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.

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.

[Assays of HbA1c and Amadori products in human biology]. / Dosage de l'HbA1c et des produits d'Amadori en biologie humaine.

Different Amadori products, formed during the early steps of the non-enzymatic glycation of proteins, may be assayed in current practice in human biology. The most important marker is HbA1c, resulting from the binding of glucose to the N-terminal extremity of HbA beta chains. HbA1c may be evaluated by various techniques (ion exchange or affinity high performance liquid chromatography, capillary electrophoresis, immunoassay, enzymatic technique) and is considered the best marker of diabetic patient survey. Due to its irreversible and cumulative formation, it provides a retrospective information on the glycemic balance over the four to eight weeks preceding blood collection. It benefits from an international standardization, based on a reference method using liquid chromatography coupled to capillary electrophoresis or mass spectrometry, maintained by an international network of reference laboratories. When HbA1c assay cannot be used (anemia, hemolysis, hemoglobinopathy) or when a shorter period of glycemic equilibrium must be evaluated (child and adolescent, pregnancy, therapeutic changes), other Amadori products may be assayed, like plasma fructosamine (all plasma glycated proteins) or glycated albumin. Nevertheless, these assays are less used in practice, because their semiological value has been less evidenced. Besides, fructosamine assay lacks specificity, and glycated albumin assay has been described recently. An expanding use of HbA1c assay is expected, especially for the diagnosis of diabetes mellitus and the evaluation of other risks, especially cardiovascular ones.

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).

A young adult with sarcosinemia. No benefit from long duration treatment with memantine.

Sarcosinemia is a rare inborn error of metabolism that is characterised by an increased level of sarcosine (N-methylglycine) in the plasma and urine. The enzymatic block results from a deficiency of sarcosine dehydrogenase (SarDH), a liver mitochondrial matrix enzyme that converts sarcosine into glycine. Although this condition may remain inapparent until later life, it has been reported in rare cases to lead to neurodevelopmental disability. A 19-year-old male with sarcosinemia presented with dystonia, developmental delay and cognitive impairment. Magnetic resonance imaging revealed vermian hypotrophy. A 2-year pharmacological treatment with memantine was negative on the clinical signs. In this case, it was concluded that the metabolic block leading to sarcosinemia was responsible of a pathologic condition with mental deficiency and complex neurological signs. A maternal isodisomy discovered in the vicinity of SarDH gene could contribute to this pathology. Deficit of SarDH may be considered as a differential diagnosis of growth failure during prenatal stages and respiratory failure at birth following a slowly progressive developmental delay.

Evaluation of the In2it analyzer for HbA1c determination.

AIM: The prominent role of HbA(1c) in the follow-up of glycaemic balance in patients with diabetes mellitus necessitates the use of robust and reliable methods of assay. The purpose of this study was to evaluate the In2it analyzer, a new device allowing HbA(1c) evaluation within 10 min, using 10 microL of blood, in the laboratory or clinical unit, using an affinity-based method. METHODS: The analytical performance of the In2it analyzer was tested for precision, interference and linearity, and correlated with two other analyzers - the high-performance liquid chromatography (HPLC)-based Variant II analyzer in a laboratory, and the immunology-based DCA 2000 analyzer in a clinical unit - for practicability and its compliance with good laboratory practices. RESULTS: HbA(1c) assay is linear from 4 to 14%, with coefficients of variations ranging from 2.4 to 3.9%. In2it correlation was satisfactory with both the HPLC Variant II (r(2)=0.974, P<0.001) and DCA 2000 (r(2)=0.794, P<0.001) analyzers although, with the latter, unpredictable differences were randomly observed. However, the method is free of interference from common haemoglobin variants, labile glycated haemoglobin and carbamylated haemoglobin, hyperbilirubinaemia (<520 micromol/L) and hypertriglyceridaemia (<6 mmol/L). The practicability of the analyzer is good. However, software specifications need to be upgraded, especially for quality-control management, traceability of results and data safety. CONCLUSION: The In2it analyzer is suitable for HbA(1c) assay in small laboratory series and for point-of-care testing, and its analytical performance is satisfactory overall. However, several issues related to software need to be improved for optimal application. Also, special attention should be paid concerning the possibility of underestimation of results in cases of high hypertriglyceridaemia.

