Glycated bovine serum albumin for use in feeding trials with animal models - In vitro methodology and characterization of a glycated substrate for modifying feed pellets.

This study investigated different methods to produce Nε-carboxymethyl-lysine (CML)-enriched bovine serum albumin (BSA) as alternatives to the classical approach using glyoxylic acid (GA) and sodium cyanoborohydride (NaBH3CN) which results in toxic hydrogen cyanide (HCN). The reaction of GA (6 mmol/L) and NaBH3CN (21 mmol/L) to produce CML remained the most effective with CML yields of 24-35%, followed by 13-24% using 300 mmol/L glyoxal (GO). GA promoted specific modification of lysine to CML, and fewer structural modifications of the BSA molecule compared with GO, as evidenced by fluorescence and proteomic analyses. GO promoted greater arginine modification compared with GA (76 vs 23%). Despite structural changes to BSA with GO, murine fecal clearance of CML was similar to literature values. Hence, BSA glycation with 300 mmol/L glyoxal is a suitable alternative to GA and NaBH3CN for generating CML-enriched protein free of HCN, but a CML-only fortification model remains to be described.

Serum carboxymethyllysine concentration is associated with erosive hand osteoarthritis.

OBJECTIVE: Carboxymethyllysine (CML) and homocitrulline (HCit) are the products of two non-enzymatic post-translational modifications of protein, a process related to age. We investigated whether serum CML and HCit concentrations were associated with hand osteoarthritis (HOA), especially erosive HOA. DESIGN: Serum CML and HCit were measured by using liquid chromatography coupled with tandem mass spectrometry at inclusion in 386 patients included in the DIGItal Cohort Design (DIGICOD) cohort. We investigated whether serum CML and/or HCit concentrations were associated with erosive HOA or with HOA clinical and radiological features. Moreover, we compared the tissular concentrations of CML and HCit in OA and non-OA cartilage from proximal interphalangeal and metacarpo-phalangeal (MCP) joints from human cadaveric donors. RESULTS: Median (IQR) serum CML concentration was lower in patients with erosive HOA than those with non-erosive HOA (178.7 [157.1-208.8] vs 194.7 [168.9-217.1] µmol/mol Lys, P = 0.002), but median HCit concentration did not differ between the groups (193.9 [162.9-232.0] vs 193.9 [155.9-224.6] µmol/mol Lys). Cartilage HCit and CML concentrations were not correlated with clinical features. Serum CML concentration was higher in OA than non-OA MCPs (7.0 vs 4.0 mmol/mol Lys, P = 0.01). CONCLUSIONS: Serum CML concentration was lower in erosive HOA than non-erosive HOA, and cartilage CML concentration was higher in OA than non-OA cartilage. These results encourage further studies to test whether serum CML could be a new prognostic biomarker in HOA.

[Involvement of inorganic phosphate starvation in Pseudomonas aeruginosa bacterial virulence]. / Implication de la carence en phosphates inorganiques dans la virulence bactérienne de Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a bacterium causing a wide spectrum of nosocomial and opportunistic respiratory infections. As an element essential for bacterial metabolism , phosphorus is incorporated as an inorganic phosphate and regulated by a two-component PhoB-PhoR system. Recently, it has been shown that as a result of overexpression of virulence factors, including the PhoB transcription factor, P. aeruginosa exhibited increased virulence in phosphate-deficient conditions. Exploration of the relationship between phosphate homeostasis and P. aeruginosa virulence could effectively contribute to the development of new, simple and innovative therapeutic strategies.

Carbamylation is a competitor of glycation for protein modification in vivo.

