymmetric dimethylarginine (ADMA) occur physiologically in human blood proteins [1–4]. The mechanisms leading to MMA, SDMA and ADMA formation and metabolism are incompletely understood. NG-Methylated arginines appear to be exclusively formed through proteolysis of arginine residues in proteins NG-methylated by protein-arginine methyltransferases . ADMA is a biomarker heralding cardiovascular risk , possibly due to its inhibitory action on endothelial nitric oxide synthase . Identiﬁcation and quantiﬁcation of ADMA-containing proteins in blood and tissues are emerging tasks in this research area. Mouse kidney tissue contains considerable ADMA amounts of about 100 nmol ADMA/g protein . Previously, we observed that ADMA is virtually absent in human serum albumin, one of the most abundant proteins in human blood. We estimated that human serum albumin carries about 0.3 nmol ADMA/g . With respect to whole plasma proteins, we calculated an ADMA content of about 1.6 nmol ADMA/g total plasma proteins . Zinellu et al. reported that whole blood proteins contain approximately 4 lmol ADMA/g protein . The discrepancy may suggest that protein-incorporated ADMA derives almost exclusively from red blood cells proteins, such as hemoglobin, which is the most abundant protein in erythrocytes and contains 12 arginine residues. Results by Zinellu et al. [1,11] and by Billecke et al. [2–4] suggest that red blood cell proteins contain considerable ADMA amounts. We reasoned that hemoglobin may be a potential substrate for protein-arginine methyltransferases, thus, potentially serving as a source of circulating ADMA. In theory, hemoglobin could accommodate as much as 185 lmol ADMA/g. In the present study, we investigated the physiological occurrence of ADMA in human hemoglobin. We determined the ADMA content of commercially available human hemoglobin by HCl-catalyzed hydrolysis, i.e., by heating aqueous HCl acid solutions of hemoglobin isolated from human blood for 24 h at 110 °C, as reported earlier for human serum albumin [9,10]. As a control, we incubated HCl acid solutions of hemoglobin for 24 h at 22 °C. Then, hydrolysates and control samples were ultraﬁltered and 10 lL aliquots of the ultraﬁltrates were analyzed for ADMA and arginine by GC–MS/MS as previously described . Because ADMA analysis by this method is sensitive to peptides, proteins and constituents of ultraﬁltration cartridges , ultraﬁltration was performed both of the control (i.e., nonhydrolyzed hemoglobin) and the hydrolysates. We applied trideutero (d3)-ADMA and d3-arginine as internal standards at concentrations of 10 nM and 1 mM, respectively. The results of this 1089-8603/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.niox.2012.03.013
experiment are shown in Fig. 1. We obtained almost identical values for ADMA from hemoglobin (0.16 mM) hydrolyzed at both temperatures. ADMA concentrations measured in all ultraﬁltrate samples ranged between 1 and 6 nM. Arginine concentration was 1.71 ± 0.07 mM in the samples incubated at 110 °C, but only 0.06 ± 0.2 mM in the samples incubated at 22 °C. These observations together with the fact that one hemoglobin molecule contains 12 arginine moieties indicate that human hemoglobin was hydrolyzed almost quantitatively (by 90%) at 110 °C and is practically free of ADMA. Oxidative stress may trigger proteolysis  and could contribute to free ADMA through increased proteolysis of ADMA-containing proteins. We recently observed that nitrite, nitric oxide’s autooxidation product, inhibits erythrocytic catalase activity and induces oxidative stress in native and more so in lyzed red blood cells . Hemolysates from a healthy volunteer were incubated for 30 min either in an ice bath or in a thermostated reaction device
Fig. 1. Measurement of the extent of incorporation of ADMA (A) and arginine (B) in commercially available human hemoglobin (Sigma, Munich, Germany; H7379) after incubation in aqueous HCl acid solutions. Aliquots (each 100 lL) of an aqueous solution of Hb (113 g/L) were added to 1000 lL aliquots of a 6 M HCl from Merck (Darmstadt, Germany) in 1.3 mL glass vials which were locked gas-tightly and incubated for 24 h at 110 °C (n = 5) or 22 °C (n = 5). Then, samples were ultraﬁltered by centrifugation at room temperature (8000g, 30 min) using 10 kDa Vivaspin cartridges (Sartorius, Göttingen, Germany), two 10 lL aliquots of the ultraﬁltrates were taken for derivatization and analysis using d3-ADMA (10 nM) and d3-arginine (1 mM) as the internal standards, respectively. ADMA and arginine were quantitated by GC–MS/MS . Data are shown as mean ± SD. Unpaired t test was used to test statistical signiﬁcance.
Letter to the Editor / Nitric Oxide 27 (2012) 72–74
Fig. 4. Relationship between the ADMA concentration measured by GC–MS/MS in (y) and the ADMA concentration added to (x) lyzed RBCs. ADMA was measured in 10 lL aliquots of the ultraﬁltrates (10 kDa) and d3-ADMA was used as the internal standard (1 lM). Data are shown as mean ± SD from three independent analyses. The y axis intercept and slope values of the regression line indicate a mean basal ADMA concentration of 324 nM and a mean recovery (accuracy) value of 94.1% for added ADMA concentrations. .
