Nitric Oxide Synthesis Metabolites-As Potential Markers in Chronic Kidney Disease in Children.

Nitric oxide (NO) is an important signaling molecule for many physiological and pathological processes. Diseases associated with abnormal NO synthesis include cardiovascular diseases, insulin-dependent diabetes, or chronic kidney disease (CKD). The aim of the paper was to evaluate NO synthesis metabolites, i.e., asymmetric dimethylarginine (ADMA), symmetric dimethylarginine (SDMA), dimethylamine (DMA), arginine, citrulline in plasma of patients with different severity of CKD and to seek possible links between these parameters and the development of this disease. Forty-eight CKD children and thirty-three age-matched controls were examined. Patients were divided into groups depending on the CKD stages (Group II-stage II, Group III-stage III, Group IV-stage IV, and Group RRT children on dialysis). To determine the concentrations of the above-mentioned metabolites in plasma liquid chromatography-mass spectrometry was used. There were significant differences observed in levels of ADMA, SDMA, DMA, and citrulline between control vis CKD groups (p values ranging from <0.001 to 0.029). Plasma arginine concentration was also higher in CKD patients compared to the control group but statistically insignificant. ADMA levels in CKD children were statistically significantly higher in relation to particular stages of CKD (RRT vis II stage of CKD: p = 0.01; RRT vis III-IV stages of CKD: p < 0.046). Citrulline levels in CKD children were statistically significantly higher in RRT group vis control (p < 0.001). Children with CKD develop disturbances in most metabolites of NO synthesis. Dialysis children treated show the greatest disturbances of plasma ADMA and citrulline levels. ADMA seems to be a good indicator of the gradual progression of the CKD, which is proved by the negative correlation with eGFR.

Adenine Nucleotide Metabolites in Uremic Erythrocytes as Metabolic Markers of Chronic Kidney Disease in Children.

Chronic kidney disease (CKD) is associated with multifaceted pathophysiological lesions including metabolic pathways in red blood cells (RBC). The aim of the study was to determine the concentration of adenine nucleotide metabolites, i.e., nicotinamide adenine dinucleotide (NAD)-oxidized form, nicotinamide adenine dinucleotide hydrate (NADH)-reduced form, nicotinic acid mononucleotide (NAMN), ß-nicotinamide mononucleotide (NMN), nicotinic acid adenine dinucleotide (NAAD), nicotinic acid (NA) and nicotinamide (NAM) in RBC and to determine a relationship between NAD metabolites and CKD progression. Forty-eight CKD children and 33 age-matched controls were examined. Patients were divided into groups depending on the CKD stages (Group II-stage II, Group III- stage III, Group IV- stage IV and Group RRT children on dialysis). To determine the above-mentioned metabolites concentrations in RBC liquid chromatography-mass spectrometry was used. Results: the only difference between the groups was shown concerning NAD in RBC, although the values did not differ significantly from controls. The lowest NAD values were found in Group II (188.6 ± 124.49 nmol/mL, the highest in group IV (324.94 ± 63.06 nmol/mL. Between Groups II and IV, as well as III and IV, the differences were statistically significant (p < 0.032, p < 0.046 respectively). Conclusions. CKD children do not have evident abnormalities of RBC metabolism with respect to adenine nucleotide metabolites. The significant differences in erythrocyte NAD concentrations between CKD stages may suggest the activation of adaptive defense mechanisms aimed at erythrocyte metabolic stabilization. It seems that the implementation of RRT has a positive impact on RBC NAD metabolism, but further research performed on a larger population is needed to confirm it.

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.

A novel mass spectrometry-based method for simultaneous determination of asymmetric and symmetric dimethylarginine, l-arginine and l-citrulline optimized for LC-MS-TOF and LC-MS/MS.

Nitric oxide (NO) is a regulatory molecule involved in many biological processes. NO is produced by nitric oxide synthase by conversion of l-arginine to l-citrulline. l-Arginine methylated derivatives, asymmetric and symmetric dimethylarginines (asymmetric dimethylarginine, ADMA, and symmetric dimethylarginine, SDMA), regulate l-arginine availability and the activity of nitric oxide synthase. As such, they have been frequently investigated as potential biomarkers in pathologies associated with dysfunctions in NO synthesis. Here, we present a new multistep analytical methodology based on liquid chromatography combined with mass spectrometry for the accurate identification of l-arginine, l-citrulline, ADMA and SDMA. Compounds are measured as stable 2,3,4,5,6-pentafluorobenzoyl chloride derivatives, which allows for simultaneous analysis of all compounds through chromatographic separation of ADMA and SDMA using a reverse-phase column. Serum aliquots (100 µL) were spiked with isotope-labeled internal standards and sodium carbonate buffer. The derivatization process was carried out at 25°C for 10 minu using pentafluorobenzoyl chloride as derivatization reagent. Calibration demonstrated good linearity (R2 = 0.9966-0.9986) for all derivatized compounds. Good accuracy (94.67-99.91%) and precision (1.92-11.8%) were observed for the quality control samples. The applicability of the method was evaluated in a cohort of angiological patients and healthy volunteers. The method discerned significantly lower l-arginine and l-citrulline in angiologic patients. This robust and fast LC-ESI-MS method may be a useful tool in quantitative analysis of l-arginine, ADMA, SDMA and l-citrulline.