Urine Metabolomic Signature of People Diagnosed with Balkan Endemic Nephropathy and Other Types of Chronic Kidney Disease Compared with Healthy Subjects in Romania

Abstract

Metabolomic analysis methods were employed to determine biomarkers for various chronic kidney diseases (CKDs). Modern analytical methods were developed and applied successfully to find a specific metabolomic profile in urine samples from CKD and Balkan endemic nephropathy (BEN) patients. The aim was to explore a specific metabolomic profile defined by feasible/easy-to-identify molecular markers. Urine samples were collected from patients with CKDs and BEN, and from healthy subjects from endemic and nonendemic areas in Romania. Metabolomic analysis of urine samples, extracted by the liquid-liquid extraction (LLE) method, was performed by gas chromatography-mass spectrometry (GC-MS). The statistical exploration of the results was performed through a principal component analysis (PCA) evaluation. Urine samples were statistically analyzed using a classification based on six types of metabolites. Most urinary metabolites are distributed in the center of a loading plot, meaning that these compounds do not represent significant markers for BEN. One of the most frequent and higher-concentration urinary metabolites in BEN patients was p-Cresol, a phenolic compound that implies a severe injury of the renal filtration function. The presence of p-Cresol was associated with protein-bound uremic toxins, which have specific functional groups such as indole and phenyl. In prospective studies for future investigation, prevention, and disease treatment, we suggest a larger sample size, sample extraction using other methods, and analysis using other chromatography techniques coupled with mass spectrometry, which can generate a more significant data set for statistical analysis.

CKD Urine Metabolomics: Modern Concepts and Approaches

“…For analysis by gas chromatography-mass spectrometry (GC-MS), a sample must be prepared by being transferred to the gas phase through physical (micro solid-phase extraction from the vapor phase, evaporation, and steam extraction from the sample without additional concentration) or chemical (derivatization of compounds) methods. The chosen liquid-liquid extraction method without derivatization results in a limited number of volatile compounds, usually thermostable ones [ 35 ]. Clinical laboratories are much more likely to have access to tandem GC-MS.…”

“…Additionally, the uremic toxin p-cresol was detected in the urine of a group of patients ( Table 1 ). It was identified as the marker yielding the most statistically significant results for separating groups of patients and healthy individuals [ 35 ]. However, the preliminary nature of the findings represents a limitation of this work.…”

“…NMR analysis could be performed on different nuclei. Biological samples are usually analyzed on the isotopes of the most abundant elements in biomolecules: 1 H, 13 C, 14 N, and 35 P. One of the first works that showed a connection between the essential marker trimethylamine N-oxide (TMAO) and CKD used a combination of 1 H-, 13 -C-, and 14 N-NMR spectroscopy [27]. But today, only 1 H-NMR spectroscopy has gained popularity in urine metabolomics due to having the highest abundance of 1 H in all metabolites of all other nuclei and, consequently, the highest signal intensity.…”

Paper spray mass spectrometry combined with machine learning as a rapid diagnostic for chronic kidney disease

“…13 All of these factors present opportunities for misclassification of CKD that may delay diagnosis and treatment. 14–16 For these reasons, recent CKD research has focused on using untargeted metabolomic approaches to associate several up- or down-regulated compounds in plasma, 17–19 serum, 20–22 or urine 23–25 with kidney disease. This approach has gained attention because it does not rely on target analytes ( e.g.…”