Determination of uremic solutes in biological fluids of chronic kidney disease patients by HPLC assay.
ABSTRACT During chronic kidney disease (CKD), solutes called uremic solutes, accumulate in blood and tissues of patients. We developed an HPLC method for the simultaneous determination of several uremic solutes of clinical interest in biological fluids: phenol (Pol), indole-3-acetic acid (3-IAA), p-cresol (p-C), indoxyl sulfate (3-INDS) and p-cresol sulfate (p-CS). These solutes were separated by ion-pairing HPLC using an isocratic flow and quantified with a fluorescence detection. The mean serum concentrations of 3-IAA, 3-INDS and p-CS were 2.12, 1.03 and 13.03 μM respectively in healthy subjects, 3.21, 17.45 and 73.47 μM in non hemodialyzed stage 3-5 CKD patients and 5.9, 81.04 and 120.54 μM in hemodialyzed patients (stage 5D). We found no Pol and no p-C in any population. The limits of quantification for 3-IAA, 3-INDS, and p-CS were 0.83, 0.72, and 3.2 μM respectively. The within-day CVs were between 1.23 and 3.12% for 3-IAA, 0.98 and 2% for 3-INDS, and 1.25 and 3.01% for p-CS. The between-day CVs were between 1.78 and 5.48% for 3-IAA, 1.45 and 4.54% for 3-INDS, and 1.19 and 6.36% for p-CS. This HPLC method permits the simultaneous and quick quantification of several uremic solutes for daily analysis of large numbers of samples.
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ABSTRACT: 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.Analytical and Bioanalytical Chemistry 01/2013; · 3.66 Impact Factor
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ABSTRACT: The expression of the renoprotective antiaging gene Klotho is decreased in uremia. Recent studies suggest that Klotho may be a tumor suppressor, and its expression may be repressed by DNA hypermethylation in cancer cells. Here we investigated the effects and possible mechanisms by which Klotho expression is regulated during uremia in uninephrectomized B-6 mice given the uremic toxins indoxyl sulfate or p-cresyl sulfate. Cultured human renal tubular HK2 cells treated with these toxins were used as an in vitro model. Injections of indoxyl sulfate or p-cresyl sulfate increased their serum concentrations, kidney fibrosis, CpG hypermethylation of the Klotho gene, and decreased Klotho expression in renal tubules of these mice. The expression of DNA methyltransferases 1, 3a, and 3b isoforms in HK2 cells treated with indoxyl sulfate or p-cresyl sulfate was significantly increased. Specific inhibition of DNA methyltransferase isoform 1 by 5-aza-2'-deoxycytidine caused demethylation of the Klotho gene and increased Klotho expression in vitro. Thus, inhibition of Klotho gene expression by uremic toxins correlates with gene hypermethylation, suggesting that epigenetic modification of specific genes by uremic toxins may be an important pathological mechanism of disease.Kidney International 01/2012; 81(7):640-50. · 7.92 Impact Factor