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Amino Acids

, Volume 51, Issue 4, pp 627–640 | Cite as

GC–MS measurement of biological NG-hydroxy-l-arginine, a stepmotherly investigated endogenous nitric oxide synthase substrate and arginase inhibitor

  • Alexander Bollenbach
  • Stephan J. L. Bakker
  • Dimitrios TsikasEmail author
Original Article

Abstract

l-Arginine is converted by nitric oxide synthase (NOS) to l-citrulline and nitric oxide (NO). NG-Hydroxy-l-arginine (NOHA) is the isolable intermediate of this reaction. NOHA has been identified in biological samples by gas chromatography–mass spectrometry (GC–MS) and quantified by high-performance liquid chromatography (HPLC). Reportedly, NOHA concentrations in human plasma and serum range over four orders of magnitude (e.g., 2 nM–34 µM). The natural occurrence of NOHA in urine has not been reported thus far. Here, we report a validated stable-isotope dilution GC–MS method for the quantitative determination of NOHA in 10-µL aliquots of human serum and urine samples. The method is based on a two-step derivatization of NOHA to the methyl ester pentafluoropropionyl (PFP) derivatives using newly synthesized trideuteromethyl ester NOHA (d3Me-NOHA) as the internal standard and GC–MS quantification. NOHA was found to form a methyl ester-NG,Nδ,Nα-pentafluoropropionyl derivative, i.e., Me-(PFP)3 (M, 642) with the NG-hydroxy group remaining non-derivatized. Selected-ion monitoring of mass-to-charge (m/z) ratio of 458 for endogenous NOHA and m/z 461 for d3Me-NOHA in the negative-ion chemical ionization mode revealed NOHA concentrations of the order of 0.2 µM in human serum and 3 µM in urine samples. Accuracy (recovery, %) was 91.6 ± 1.6% in serum and 39.9 ± 4.5% in urine. Inorganic nitrate was found to decrease NOHA recovery from urine presumably through the reaction of the OH group of NOHA with nitric acid. Imprecision (RSD,  %) ranged between 1.4 and 14.8% in serum, and between 5.3 and 18.4% in urine in the investigated concentration range (0–15 µM NOHA). Ten healthy kidney donors excreted in the urine (mean ± SEM) 13.9 ± 1.81 µmol NOHA per day before and 10.9 ± 1.4 µmol NOHA per day after kidney donation (P = 0.24). Similar results were observed for dimethylamine (DMA), the major urinary metabolite of asymmetric dimethylarginine (ADMA). Changes in NOHA and DMA correlated positively (r = 0.718, P = 0.019). This is the first report on the occurrence and measurement of NOHA in human urine and on the effect of human unilateral nephrectomy on urinary NOHA and DMA. Healthy kidney donation may be useful as a model for kidney disease.

Keywords

Arginase l-Arginine Asymmetric dimethylarginine Dimethylamine NG-Hydroxy-l-arginine Mass spectrometry Nitrate Nitric oxide Nitric oxide synthase 

Abbreviations

ADMA

Asymmetric dimethylarginine

DDAH

Dimethylarginine dimethylaminohydrolase

DMA

Dimethylamine

EI

Electron ionization

GC–MS

Gas chromatography–mass spectrometry

IS

Internal standard

LC–MS

Liquid chromatography–mass spectrometry

LC–MS/MS

Liquid chromatography–tandem mass spectrometry

LLOQ

Lower limit of quantitation

LPS

Lipopolysaccharide

MeOH

Methanol

m/z

Mass-to-charge

NG

Guanidine nitrogen

NO

Nitric oxide

NOHA

NG-Hydroxy-l-arginine

NOS

Nitric oxide synthase

iNOS

Inducible nitric oxide synthase

OPA

o-Phthaldialdehyde

PAR

Peak area ratio

PFP

Pentafluoropropionyl

PFPA

Pentafluoropropionic anhydride

PRMT

Protein arginine methyltransferase

RSD

Relative standard deviation

SDMA

Symmetric dimethylarginine

TNF-α

Tumor necrosis factor alpha

Notes

Compliance with ethical standards

Conflicts of interest

All authors report no conflicts of interest.

Ethical statement

The Institutional Review Board approved the study protocol (METc 2008/186) which was in adherence to the Declaration of Helsinki.

Supplementary material

726_2018_2695_MOESM1_ESM.docx (273 kb)
Supplementary material 1 (DOCX 273 kb)

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Copyright information

© Springer-Verlag GmbH Austria, part of Springer Nature 2019

Authors and Affiliations

  • Alexander Bollenbach
    • 1
  • Stephan J. L. Bakker
    • 2
    • 3
  • Dimitrios Tsikas
    • 1
    Email author
  1. 1.Institute of ToxicologyCore Unit Proteomics, Hannover Medical SchoolHannoverGermany
  2. 2.Division of Nephrology, Department of Internal MedicineUniversity Medical CenterGroningenThe Netherlands
  3. 3.Groningen Kidney CenterGroningenThe Netherlands

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