Metabolic profiling of human plasma and urine in chronic kidney disease by hydrophilic interaction liquid chromatography coupled with time-of-flight mass spectrometry: a pilot study.

A typical characteristic of chronic kidney disease (CKD) is the progressive loss in renal function over a period of months or years with the concomitant accumulation of uremic retention solutes in the body. Known biomarkers for the kidney deterioration, such as serum creatinine or urinary albumin, do not allow effective early detection of CKD, which is essential towards disease management. In this work, a hydrophilic interaction liquid chromatography time-of-flight mass spectrometric (HILIC-TOF MS) platform was optimized allowing the search for novel uremic retention solutes and/or biomarkers of CKD. The HILIC-ESI-MS approach was used for the comparison of urine and plasma samples from CKD patients at stage 3 (n = 20), at stage 5 not yet receiving dialysis (n = 20) and from healthy controls (n = 20). Quality control samples were used to control and ensure the validity of the metabolomics approach. Subsequently the data were treated with the XCMS software for multivariate statistical analysis. In this way, differentiation could be achieved between the measured metabolite profile of the CKD patients versus the healthy controls. The approach allowed the elucidation of a number of metabolites that showed a significant up- and downregulation throughout the different stages of CKD. These compounds are cinnamoylglycine, glycoursodeoxycholic acid, 2-hydroxyethane sulfonate, and pregnenolone sulfate of which the identity was unambiguously confirmed via the use of authentic standards. The latter three are newly identified uremic retention solutes.

Response to Tsikas et al. Comments on Boelaert et al. Determination of Asymmetric and Symmetric Dimethylarginine in Serum from Patients with Chronic Kidney Disease: UPLC-MS/MS versus ELISA. Toxins 2016, 8, 149.

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Determination of Asymmetric and Symmetric Dimethylarginine in Serum from Patients with Chronic Kidney Disease: UPLC-MS/MS versus ELISA.

Asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide (NO) synthesis, and its structural isomer symmetric dimethylarginine (SDMA) are uremic toxins accumulating in chronic kidney disease (CKD) patients. The objective of this study was to develop and validate a robust UPLC-MS/MS method for the simultaneous determination of ADMA and SDMA in human serum. Chromatographic separation after butyl ester derivatization was achieved on an Acquity UPLC BEH C18 column, followed by tandem mass spectrometric detection. After validation, the applicability of the method was evaluated by the analysis of serum samples from 10 healthy controls and 77 CKD patients on hemodialysis (CKD5HD). Both ADMA (0.84 ± 0.19 µM vs. 0.52 ± 0.07 µM) and SDMA concentrations (2.06 ± 0.82 µM vs. 0.59 ± 0.13 µM) were significantly (p < 0.001) elevated in CKD5HD patients compared to healthy controls. In general, low degrees of protein binding were found for both ADMA and SDMA. In addition, an established commercially available ELISA kit was utilized on the same samples (n = 87) to compare values obtained both with ELISA and UPLC-MS/MS. Regression analysis between these two methods was significant (p < 0.0001) but moderate for both ADMA (R = 0.78) and SDMA (R = 0.72).

New methods and technologies for measuring uremic toxins and quantifying dialysis adequacy.

This publication reviews the currently available methods to identify uremic retention solutes, to determine their biological relevance and to quantify their removal. The analytical methods for the detection of uremic solutes have improved continuously, allowing the identification of several previously unknown solutes. Progress has been accelerated by the development of comprehensive strategies such as genomics, proteomics and the latest "omics" area, metabolomics. Those methodologies will be further refined in future. Once the concentration of solutes of interest is known based on targeted analysis, their biological relevance can be studied by means of in vitro, ex vivo, or animal models, provided those are representative for the key complications of the uremic syndrome. For this to come to pass, rigid protocols should be applied, e.g., aiming at free solute concentrations conform those found in uremia. Subsequently, the decrease in concentration of relevant solutes should be pursued by nondialysis (e.g., by influencing nutritional intake or intestinal generation, using sorbents, modifying metabolism, or preserving renal function) and dialysis methods. Optimal dialysis strategies can be sought by studying solute kinetics during dialysis. Clinical studies are necessary to assess the correct impact of those optimized strategies on outcomes. Although longitudinal studies of solute concentration and surrogate outcome studies are first steps in suggesting the usefulness of a given approach, ultimately hard outcome randomized controlled trials are needed to endorse evidence-based therapeutic choices. The nonspecificity of dialysis removal is however a handicap limiting the chances to provide proof of concept that a given solute or group of solutes has definite biological impact.