Fig. 2. Measurement of ADMA in hemolysate samples incubated for 30 min (A) and up to 6 h (B) in an ice bath or at 37 °C in the absence (A, B) and in presence (A) of varying concentrations of nitrite. After incubation, samples were ultraﬁltered by centrifugation (8000g, 4 °C, 30 min; 10 kDa), 10 lL aliquots of the ultraﬁltrates were taken for derivatization and analysis using d3-ADMA (1 lM) as the internal standard. ADMA was quantitated by GC–MS/MS . Data are shown as mean ± SD. Unpaired t test was used to test statistical signiﬁcance (A). Blood was donated by a healthy volunteer. Approval was obtained from the Ethics Committee of the Hannover Medical School.
at 37 °C in the presence of varying nitrite concentrations (0, 0.1, 0.5, 1, 2 and 5 mM). Fig. 2 shows that incubation of hemolysates at 37 °C increased free soluble ADMA concentrations compared with the ADMA concentration observed at 0 °C. Regardless of incubation temperature, up to 5 mM nitrite did not change free ADMA concentration (Fig. 2A). Fig. 2B shows that incubation of hemolysate samples at 37 °C increased free soluble ADMA concentration over time compared with the ADMA concentration observed at 0 °C. These results conﬁrm a similar incubation-induced ADMA release that was inhibited by a series of protease inhibitors . The mean free ADMA concentration in the hemolysate used ranged between 210 and 320 nM (Fig. 2B) corresponding to an approximate ADMA concentration of 420–640 nM in red blood cells. This concentration range
Fig. 3. Representative chromatograms from the GC–MS/MS analysis of ADMA in hemolysates incubated for 30 min in an ice bath (A) or at 37 °C (B) in the presence of 100 lM nitrite each. ADMA was quantitated by GC–MS/MS on a TSQ 7000 instrument using d3-ADMA (1 lM) as the internal standard . Upper and lower panels show the trace for endogenous ADMA and d3-ADMA, respectively. Note that the hemolysate was diluted with phosphate buffered saline (1:10, v/v) prior to ultraﬁltration. RT and AA indicate retention time (min) and automatically calculated peak area (arbitrary units). For further details see Fig. 2.
Letter to the Editor / Nitric Oxide 27 (2012) 72–74
is slightly higher than the mean ADMA concentration of 390 nM measured in plasma of healthy adults by the same GC–MS/MS method . Representative chromatograms from the GC–MS/MS analysis of ADMA in this experiment are shown in Fig. 3. In hemolysates, endogenous and externally added ADMA was precisely and accurately measured by GC–MS/MS (Fig. 4). Our results suggest that the increase in the ADMA concentration in hemolysates is due to nitrite-independent proteolysis (Fig. 2). Two major blood proteins, namely albumin  and hemoglobin (present study), do not carry ADMA residues in their molecules. Because we did not use proteolysis inhibitors in the present study, we cannot fully exclude ADMA formation from asymmetric NGdimethylation of soluble L-arginine by N-methyltransferases in human erythrocytes. N-Methyltransferases are abundantly present in red blood cells of humans and a variety of mammalian species . This potential pathway warrants further investigations. Acknowledgments This study was supported by the Deutsche Forschungsgemeinschaft (Grant TS60/4-1). The authors thank B. Beckmann for laboratory assistance and F.-M. Gutzki for performing GC–MS/MS analyses. References  A. Zinellu, S. Sotgia, B. Scanu, L. Deiana, C. Carru, Determination of proteinincorporated methylated arginine reference values in healthy subjects whole blood and evaluation of factors affecting protein methylation, Clin. Biochem. 41 (2008) 1218–1223.  S.S. Billecke, L.A. Kitzmiller, J.J. Northrup, S.E. Whitesall, M. Kimoto, A.V. Hinz, L.G. D´Alecy, Contribution of whole blood to the control of plasma asymmetrical dimethylarginine, Am. J. Physiol. Heart Circ. Physiol. 291 (2006) H1788–H1796.  S.S. Billecke, L.G. D‘Alecy, R. Platel, S.E. Whitesall, K.A. Jamerson, R.L. Perlman, C.A. Gadegbeku, Blood content of asymmetric dimethylarginine: new insights into its dysregulation in renal disease, Nephrol. Dial. Transplant. 24 (2009) 489–496.  L.G. D‘Alecy, S.S. Billecke, 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 (2010) 40.  J.M. Leiper, P. Vallance, The synthesis and metabolism of asymmetric dimethylarginine (ADMA), Eur. J. Clin. Pharmacol. 26 (2006) 33–38.
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Anke Böhmer Henning Großkopf Jens Jordan ⇑ Dimitrios Tsikas Institute of Clinical Pharmacology, Hannover Medical School, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany ⇑ Fax: +49 511 532 2750. E-mail address: [email protected]
(D. Tsikas) Available online 3 April 2012