AIM: Chronic kidney disease (CKD) and diabetes mellitus are two diseases that accelerate protein molecular ageing through carbamylation and glycation reactions, characterized by the binding of urea-derived isocyanic acid and of sugars on proteins, respectively. These two reactions target the same protein amino groups and, thus, compete with each other. Such competition may arise especially in diabetic patients with nephropathy. This study aimed to evaluate their potential competitive effects in vitro and under conditions reproducing CKD and/or diabetes in vivo. METHODS: Albumin was incubated in vitro with glucose, urea or cyanate. Carbamylation in vivo was enhanced in normal and diabetic (db/db) mice by either subtotal nephrectomy or cyanate consumption. Homocitrulline, carbamylated haemoglobin and furosine were measured by LC-MS/MS, fructosamine by colorimetric assay and HbA1c by immunological assay. RESULTS: Reciprocal inhibition between carbamylation and glycation was observed during albumin incubations in vitro. Besides, 5 weeks after induction of CKD in vivo, plasma homocitrulline concentrations were similar in both diabetic and non-diabetic mice, whereas fructosamine and HbA1c were decreased (-23% and -42%, respectively) in diabetic mice with CKD compared with only diabetic ones. Fructosamine and HbA1c were also decreased in cyanate-spiked water-drinking mice compared with plain water-drinking diabetic mice. CONCLUSION: Carbamylation competes with glycation in vivo, especially under conditions of high glycation. Thus, the classic markers of glycaemic control should be interpreted with caution in diabetic patients with CKD because of this competitive effect.

Association of peripheral neuropathy with circulating advanced glycation end products, soluble receptor for advanced glycation end products and other risk factors in patients with type 2 diabetes.

BACKGROUND: The pathogenesis of diabetic peripheral neuropathy remains uncertain and nonenzymatic glycoxidation is one of the contributing mechanisms. The aim of this study was to assess the respective relationship of diabetic peripheral neuropathy with glycoxidation, compared with other identified risk factors, in patients with type 2 diabetes. METHODS: We included 198 patients with type 2 diabetes and high risk for vascular complications. Circulating concentrations of three advanced glycation end products (carboxymethyllysine, methyl-glyoxal-hydroimidazolone-1, pentosidine) and of their soluble receptor (sRAGE) were measured. Peripheral neuropathy was assessed by the neuropathy disability score and by the monofilament test and defined as either an abnormal monofilament test and/or a neuropathy disability score ≥6. Multivariate regression analyses were performed adjusting for potential confounding factors for neuropathy: age, gender, diabetes duration, current smoking, systolic blood pressure, waist circumference, height, peripheral arterial occlusive disease, glycated haemoglobin, estimated glomerular filtration rate and lipid profile. RESULTS: Prevalence of peripheral neuropathy was 20.7%. sRAGE and carboxymethyllysine were independently and positively associated with the presence of peripheral neuropathy. No significant association was found between peripheral neuropathy and methyl-glyoxal-hydroimidazolone-1 or pentosidine. Waist circumference, height and peripheral arterial occlusive disease were independently associated with peripheral neuropathy. CONCLUSIONS: Carboxymethyllysine and sRAGE were independently associated with peripheral neuropathy in patients with type 2 diabetes. Although the conclusions are limited by the absence of a healthy control population, this study confirms the relationship between advanced glycoxidation and diabetic peripheral neuropathy, independently of other risk factors.

Interference of the most frequent haemoglobin variants on quantification of HbA1c: comparison between the LC-MS (IFCC reference method) and three routinely used methods.

AIM: Assaying HbA1c in patients with haemoglobin variants has long been a technical challenge, despite methodological advances that have progressively limited the problem. The purpose of this study was to evaluate the impact of the most frequent haemoglobin variants on three routine separation methods compared with the IFCC reference method. PATIENTS: Blood samples from heterozygous patients (AS, AC, AD, AE) were analyzed using the IFCC reference method (LC-MS), and the results compared with those obtained by capillary electrophoresis (CAPILLARYS 2 Flex Piercing, Sebia) and two HPLC methods using cation-exchange (Variant II, Bio-Rad) and affinity chromatography (Ultra(2), Primus). RESULTS: HbA1c values obtained by the IFCC reference method were comparable to those obtained by the three tested methods whatever the haemoglobin variant. Mean relative biases did not exceed the threshold of 7% (above which differences are generally considered clinically significant), although some individual values were above this limit with Variant II in samples with HbS and for all three methods in samples with HbE. CONCLUSION: This comparative study of the LC-MS reference method and three field methods has demonstrated that these assays are not clinically influenced by the presence of the most common haemoglobin variants. The present results also confirm that the interpretation of HbA1c values in patients with Hb variants remains complex and depends on the assays used and should, in some cases, take into account parameters other than analytical ones (such as differences in glycation rates and half-lives of haemoglobin variants).

[Comparison of two osmometers at the clinical chemistry laboratory]. / Evaluation de deux osmomètres au laboratoire de biologie clinique.

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.