Search

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.

Background: HbA1c is a key biomarker for the monitoring of glycemic balance in diabetic patients. It may be measured by various methods, including HPLC and immunoassays. Here we report the results of the first evaluation of Capillarys 2 Flex Piercing®, a new analyzer using capillary electrophoresis for the separation and the quantification of HbA1c., Methods: We have evaluated the analytical performances of the method as well as the influence of the most frequent analytical interferences regarding HbA1c assays (i.e., labile HbA1c, carbamylated hemoglobin, high HbF and hemoglobin variants)., Results: Intra-assay and between-assays CVs were respectively lower than 1.98% and 2.68% on the pre-market version of the instrument, and lower than 1.62% and 1.45% on the commercial version. The linearity was excellent for HbA1c values ranging from 19 mmol/mol (3.9%) to 161 mmol/mol (16.9%) (r=0.999). The results were well correlated with those obtained by the HPLC method routinely used in the laboratory (Variant II® NU Kit - Bio-Rad): HbA1c[Capillarys 2]=0.9452×HbA1c[Variant II]+1.7279 (r=0.994, n=500). The use of titrated quality control samples indicated a good accuracy of the method. Neither the presence of HbF (until 15%), labile HbA1c or carbamylated hemoglobin, nor that of some typical hemoglobin variants, such as hemoglobin S, D, C and E, affected HbA1c measurement., Conclusions: This evaluation showed that the analytical performances of Capillarys 2 Flex Piercing® analyzer for HbA1c assay fulfilled quality criteria requested for clinical use, and allowed to recommend its implementation in clinical chemistry laboratories for routine practice.

Homocitrulline (HCit), an amino acid formed by the carbamylation of ε-amino groups of lysine residues, is considered a promising biomarker for monitoring diseases such as chronic renal failure and atherosclerosis. This paper describes a tandem mass spectrometric method for total, protein-bound and free HCit measurement in plasma samples. HCit was separated from other plasma components by hydrophilic interaction liquid chromatography. Detection was achieved by monitoring transitions of 190.1 > 127.1 and 190.1 > 173.1 for HCit, and 183.1 > 120.2 for d(7)-citrulline used as internal standard. This method allowed HCit quantification within 5.2 min and was precise (inter-assay CV < 5.85%), accurate (mean recoveries ranging from 97% to 106%), and exhibited a good linearity from 10 nmol/L to 1.6 μmol/L. Plasma samples from control and uremic mice (n = 10) were analyzed. In control mice, mean total plasma HCit concentration was 0.78 ± 0.12 μmol/mol amino acids, whereas it was increased 2.7-fold in uremic mice plasma, reaching 2.10 ± 0.50 μmol/mol amino acids (p < 0.001). In conclusion, this method exhibits good analytical performances and meets the criteria of sensitivity suitable for HCit concentration assessment in plasma samples.

Background: Carbamylation is a posttranslational modification of proteins resulting from the nonenzymatic reaction between isocyanic acid and specific free functional groups. This reaction alters protein structural and functional properties and thus contributes to molecular ageing. Many studies have shown the involvement of carbamylated proteins in diseases, especially in chronic renal failure and atherosclerosis., Content: In this review we describe the biochemical basis of the carbamylation process and its role in protein molecular ageing. We summarize the current evidence of protein carbamylation involvement in disease, identify available biomarkers of the carbamylation process and their related analytical methods, and discuss the practical relevance of these biomarkers., Summary: Carbamylation-induced protein alterations are involved in the progression of various diseases, because carbamylation-derived products (CDPs) are bioactive compounds that trigger specific and inappropriate cellular responses. For instance, carbamylation may promote hormone and enzyme inactivation, and carbamylated proteins, as diverse as collagen or LDLs, induce characteristic biochemical events of atherosclerosis progression. CDPs are potential biomarkers to monitor diseases characterized by an increased rate of carbamylation (e.g., chronic renal failure and atherosclerosis). Different methods (e.g., liquid chromatography-tandem mass spectrometry and immunoassays) to measure specific carbamylated proteins or general markers of carbamylation, such as protein-bound homocitrulline, have been described. Their use in clinical practice must still be validated by appropriate clinical studies.

