Quantitative Analysis of l-Arginine, Dimethylated Arginine Derivatives, l-Citrulline, and Dimethylamine in Human Serum Using Liquid Chromatography–Mass Spectrometric Method

Abstract

Nitric oxide (NO) is a small molecule involved in the regulation of many physiological processes. It plays a crucial role in the regulation of nervous system, immune and inflammatory responses, and blood flow. NO is synthesized by nitric oxide synthase (NOS) during two-step oxidation of l-arginine to l-citrulline. Intermediates and derivatives of NO metabolism, such as l-arginine, l-citrulline, asymmetrical dimethylarginine (ADMA), symmetrical dimethylarginine (SDMA), and dimethylamine (DMA), are investigated as potential biomarkers. In this article, we present a novel analytical method that allowed for simultaneous analysis of l-arginine, ADMA, SDMA, l-citrulline, and DMA, in a single-step extraction and derivatization using benzoyl chloride. In brief, aliquots of serum were mixed with internal standard solution mixture (50 µM D6-DMA, 20 µM D7-ADMA, and 100 µM D7-arginine) and 0.025 M borate buffer, pH 9.2 (10:1:5). The derivatization process was performed at 25 °C for 5 min using 10% benzoyl chloride. A reverse phase column was used for chromatographic separation. Quantitation was performed using following ions (m/z): 279.1457, 286.1749, 307.1717, 314.2076, 280.1297, 150.0919, and 156.1113 for l-arginine, D7-arginine, ADMA, SDMA, D7-ADMA, l-citrulline, DMA, and D6-DMA, respectively. The method was validated, and its assay linearity, accuracy and precision, recovery, and limits of detection (1.7 µM l-arginine, 0.03 µM ADMA, 0.02 µM SDMA, 0.36 µM l-citrulline, 0.06 µM DMA) and quantification (3.2 µM l-arginine, 0.08 µM ADMA, 0.05 µM SDMA, 1.08 µM l-citrulline, 0.19 µM DMA) were determined. The method is sensitive, reliable, repeatable, and reproducible. It can be applied in the routine clinical/diagnostic laboratory.Graphical abstract

L-Arginine/Nitric Oxide Pathway Is Altered in Colorectal Cancer and Can Be Modulated by Novel Derivatives from Oxicam Class of Non-Steroidal Anti-Inflammatory Drugs

“…Metabolite concentration in cell cultures was quantified using a method recently developed [ 85 ] and routinely used in our laboratory [ 6 , 7 , 86 , 87 , 88 ] with small modifications.…”

Transcriptional and Metabolomic Analysis of L-Arginine/Nitric Oxide Pathway in Inflammatory Bowel Disease and Its Association with Local Inflammatory and Angiogenic Response: Preliminary Findings

“…Serum concentrations of L-arginine/NO pathway metabolites were measured on a Xevo G2 quadrupole time-of-flight (Q-TOF) instrument (Waters, Milford, MA, USA) by stable isotope dilution liquid chromatography tandem mass spectrometry (LC-MS/MS), using a method developed in the laboratory of Medical Biochemistry Department [68]. In short, 100 µL of sera or calibration sample, 10 µL of internal standard solution in water (20 µM D7-ADMA, 50 µM D6-DMA, and 100 µM D7-arginine), and 50 µL of borate buffer (0.025 M Na 2 B 4 O 7 ·10H 2 O, 1.77 mM sodium hydroxide (NaOH), pH = 9.2) were vortexed (1 min, 1100 rpm, 25 • C) in polypropylene microtubes.…”

L-Arginine/NO Pathway Metabolites in Colorectal Cancer: Relevance as Disease Biomarkers and Predictors of Adverse Clinical Outcomes Following Surgery

“…Serum arginine depletion has been attributed to the upregulated amino acid uptake by tumors and linked with their accelerated growth, with concomitant obstruction of an anti-tumor immune response [ 12 ]. The decrease in systemic arginine might be an early event in colonic carcinogenesis as it is also seen in active IBD [ 16 ], a condition associated with an increased risk of CRC [ 24 ]. ADMA and SDMA have been shown to accumulate in tumor tissues as well [ 13 , 23 ], and are elevated in the urine of CRC-bearing mice, together with arginine and citrulline [ 23 ].…”

“…Calibration standards and serum samples were prepared in accordance with the method recently developed in our laboratory [ 17 ] and validated on patients with cancer and inflammatory conditions [ 16 , 18 , 19 , 20 ]. The 100 µL aliquots of the calibration standards or sera were mixed with 10 µL of an internal standard solution (50 µM D6-DMA, 20 µM D7-ADMA, and 100 µM D7-arginine, respectively) and 50 µL of a borate buffer (0.025 M Na 2 B 4 O 7 × 10H 2 O, 1.77 mM NaOH, pH = 9.2) and vortexed (1 min, 25 °C).…”