Determination of Homoarginine, Arginine, NMMA, ADMA, and SDMA in Biological Samples by HPLC-ESI-Mass Spectrometry

Luigi Servillo,Rosario Casale,Nunzia D'Onofrio,Maria Balestrieri,Domenico Cautela,Alfonso Giovane,Domenico Castaldo

doi:10.3390/ijms141020131

Abstract

NG,NG-dimethyl-l-arginine (ADMA) and NG-methyl-l-arginine (NMMA) are endogenous inhibitors of nitric oxide synthase (NOS). In contrast, NG,N′G-dimethyl-Larginine (SDMA) possesses only a weak inhibitory potency towards neuronal NOS and it is known to limit nitric oxide (NO) production by competing with l-arginine for cellular uptake. The inhibition of NOS is associated with endothelial dysfunction in cardiovascular diseases as well in chronic renal failure. l-Homoarginine (HArg), a structural analog of l-arginine (Arg), is an alternative but less efficient substrate for NOS. Besides, it inhibits arginase, leading to an increased availability of l-arginine for NOS to produce NO. However, its relation with cardiovascular disease remains unclear. To date, several analytical methods for the quantitative determination of Arg, HArg, NMMA, AMDA, and SDMA in biological samples have been described. Here, we present a simple, fast, and accurate HPLC-ESI-MS/MS method which allows both the simultaneous determination and quantification of these compounds without needing derivatization, and the possibility to easily modulate the chromatographic separation between HArg and NMMA (or between SDMA and ADMA). Data on biological samples revealed the feasibility of the method, the minimal sample preparation, and the fast run time which make this method very suitable and accurate for analysis in the basic and clinical settings.

Highlights

The methylarginines, NG-methyl-L-arginine, NG,NG-dimethyl-Larginine, and NG,N'G-dimethyl-L-arginine as well as L-arginine (Arg) and L-homoarginine (HArg) are important players of the nitric oxide (NO) metabolism [1]
Free ADMA, SDMA, and NMMA are supposed to be produced in the human body through the methylation of protein arginine residues by protein arginine methyltransferases (PRMT) and released during proteolysis of the methylated proteins [4]
To maintain a steady pool size, free methylarginines are metabolized by dimethylarginine dimethylaminohydrolase (DDAH) enzymes

Summary

Introduction

The methylarginines, NG-methyl-L-arginine (monomethylarginine; NMMA), NG,NG-dimethyl-Larginine (asymmetric dimethylarginine, ADMA), and NG,N'G-dimethyl-L-arginine (symmetric dimethylarginine; SDMA) as well as L-arginine (Arg) and L-homoarginine (HArg) are important players of the nitric oxide (NO) metabolism [1]. To maintain a steady pool size, free methylarginines are metabolized by dimethylarginine dimethylaminohydrolase (DDAH) enzymes An imbalance in this pool, due to PRMT or DDAH dysfunction, might increase cardiovascular risk. This method, performed with a short silica column (SupelcosilTM LC-Si. 3.3 cm × 4.6 mm i.d., 3 μm particle size) utilizes isocratic elution conditions which represent a noticeable advantage in terms of baseline stability during the chromatography with MS detection and spare time for skipping column re-equilibration after each analysis [11]. We report a novel and feasible application of the HPLC-ESI-mass spectrometry detection using the SupelcosilTM LC-Si column which allows an accurate and combined determination of NMMA, SDMA, ADMA, Arg, and HArg in biological fluids such as plasma and urine

Results and Discussion

Reagents

Sample Preparation

Conclusions