The evaluation of glycated hemoglobin (HbA(1c)) is used in daily practice for monitoring the diabetes mellitus. HbA(1c) may be assayed by different methods, among which high-pressure liquid chromatography (HPLC). We have evaluated a new kit 2.0 available on HPLC Variant II(®) Turbo analyzer (Bio-Rad), which is characterized by an elution buffer containing sodium perchlorate. The correlation with the routine method used in our laboratory (HPLC Variant II(®) analyzer equipped with the NU kit) is good (r² = 0.997). Intra- and inter-assay coefficients of variation are respectively lower than 0.6 and 1.6%. Linearity is excellent from 3.2% (11 mmol/mol) to 18.3% (177  mmol/mol). There is no inter-sample contamination. This method provides a result quickly (96  seconds), but does not allow separating labile HbA(1c) from carbamylated hemoglobin, contrary to the NU kit. As the control of analytical quality is a major concern for validation and clinical use of HbA(1c) results, the characteristics of this new kit make it a well-suited tool for daily laboratory practice.

HbA(1c) represents a key parameter in the monitoring of glycemic balance in diabetic patients. It may be assayed by various methods, among which high-pressure liquid chromatography (HPLC). We have evaluated a new HPLC analyzer, Glycomat 3000 (Biocode Hycel). HbA(1c) values are well correlated (r(2) = 0.994) with those obtained by the HPLC analyzer used routinely in our laboratory (Variant II, Bio-Rad). However, the precision of assays can be at the limit of acceptability (CV of repeatability between 0.7 and 1.4% and CV of reproducibility between 3.3 and 4.1%) if strict conditions of calibration and quality control are not implemented. The assay is sensitive to the interference of modified fractions of hemoglobin (labile HbA(1c) and carbamylated Hb) in the clinical range, and to the presence of HbF. Besides, Glycomat 3000 is a friendly and easy to use analyzer.

HbA(1c) assay by high-pressure liquid chromatography (HPLC) remains submitted to various analytical interferences. We have evaluated the behaviour of the analyzers G8 (Tosoh Bioscience) and Variant II (BioRad) towards the most common interferences (i.e. labile fraction of HbA(1c), carbamylated hemoglobin, bilirubin, triglycerides, hemoglobin variants). This comparative study showed that the influence of these interferences varied according to the analyzer and depended on various settings such as the chromatographic resolution and the peak integration mode. However, this influence remains limited when strict and analyzer-specific rules of technical validation have been previously determined. The outcome of the study underlines the importance of a detailed interpretation of chromatograms during the biological validation process, and recommends specific procedures in case of interferences, in order to ensure the reliability of HbA(1c) results.

Background: During their biological life, proteins are exposed in a cumulative fashion to irreversible nonenzymatic, late posttranslational modifications that are responsible for their molecular aging. It is now well established that these damaged proteins constitute a molecular substratum for many dysfunctions described in metabolic and age-related diseases, such as diabetes mellitus, renal insufficiency, atherosclerosis, or neurodegenerative diseases. Accordingly, the specific end products derived from these reactions are considered potentially useful biomarkers for these diseases., Content: The aim of this review is to give an overview of nonenzymatic posttranslational modifications of proteins and their influence in vivo, take inventory of the analytical methods available for the measurement of posttranslational modification-derived products, and assess the potential contribution of new technologies for their clinical use as biological markers of protein molecular aging., Summary: Despite their clinical relevance, biomarkers of posttranslational modifications of proteins have been studied only in the context of experimental clinical research, owing to the analytical complexity of their measurement. The recent implementation in clinical chemistry laboratories of mass spectrometry-based methods that provide higher specificity and sensitivity has facilitated the measurement of these compounds. These markers are not used currently by clinicians in routine practice, however, and many challenges, such as standardization, have to be confronted before these markers can be used as efficient tools in the detection and monitoring of long-term complications of metabolic and age-related diseases.

