Targeted arginine metabolomics: A rapid, simple UPLC-QToF-MSE based approach for assessing the involvement of arginine metabolism in human disease
Background Nitric oxide synthase (NOS) mediated conversion of arginine (ARG) to citrulline
(CIT) is a key pathway for nitric oxide synthesis. ARG is also metabolised by alternate
pathways to ornithine (ORN), homoarginine (HMA), N G-monomethyl-L-arginine (MMA), NG,
N G-dimethyl-L-arginine (ADMA) and NG, NG′-dimethyl-L-arginine (SDMA), all of which
have the capacity to alter NOS activity. Simultaneous assessment of these analytes, when
assessing the impact of arginine metabolism in human disease states, is desirable. Methods …
(CIT) is a key pathway for nitric oxide synthesis. ARG is also metabolised by alternate
pathways to ornithine (ORN), homoarginine (HMA), N G-monomethyl-L-arginine (MMA), NG,
N G-dimethyl-L-arginine (ADMA) and NG, NG′-dimethyl-L-arginine (SDMA), all of which
have the capacity to alter NOS activity. Simultaneous assessment of these analytes, when
assessing the impact of arginine metabolism in human disease states, is desirable. Methods …
Background
Nitric oxide synthase (NOS) mediated conversion of arginine (ARG) to citrulline (CIT) is a key pathway for nitric oxide synthesis. ARG is also metabolised by alternate pathways to ornithine (ORN), homoarginine (HMA), NG-monomethyl-L-arginine (MMA), NG,NG-dimethyl-L-arginine (ADMA) and NG,NG′-dimethyl-L-arginine (SDMA), all of which have the capacity to alter NOS activity. Simultaneous assessment of these analytes, when assessing the impact of arginine metabolism in human disease states, is desirable.
Methods
Analytes (ARG, ADMA, SDMA, MMA, HMA, CIT and ORN) were isolated from human plasma by solvent extraction, evaporated and reconstituted. Ultra-performance liquid chromatography (UPLC) was performed on a 150 mm × 2.1 mm T3 HSS column using a gradient mobile phase comprising ammonium formate (10 mM, pH 3.8) in methanol (1% to 63%). Analytes were detected by time-of-flight mass spectrometry (Q-ToF-MS) in positive ion mode with electrospray ionisation (ESI +). Data were collected using MSE.
Results
Solvent extraction provided high recovery (> 95%). UPLC-QToF-MSE facilitated the separation and quantification of the 7 analytes in an analysis time of 6 min. The approach has high sensitivity; LOQ range from 0.005 μM (NMMA) to 0.25 μM (ARG and ORN), and good precision; intra- and inter-day %RSD are < 6% for all analytes.
Conclusions
This approach provides the capacity to quantify 7 key compounds involved in ARG metabolism in a small sample volume, with a short total analysis time. These characteristics make this approach ideal for undertaking a comprehensive characterisation of this pathway in large data sets (e.g. population studies).
Elsevier