Uremia-related oxidative stress in leukocytes is not triggered by ß2-microglobulin.

BACKGROUND: Chronic kidney disease (CKD) is characterized by low-grade inflammation and increased risk for cardiovascular disease. The interest in ß2-microglobulin (B2M) as a marker for cardiovascular outcome with and without CKD has grown. Clinical studies suggested that B2M could be involved in the pathogenesis of vascular disease, for which chronic leukocyte activation is a pathogenic factor. We investigated whether B2M is proinflammatory by inducing oxidative burst in leukocytes. METHODS: Oxidative burst was measured at baseline and after stimulation with N-formyl-methionine-leucine-phenylalanine (fMLP), Escherichia coli, or phorbol-12-myristate-acetate (PMA) in the whole blood of healthy volunteers in the absence (saline) and presence of human B2M (hB2M; 10 and 50 mg/L) versus uremic whole blood. Because of suspicion of contamination, hB2M was dialyzed for purification and purified B2M (dB2M) and dialysates were tested in the burst test. As a comparator, reactive oxygen species (ROS) in response to lipopolysaccharide (LPS) was measured. RESULTS: Unpurified hB2M strongly enhanced ROS in monocytes and granulocytes after E. coli and PMA and moderately after fMLP stimulation compared with control (P < .01) and uremia (P < .01) whereas at baseline hB2M only induced ROS in granulocytes (P < .05). After purification, dB2M no longer increased burst activity, suggesting that contamination was responsible for the initial effect. An endotoxin concentration of less than 1.5 EU/mL, as observed in hB2M, could not induce oxidative stress. CONCLUSION: This study suggests that B2M, a traditional marker for middle molecule retention and a novel marker for cardiovascular outcome, may not by itself cause vascular damage by influencing inflammatory response due to induction of leukocyte free radical production. However, an effect on other cell types involved cannot be excluded. Our data further reveal that this type of research might be skewed by non-LPS contaminants, and that care should be taken to exclude this bias.

A novel UPLC-MS-MS method for simultaneous determination of seven uremic retention toxins with cardiovascular relevance in chronic kidney disease patients.

Chronic kidney disease (CKD) is a devastating illness characterized by accumulation of uremic retention solutes in the body. The objective of this study was to develop and validate a simple, rapid, and robust UPLC-MS-MS method for simultaneous determination, in serum, of seven organic acid uremic retention toxins, namely uric acid (UA), hippuric acid (HA), indoxylsulfate (IS), p-cresylglucuronide (pCG), p-cresylsulfate (pCS), indole-3-acetic acid (IAA), and 3-carboxy-4-methyl-5-propyl-2-furanpropionic acid (CMPF). Isotopically labeled internal standards (d(5)-HA; 1,3-(15)N(2)-UA, and d(5)-IAA) were used to correct for variations in sample preparation and system performance. Separation on a C18 column was followed by negative electrospray ionization and tandem mass spectrometric detection. Accuracy was below the 15 % threshold. Within-day precision varied from 0.60 to 4.54 % and between-day precision was below 13.33 % for all compounds. The applicability of the method was evaluated by analyzing 78 serum samples originating both from healthy controls and from patients at different stages of CKD. These results were compared with those obtained by use of conventional HPLC-PDA-FLD methods. A good correlation was obtained between both methods for all compounds.