Early detection of liver injuries by the Serum enhanced binding test sensitive to albumin post-transcriptional modifications.

Early and sensitive biomarkers of liver dysfunction and drug-induced liver injury (DILI) are still needed, both for patient care and drug development. We developed the Serum Enhanced Binding (SEB) test to reveal post-transcriptional modifications (PTMs) of human serum albumin resulting from hepatocyte dysfunctions and further evaluated its performance in an animal model. The SEB test consists in spiking serum ex-vivo with ligands having specific binding sites related to the most relevant albumin PTMs and measuring their unbound fraction. To explore the hypothesis that albumin PTMs occur early during liver injury and can also be detected by the SEB test, we induced hepatotoxicity in male albino Wistar rats by administering high daily doses of ethanol and CCl4 over several days. Blood was collected for characterization and quantification of albumin isoforms by high-resolution mass spectrometry, for classical biochemical analyses as well as to apply the SEB test. In the exposed rats, the appearance of albumin isoforms paralleled the positivity of the SEB test ligands and histological injuries. These were observed as early as D3 in the Ethanol and CCl4 groups, whereas the classical liver tests (ALT, AST, PAL) significantly increased only at D7. The behavior of several ligands was supported by structural and molecular simulation analysis. The SEB test and albumin isoforms revealed hepatocyte damage early, before the current biochemical biomarkers. The SEB test should be easier to implement in the clinics than albumin isoform profiling.

Posttranslational-modifications of human-serum-albumin analysis by a top-down approach validated by a comprehensive bottom-up analysis.

The posttranslational modifications (PTM) of human serum albumin (HSA) can result in the development of isoforms that have been identified as potential biomarkers for advanced hepatic diseases. However, previous approaches using top-down (TD) analysis to identify isoforms based on molecular weight may have resulted in misidentifications. The nature of the identified isoforms has never been confirmed in previous works. Here, we aimed to critically evaluate TD for the characterization and determination of HSA isoforms in patients and make an inventory of HSA-PTM. Serum samples from control subjects and patients with liver dysfunctions were analyzed using both top-down (TD) and bottom-up (BU) approaches. TD analysis involved using a LC-TOF-MS system to obtain a multicharged spectrum of HSA, which was deconvoluted to identify isoforms. Spectra were then used for relative quantitation analysis of albumin isoform abundances based on trapezoidal integration. For BU analysis, serums were reduced +/- alkylated, digested with trypsin and analyzed in the Q-TOF, data-dependent acquisition (DDA) mode to generate a SWATH-MS high-resolution mass spectral library of all HSA peptides. Tryptic digests of another set of serum samples were then analyzed using data-independent acquisition (DIA) mode to confirm the presence of HSA isoforms and their modification sites. TD detected 15 isoforms corresponding to various modifications, including glycation, cysteinylation, nitrosylation, and oxidation (di- and tri-). In BU, the spectral library containing 127 peptides allowed for the characterization of the important isoforms with their modified sites, including some modifications that were only characterized in BU (carbamylation, deamidation, and amino-acid substitution). The method used for determining isoforms offered acceptable reproducibility (intra-/inter-assay CVs < 15%) for all isoforms present at relative abundances higher than 2%. Overall, the study found that several isoforms could be missed or misidentified by TD. However, all HSA isoforms identified by TD and reported to be relevant in liver dysfunctions were confirmed by BU. This critical evaluation of TD approach helped design an adequate and reliable method for the characterization of HSA isoforms in patients and offers the possibility to estimate isoform abundances within 3 min. These findings have significant implications for the diagnosis and treatment of liver dysfunctions.

Effect of Sevelamer and Nicotinamide on Albumin Carbamylation in Patients with End-Stage Kidney Disease.

BACKGROUND AND OBJECTIVE: In end-stage kidney disease, high urea levels promote the carbamylation of lysine side chains on a variety of proteins, including albumin. Albumin carbamylation has been identified as a risk factor for mortality and sevelamer led to a decrease in urea levels in dialysis patients. In the present secondary analysis of the NICOREN trial, we investigated the putative impacts of sevelamer and nicotinamide on albumin carbamylation, and the potential correlation between carbamylation and vascular calcifications. METHODS: All possible carbamylation of circulating albumin were screened for with high-resolution liquid chromatography-tandem mass spectrometry. Levels of three carbamylated peptides were then measured as a guide to the extent of albumin carbamylation. Carbamylation was measured at baseline in 55 patients included in the NICOREN trial and 29 patients at 24 weeks of treatment. Calcifications on plain radiographs were quantified as the Kauppila score and the Adragao score. RESULTS: Baseline albumin carbamylation was present at three different sites in subjects with end-stage kidney disease. At baseline, we observed only a correlation between urea and the KQTA carbamylation site in these patients. Albumin carbamylation levels did not decrease after 24 weeks of treatment with either sevelamer or nicotinamide. Furthermore, the proportion of carbamylated serum albumin was not correlated with vascular calcification scores in this population. CONCLUSIONS: Our results confirmed the presence of carbamylated albumin in patients with end-stage kidney disease and demonstrated the presence of carbamylation beyond the LRVP residues. The results also demonstrated the lack of impact of sevelamer or nicotinamide on albumin carbamylation levels. Therapeutic strategies to lower carbamylation load should probably be focused on direct anti-carbamylation processes and/or potentially anti-inflammatory therapies.