[Incidence of sample storage temperature on HbA 1c determination by high performance liquid chromatography method]. / Influence de la température de conservation sur le dosage d'HbA 1c par méthode de chromatographie liquide haute performance.

We have evaluated the conservation prior to HbA(1c) determination of three whole blood samples stored at -80 degrees C, -20 degrees C, 4 degrees C and 20 degrees C, for a maximal duration of one year. HbA(1c) was measured by an ion-exchange high performance liquid chromatography (HPLC) method (Variant II). We have analyzed the HbA(1c) value and the quality of the chromatographic separation for each sample. Storage of whole blood samples at -80 degrees C is good for at least one year. Storage at 4 degrees C is correct for two weeks, without major sample degradation. A more important and earlier degradation occurs at -20 degrees C. The conservation at 20 degrees C (room temperature) is very short. In conclusion, the temperatures of 4 and -80 degrees C are of interest for whole blood storage before HbA(1c) measurement, respectively for short and long term conservations. The temperatures of 20 and -20 degrees C are not recommended.

[Propositions for the standardized expression of HbA 1c results]. / Propositions pour l'expression standardisée des résultats d'HbA 1c.

The 2007 international consensus about the standardization of HbA(1c) determination and expression of results is progressively implemented in most countries. In France, a common working group of the Société française de biologie clinique (SFBC) and the Société francophone de diabétologie (SFD) has expressed the following recommendations. HbA(1c) results are expressed in percentage of total hemoglobin and in mmol HbA(1c)/mol Hb, but are not converted into estimated average glucose. A table indicating the correspondence between HbA(1c) and estimated average glucose may be given with the results, subject to precautions of interpretation at the individual level.

[Evaluation of prescription and interpretation of microalbuminuria by general practitioners]. / Evaluation de la prescription et de l'interprétation des dosages de microalbuminurie en médecine générale.

Microalbuminuria is well recognized as an independent marker of early renal failure in patients with diabetes mellitus and hypertension. We describe here the french results of an international study on the use and interpretation of microalbuminuria by general practitioners. A case history based questionnaire upon a type 2 diabetic patient was sent to 600 general practitioners in the Champagne-Ardenne Region to identify their habits in terms of prescription and of results interpretation. The analysis of the results shows a great variability of practices, regarding the procedures of urine collection, the units used, or the decision limits. These discrepancies can lead to inappropriate care of the patient. Even though national recommendations on the use of MA have been made, this study highlights the necessity for general practitioners to refer to concerted and consensual practices.

[Evaluation of Variant II analyzer equipped with the new 270-2101 NU kit (Bio-Rad) for HbA 1c assay]. / Evaluation de l'analyseur Variant II équipé de la nouvelle trousse 270-2101 NU (Bio-Rad) pour le dosage de l'HbA 1c.

HbA(1c) represents a key parameter in the follow-up of glycemic balance in diabetic patients. It may be assayed by different methods, among which high-pressure liquid chromatography (HPLC). We have evaluated a new method available on HPLC Variant II analyzer (BioRad) equipped with the new kit 270-2101 NU. Chromatographic separation is improved, allowing a better identification of peaks. Intra- and inter-assay coefficients of variation are respectively lower than 1.1% and 1.8%. Linearity is excellent from 3.2% to more than 18%. The correlation with the previous method (kit 270-2101) is good: y (% HbA(1c) new kit) = 0.944x (% HbA(1c) previous kit) + 0.299, r(2) = 0.995. There is no inter-sample contamination. This method is less sensitive to interferences frequently found in practice (labile glycated hemoglobin, carbamylated haemoglobin) than the previous one. Validation is possible in more circumstances when an abnormal hemoglobin is present (especially in case of hemoglobin D or E). As the control of analytic quality is a major element for validation and clinical use of HbA(1c) results, the characteristics of this new method make it a well-suited tool for daily laboratory practice.