The methods of HbA1c assay using ion exchange high pressure liquid chromatography (HPLC) allow the detection of the most common hemoglobin variants. This observation highlights the different behaviour of two HPLC analyzers in the presence of Tatras hemoglobin. By one of the analyzer (Variant II, Bio-Rad) this variant is detected, but not by the other (G8, Tosoh Biosciences). As HbA1c result is crucial for the therapeutic decision, it is important that biologists know the characteristics of the method they use, in order to detect the possible occurence of an hemoglobinopathy and to ensure the best interpretation of the result.

Our purpose was to identify the sequence of omega-amidase, which hydrolyses the amide group of alpha-ketoglutaramate, a product formed by glutamine transaminases. In the Bacillus subtilis genome, the gene encoding a glutamine transaminase (mtnV) is flanked by a gene encoding a putative 'carbon-nitrogen hydrolase'. The closest mammalian homolog of this putative bacterial omega-amidase is 'nitrilase 2', whose size and amino acid composition were in good agreement with those reported for purified rat liver omega-amidase. Mouse nitrilase 2 was expressed in Escherichia coli, purified and shown to catalyse the hydrolysis of alpha-ketoglutaramate and other known substrates of omega-amidase. No such activity was observed with mouse nitrilase 1. We conclude that mammalian nitrilase 2 is omega-amidase.

Oxidative functions of polymorphonuclear neutrophils (PMNs), which play a deciding role in the phagocytosis process, are stimulated by extracellular matrix proteins such as type I collagen. Previous studies have demonstrated the involvement of a DGGRYY sequence located within the alpha(1) chain C-terminal telopeptide in type I collagen-induced PMN activation, but so far the mechanism has not been completely elucidated. We have recently demonstrated that collagen carbamylation (i.e. post-translational binding of cyanate to lysine epsilon-NH(2) groups) impairs PMN oxidative functions, suggesting the potential involvement of lysine residues in this process. The present study was devoted to the identification of lysine residues involved in the collagen-induced activation of PMNs. The inhibition of PMN activation by collagen in the presence of 6-amino-hexanoic acid, a structural analogue of lysine residues, confirmed the involvement of specific lysine residues. Modification of lysine residues by carbamylation demonstrated that only one residue, located within the alpha(1)CB6 collagen peptide, was involved in this mechanism. A recombinant alpha(1)CB6 peptide, designed for the substitution of lysine 1047 by glycine, exhibited decreased activity, demonstrating that the lysine residue at position 1047 within the collagen molecule played a significant role in the mechanism of activation. These results help to understand in more detail the collagen-mediated PMN activation mechanism and confirm the prominent involvement of lysine residues in interactions between extracellular matrix proteins and inflammatory cells.

Measurement of osmolality and calculation of osmolar gap are useful diagnostic tools in pathological situations such as hyponatremia, or intoxication by methanol or ethylene glycol. It is thus necessary to handle reliable systems of osmolality measurement. The aim of this study was to compare performances of two currently available osmometers, Fiske 210 and Advanced 3300 devices, both of them being distributed by Radiometer S.A.S. society, in order to determine the best criteria for purchase. This study showed very good performances of repeatability and reproductibility for both analyzers (CV< 2.1%) and a good correlation of results between them and with the osmometer routinely used in the laboratory. Other criteria such as a more suitable praticability for our needs and a better quality/price ratio orientated our choice towards Fiske 210 osmometer.

Carbamylation is a post-translational modification of proteins characterized by the binding of cyanate to amino groups, increased in renal failure. Pathophysiological consequences of carbamylation and adverse effects of carbamylated proteins on cell functions are poorly understood. We studied the influence of carbamylated albumin on polymorphonuclear neutrophil (PMN) O(2)(-) production. Carbamylated albumin significantly decreased O(2)(-) production in PMNs stimulated by type I collagen, but not by phorbol 12-myristate 13-acetate or tumor necrosis factor-alpha. This effect was related to inhibition of p(125)FAK phosphorylation. Such an alteration of neutrophil oxidative functions might explain characteristic complications of renal failure, such as increased occurrence of inflammation or infections.

