Abstract:
:Low concentrations of L-homoarginine (hArg) in plasma or serum and urine have recently emerged as a novel cardiovascular risk factor. Previously, we reported gas chromatography-mass spectrometry (GC-MS) and GC-tandem MS (GC-MS/MS) methods for the quantitative determination of hArg and Arg in plasma, serum, urine and other biological samples. In these methods, plasma and serum are ultrafiltered by means of commercially available cartridges (10 kDa), and 10-µL ultrafiltrate aliquots are subjected to a two-step derivatization procedure, yielding the methyl ester tri(N-pentafluoropropionyl) derivatives. De novo prepared trideuteromethyl ester hArg (d3Me-hArg) was used as an internal standard. To make the hArg analysis in plasma more convenient, straightforward and cheaper we performed two key modifications: (1) precipitation of plasma proteins by methanol and (2) use of newly prepared and d3Me-hArg as the internal standard. The method was validated and used for the quantitative determination of hArg in human plasma by GC-MS after electron-capture negative-ion chemical ionization (ECNICI) using methane as the reactant gas. Intra-assay accuracy (recovery) and imprecision (relative standard deviation) were within generally accepted ranges (93-109 and 2.3-10 %, respectively). Furthermore, we extended the applicability of this method to guanidinoacetate (GAA). This is of particular importance because hArg and GAA are produced from Arg by the catalytic action of arginine:glycine amidinotransferase (AGAT) also known as glycine:arginine transamidinase (GATM). Using this method, we quantitated simultaneously hArg, Arg and GAA in the selected-ion monitoring mode in 10-µL aliquots of plasma. In plasma samples of 17 non-medicated healthy young men, the concentration of hArg, GAA and Arg was determined to be (mean ± SD) 1.7 ± 0.6, 2.6 ± 0.8, 91 ± 29 µM, respectively. The correlation between hArg and Arg was borderline (r = 0.47, P = 0.06). GAA strongly correlated with Arg (r = 0.82, P < 0.0001) but did not correlate with hArg (r = 0.17, P = 0.52). The plasma concentrations of hArg, GAA and Arg measured in 9 patients suffering from stroke or transitory ischemic attack were 1.8 ± 0.6, 2.7 ± 0.4 and 82 ± 17 µM. The ratio values of the hArg, GAA and Arg concentrations measured after removal of plasma proteins by methanol precipitation or ultrafiltration were 0.94 ± 0.1, 0.94 ± 0.08, and 0.88 ± 0.07, respectively. Simultaneous measurement of hArg and GAA in human plasma may allow assessment of AGAT activity in vivo with respect both to GAA and to hArg and their relationship in health, disease, nutrition and pharmacotherapy.
journal_name
Amino Acidsjournal_title
Amino acidsauthors
Hanff E,Kayacelebi AA,Yanchev GR,Maassen N,Haghikia A,Tsikas Ddoi
10.1007/s00726-015-2120-0subject
Has Abstractpub_date
2016-03-01 00:00:00pages
721-732issue
3eissn
0939-4451issn
1438-2199pii
10.1007/s00726-015-2120-0journal_volume
48pub_type
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