Aurore Desmons

Homocitrulline: a new marker for differentiating acute from chronic renal failure

Abstract Background: Carbamylation is a non-enzymatic post-translational modification of proteins characterized by the addition of isocyanic acid to amino groups. As isocyanic acid mainly originates from the spontaneous dissociation of urea, carbamylation rate is increased during renal failure. The aim of the study was to evaluate serum homocitrulline (HCit), which results from the carbamylation of ε-amino groups of lysine (Lys) residues, in acute renal failure (ARF) and to determine if it could be useful for differentiating acute from chronic renal failure (CRF). Methods: In total, 213 patients with renal failure referred to the nephrology department of the university hospital of Reims were included. Patients were classified into three groups: patients with ARF (ARF group, n=39), patients with CRF complicated with ARF (A/CRF group, n=29) and patients with CRF (CRF group, n=145). Serum HCit concentrations were measured by LC-MS/MS. Concentration kinetics of HCit and urea were studied in patients suffering from ARF. The HCit thresholds distinguishing ARF and CRF were investigated. Results: HCit concentrations increased in ARF patients reaching a peak delayed compared to urea concentration peak. HCit concentrations were positively correlated with urea concentrations (r=0.51) and with the time elapsed since the estimated onset of ARF (r=0.57). Serum HCit concentrations were higher (p<0.05) in CRF group compared to ARF group. The receiver operating characteristic curve analysis showed that HCit concentrations <289 μmol/mol Lys were predictive of ARF (Sensitivity: 83%, Specificity: 72%, AUC: 0.856). Conclusions: Our results demonstrate that HCit is a promising biomarker for distinguishing between ARF and CRF patients.

Homocitrulline as marker of protein carbamylation in hemodialyzed patients

BACKGROUND Homocitrulline (HCit) is a carbamylation-derived product (CDP) that has been identified as a valuable biomarker of morbidity and mortality in patients with chronic kidney disease (CKD). The aim of this study was to determine whether initiation of hemodialysis therapy (HD) could induce variations of HCit concentrations in CKD patients. METHODS Serum HCit concentrations were determined by LC-MS/MS in CKD patients (n=108) just before (M0) and six months (M6) after the initiation of HD therapy. RESULTS Mean HCit concentrations reached 1000μmol/mol Lysine before initiation of HD therapy and decreased by 50% within 6months after HD onset. HCit concentrations remained stable over time as assessed during a 24-months follow-up period. HCit was mostly found in its protein-bound form in HD patients. HCit concentrations obtained at M0 were positively correlated with urea (r=0.58) and carbamylated hemoglobin (r=0.41), and are likely to be promising predictive markers of mortality. However, no correlations were found between HCit concentrations and Kt/V values, suggesting that HCit is not a marker of HD efficiency. CONCLUSION HCit concentrations reflect the intensity of protein carbamylation and are stable over time during HD treatment, making HCit a reliable biomarker in the follow-up of CKD patients.

Analytical performances of the D-100TM hemoglobin testing system (Bio-Rad) for HbA1c assay

Abstract Background: Glycated hemoglobin (HbA1c) is widely used for the monitoring of glycemic balance in diabetic patients and has also been proposed as a tool for the diagnostic of diabetes mellitus. Accordingly, HbA1c quantification must be performed using robust, reliable and efficient methods. Here are reported the results of the evaluation of a new high performance liquid chromatography (HPLC) system for HbA1c quantification, the D-100TM system from Bio-Rad Laboratories. Methods: The analytical performances of the method as well as the influence of the most frequent interferences regarding HbA1c assays (e.g., labile HbA1c, carbamylated hemoglobin, high HbF) have been tested. Results: Intra- and between-assay CVs were respectively lower than 0.93% and 1.46% (HbA1c results expressed in NGSP units) and lower than 1.67% and 2.27% (HbA1c results expressed in IFCC units). The linearity proved to be excellent from 15 mmol/mol (3.5%) to 184 mmol/mol (19.0%) (r=0.999). The results were well correlated with those obtained by another HPLC method (VARIANTTM II Hemoglobin A1c Program reorder pack 270-2101NU-Bio-Rad): HbA1c[VARIANTTM II, mmol/mol]=1.013×HbA1c[D-100TM, mmol/mol]+0.637 (r=0.993, n=2000). The D-100TM system provided results consistent with IFCC-assigned external quality control samples and the presence of labile HbA1c, carbamylated hemoglobin and HbF did not interfere with HbA1c measurement. Conclusions: The D-100 TM system proved to be a robust and reliable method for HbA1c measurement suitable for routine practice in clinical chemistry laboratories.