Carbamylation is a post-translational modification due to nonenzymatic binding of cyanate, a by-product of urea, on free amino groups of proteins. Post-translational modifications are known to induce alterations in structural and functional properties of proteins, thus disturbing protein-protein or cell-protein interactions. We report the impact of carbamylation on type I collagen sensitivity to enzymatic proteolysis. Type I collagen was extracted from rat tail tendons and carbamylated by incubation with 0.1 M potassium cyanate at 37 degrees C for 2, 6 or 24 h. Degradation assays revealed that carbamylated collagen exhibited a greater resistance to collagenases (i.e. bacterial collagenase, matrix metalloproteinase(MMP)-1, MMP-8 and MMP-13), together with an increased sensitivity to MMP-2. Evaluation of collagen triple helix conformation by polarimetry indicated that local destabilizations of triple helix structure related to carbamylation could be responsible for the observed differences in sensitivity. These results confirm the crucial role of triple helix integrity in the degradation of type I collagen by MMPs, and support the deleterious impact of post-translational modifications in vivo by altering the balanced remodeling of collagen within connective tissue.

Carbamylation by urea-derived cyanate is a posttranslational modification of proteins increasing during chronic renal insufficiency, which alters structural and functional properties of proteins and modifies their interactions with cells. We report here the major structural alterations of type I collagen induced by carbamylation. Biophysical methods revealed that carbamylated collagen retained its triple-helical structure, but that slight changes destabilized some regions within the triple helix and decreased its ability to polymerize into normal fibrils. These changes were associated with the incapacity of carbamylated collagen to stimulate polymorphonuclear neutrophil oxidative functions. This process involved their interaction with LFA-1 integrin, but no subsequent p(125)FAK phosphorylation. Carbamylation of collagen might alter interactions between collagen and inflammatory cells in vivo and interfere with the normal remodeling of extracellular matrix, thus participating in the pathophysiological processes occurring during renal insufficiency.

Carbamylation refers to chemical modification of protein side chains by cyanate derived e.g. from urea. It alters their structural and functional properties. We have studied the influence of the carbamylation of type I collagen in vitro on its interactions with elutriated human monocytes, and its potential role in atherosclerosis. Adhesion of monocytes onto carbamylated collagen was significantly enhanced compared to native collagen. There was no change in superoxide anion production. On the other hand, there was an increase in the production and the activation of matrix metalloproteinase-9. No effect was found on tissue inhibitor of metalloproteinase-1 production. Thus, the presence of carbamylated collagen may stimulate the remodelling of extracellular matrix mediated by activated monocytes. Such alterations may contribute to enhanced atherosclerosis in renal insufficiency, a pathological condition associated with elevated levels of carbamylation.

Background: The dilatation and tortuosity observed in varicose veins provide evidence for progressive venous wall remodeling associated with abnormalities of smooth muscle cells and extracellular matrix. The present study was designed to examine if the phenotypic modulations observed in the venous smooth muscle cells of patients with varicose veins were also present in their dermal fibroblasts., Methods and Results: Collagen type I (collagen I), type III (collagen III), and type V (collagen V) were compared in dermal fibroblasts derived from the skin of control subjects and patients with varicose veins. The synthesis of collagen I, the release of its metabolites, and the expression of its mRNA were increased in fibroblasts from patients with varicose veins, whereas the synthesis of collagen III was decreased but not correlated with a decrease in mRNA expression and in metabolite release. Matrix metalloproteinases (MMP1, 2, 7, 8, 9, and 13) and their inhibitors (TIMP1 and 2) were quantified in both cell types; only the production of proMMP2 was increased in cells derived from patients with varicose veins., Conclusions: These findings suggest that the synthesis of collagen I and III is dysregulated in dermal fibroblasts derived from patients with varicose veins. These results are comparable with those observed in smooth muscle cells derived from varicose veins, thus suggesting a systemic alteration of tissue remodeling in subjects with varicose veins.