[Interference of labile glycated hemoglobin on HbA(1c) assay by a high pressure liquid chromatography method]. / Interférence de l'hémoglobine glyquée labile sur le dosage de l'HbA(1c) par une méthode de chromatographie liquide haute performance.

HbA(1c) assay by high pressure liquid chromatography remains submitted to interferences, among which that of labile HbA(1c) in 1 to 2% of samples. We have evaluated the interference of labile HbA(1c) on HbA(1c) assay using Variant II analyzer (Biorad), by in vitro formation of labile glycated haemoglobin and by evaluation of two protocols of elimination of labile HbA(1c) (wash and incubation of red blood cells in saline solution, or incubation in the wash/dilution solution of the analyzer). Levels of labile HbA(1c) higher than 4.5 % lead to underestimation of HbA(1c). The different protocols tested proved efficient and were adapted to routine conditions. The fastest method is the incubation of red blood cells in the wash/dilution solution for at least two hours, or more if labile fraction is unusually high.

Type I collagen induces tissue factor expression and matrix metalloproteinase 9 production in human primary monocytes through a redox-sensitive pathway.

BACKGROUND: Tissue factor (TF), the main trigger of coagulation cascade, is a major component of the atherosclerotic plaque. Matrix metalloproteinases (MMPs) are recognized as key mediators of extracellular matrix remodeling during inflammation. It was recently emphasized that both TF and MMP-9 were overexpressed in atherosclerotic plaques, suggesting a role of both molecules in plaque instability and thrombogenicity. OBJECTIVE: The present study was designed to determine whether human monocytes could co-express TF and MMP-9 when the cells interact with type I collagen, a major component of the extracellular matrix and atherosclerotic plaque. METHODS: Human monocytes were isolated by elutriation and incubated in collagen I-coated plates. Tissue factor and MMP-9 expression were examined using real-time reverse transcription-polymerase chain reaction, flow cytometry, western blot and zymography. The activation of nuclear factor-kappa B (NF-kappaB) and the role of reactive oxygen species (ROS) in TF and MMP-9 production was studied using gel shift experiments, antioxidants pyrrolidine dithiocarbamate (PDTC) and N-acetyl-cysteine (NAC), and apocynin (a specific inhibitor of the NADPH oxidase). RESULTS: Type I collagen induced TF expression and increased MMP-9 production. In addition, the pro-inflammatory tumor necrosis factor-alpha (TNF-alpha), produced in response to collagen I, increased MMP-9 production. PDTC and NAC inhibited NF-kappaB activation during monocyte interaction with collagen I. Finally, both antioxidants and apocynin decreased the expression of TF, TNF-alpha, and MMP-9. CONCLUSIONS: These results indicate a new mechanism in the monocyte expression of TF and MMP-9 in response to collagen I involving a ROS-dependent pathway linked to the activation of the NADPH oxidase.

[Immunoquantification of serum lipoprotein(a) in healthy Ivorian subject: a comparative evaluation of two methods]. / Valeurs sériques de la lipoprotéine(a) chez l'Ivoirien présumé sain: étude comparée de deux méthodes de dosage immunochimiques.

We have determined the concentration of Lp(a) in an Ivory Coast population (n=102) using two immunochemical methods: Laurell's monodimensional electro-immunodiffusion (EID) and immunonephelometry (IN). Within-run and between-run precision was respectively 3.07% and 3.97% by IN and 1.52% and 4.48% by EID method. As regard the exactitude, the bias goals in two methods were 3.5% and 3.0% respectively with IN and EID. The two methods were correlated (r=0.84; p=0.006). Mean values of Lp(a) were significantly (p=0.0007) higher by IN than EID: 0.48+/-0.34 g/L versus 0.32+/-0.19 g/L. The Lp(a) distributions were non-Gaussian, skewed towards high values, with median value of 0.47 g/L and 0.32 g/L respectively for IN and EID methods. Therefore, we conclude that although both methods showed a satisfactory precision, and results were correlated, Lp(a) values were higher by INP. Furthermore, mean values of Lp(a) in presumed healthy Ivorian is higher than in Caucasians.

[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.