Post-mortem diagnosis, of cytomegalovirus and varicella zoster virus co-infection by combined histology and tissue molecular biology, in a sudden unexplained infant death

Abstract Background An autopsy case of a two-month-old male infant who suddenly and unexpectedly died during his sleep, eight days after the onset of benign varicella. Objectives To describe post-mortem combined histological and tissue molecular biological techniques for the diagnosis of cytomegalovirus and varicella zoster virus co-infection as a cause of death. Study design Real-time quantitative PCR and RT-PCR assays for Herpesviruses, respiratory viruses, Adenovirus, Enterovirus and Parvovirus B19 were performed on multi-organ frozen samples and paraffin-embedded tissues in combination with histology. Results Cytomegalovirus and varicella zoster virus were detected by molecular biology with highest viral loads detected in the lungs (4.6×107 and 1.9×105 genome copies per million of cells, respectively). Pulmonary extensive necrotizing inflammation and immunohistochemistry correlated to virological data. Virological molecular biology was negative on paraffin-embedded tissues. Conclusions This case shows that thorough quantitative virological investigations on frozen tissues must be performed in combination with histology and immunohistochemistry for the determination of the cause of a sudden unexplained infant death.

Measurement of Homocitrulline, A Carbamylation‐derived Product, in Serum and Tissues by LC‐MS/MS

Carbamylation corresponds to the non‐enzymatic binding of isocyanic acid to protein amino groups and participates in protein molecular aging, characterized by the alteration of their structural and functional properties. Carbamylated proteins exert deleterious effects in vivo and are involved in the progression of various diseases, including atherosclerosis and chronic kidney disease. Therefore, there is a growing interest to evaluate the carbamylation rate of blood or tissue proteins, since carbamylation‐derived products (CDPs) constitute valuable biomarkers in these contexts. Homocitrulline, formed by isocyanic acid covalently attaches to the ε‐NH2 group of lysine residue side chain, is the most characteristic CDP. Sensitive and specific quantification of homocitrulline requires mass spectrometry‐based methods. This unit describes a liquid chromatography–tandem mass spectrometry (LC‐MS/MS) method for the quantification of homocitrulline, with special emphasis on pre‐analytical steps that allow quantification of total or protein‐bound homocitrulline in serum or tissue samples.

Labile glycated haemoglobin and carbamylated haemoglobin are still critical points for HbA1c measurement

Introduction Haemoglobin A1c (HbA1c) is a key analyte for the monitoring of glycemic balance in diabetic patients and is used for diabetes diagnosis in many countries. The potential interference of carbamylated haemoglobin (cHb) and labile glycated haemoglobin (LA1c) on HbA1c assays must remain a matter of vigilance. Such a situation has occurred in our laboratory with a kit replacement on the Bio-Rad Variant™ II testing system, a cation-exchange high performance liquid chromatography (HPLC) system. With this method, LA1c and cHb coeluted in a same peak which may have different consequences on HbA1c values. Materials and methods The influence of increasing LA1c and cHb values on HbA1c results was studied with in vitro glycation and carbamylation of samples. Samples from patients with high and normal blood urea concentrations were assayed by HPLC and immunological assay. Results We observed that the degree of interference greatly varied depending on the nature of the interfering Hb fractions found under the so-called “LA1c peak”. Thus, we have decided to apply a decision tree using “LA1c” thresholds depending on: (i) the retention time, (ii) the shape of the peak, (iii) other analytes, like urea. If the peak recognized as “LA1c” is mainly formed by LA1c, we consider that there is no interference until 4%. If the peak is mainly formed by cHb, we consider an interference threshold equal to 2%. Conclusions This situation reminds that cHb and LA1c remain critical issues in chromatography-based HbA1c assays and that adapted criteria must be set up for result interpretation.

[Interest of the study of a not identified peak on a hemoglobin chromatogram: delta variant or inter-sample contaminations?].

D-10(®) (Bio-Rad) analyzer using cationic exchange high performance chromatography (HPLC) allows the detection of the main hemoglobin variants. This observation shows the presence of a peak on chromatogram with a low intensity and no quantifiable which can lead to different diagnosis. Inter-sample contaminations can be confused with the presence of an hemoglobin variant. This case highlights the importance of the knowledge of technicals limits for validation and clinical use of results.