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.

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.

[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.

Early- and life-long intake of dietary advanced glycation end-products (dAGEs) leads to transient tissue accumulation, increased gut sensitivity to inflammation, and slight changes in gut microbial diversity, without causing overt disease.

Dietary advanced glycation end-products (dAGEs) accumulate in organs and are thought to initiate chronic low-grade inflammation (CLGI), induce glycoxidative stress, drive immunosenescence, and influence gut microbiota. Part of the toxicological interest in glycation products such as dietary carboxymethyl-lysine (dCML) relies on their interaction with receptor for advanced glycation end-products (RAGE). It remains uncertain whether early or lifelong exposure to dAGEs contributes physiological changes and whether such effects are reversible or permanent. Our objective was to examine the physiological changes in Wild-Type (WT) and RAGE KO mice that were fed either a standard diet (STD - 20.8 ± 5.1 µg dCML/g) or a diet enriched with dCML (255.2 ± 44.5 µg dCML/g) from the perinatal period for up to 70 weeks. Additionally, an early age (6 weeks) diet switch (dCML→STD) was explored to determine whether potential harmful effects of dCML could be reversed. Previous dCML accumulation patterns described by our group were confirmed here, with significant RAGE-independent accumulation of dCML in kidneys, ileum and colon over the 70-week dietary intervention (respectively 3-fold, 17-fold and 20-fold increases compared with controls). Diet switching returned tissue dCML concentrations to their baseline levels. The dCML-enriched diet had no significative effect on endogenous glycation, inflammation, oxidative stress or senescence parameters. The relative expression of TNFα, VCAM1, IL6, and P16 genes were all upregulated (∼2-fold) in an age-dependent manner, most notably in the kidneys of WT animals. RAGE knockout seemed protective in this regard, diminishing age-related renal expression of TNFα. Significant increases in TNFα expression were detectable in the intestinal tract of the Switch group (∼2-fold), suggesting a higher sensitivity to inflammation perhaps related to the timing of the diet change. Minor fluctuations were observed at family level within the caecal microbiota, including Eggerthellaceae, Anaerovoracaceae and Marinifilaceae communities, indicating slight changes in composition. Despite chronic dCML consumption resulting in higher free CML levels in tissues, there were no substantial increases in parameters related to inflammageing. Age was a more important factor in inflammation status, notably in the kidneys, while the early-life dietary switch may have influenced intestinal susceptibility to inflammation. This study affirms the therapeutic potential of RAGE modulation and corroborates evidence for the disruptive effect of dietary changes occurring too early in life. Future research should prioritize the potential influence of dAGEs on disease aetiology and development, notably any exacerbating effects they may have upon existing health conditions.

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.

[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.

[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.

[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.

[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.

[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.

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.

In vivo stimulation of connective tissue accumulation by the tripeptide-copper complex glycyl-L-histidyl-L-lysine-Cu2+ in rat experimental wounds.

The tripeptide-copper complex glycyl-L-histidyl-L-lysine-Cu2+ (GHK-Cu) was first described as a growth factor for differentiated cells. Recent in vitro data showed that it possesses several properties of a potential activator of wound repair. We investigated the effects of GHK-Cu in vivo, using the wound chamber model described previously (Schilling, J.A., W. Joel, and M.T. Shurley, 1959. Surgery [St. Louis]. 46:702-710). Stainless steel wire mesh cylinders were implanted subcutaneously on the back of rats. The animals were divided into groups that received sequential injections into the wound chamber of either saline (control group) or various concentrations of GHK-Cu. At the end of the experiments, rats were killed, wound chambers were collected, and their content was analyzed for dry weight, total proteins, collagen, DNA, elastin, glycosaminoglycans, and specific mRNAs for collagens and TGF beta. In the GHK-Cu-injected wound chambers, a concentration-dependent increase of dry weight, DNA, total protein, collagen, and glycosaminoglycan contents was found. The stimulation of collagen synthesis was twice that of noncollagen proteins. Type I and type III collagen mRNAs were increased but not TGF beta mRNAs. An increase of the relative amount of dermatan sulfate was also found. A control tripeptide, L-glutamyl-L-histidyl-L-proline, had no significant effect. These results demonstrate that GHK-Cu is able to increase extracellular matrix accumulation in wounds in vivo.

[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.

[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.

[Oxidative stress and protein glycation in diabetes mellitus]. / Stress oxydant et glycation des protéines au cours du diabète sucré.

Metabolic alterations of diabetes mellitus are not only informative biological signs, but also factors of degenerative complications. Thus, hyperglycaemia increases non enzymatic glycation, characterized by the binding of simple oses (glucose) or by-products to amino groups of proteins. This reaction leads to the formation of complex compounds, advanced glycation end products (AGEs), which alter structure and functions of proteins. Glycation and oxidative stress are closely linked, and both phenomena are referred to as "glycoxidation". All steps of glycoxidation generate oxygen free radical production, some of them being common with lipidic peroxidation pathways. Besides, glycated proteins activate membrane receptors such as RAGE through AGEs, and induce an intracellular oxidative stress and a pro-inflammatory status. So, glycated proteins may modulate functions of cells involved in oxidative metabolism and induce inappropriate responses. Finally, some oxidative products (reactive aldehydes such as methylglyoxal) or lipid peroxidation products (malondialdehyde) may bind to proteins and amplify glycoxidation-generated lesions. The knowledge of glycoxidation mechanisms may lead to new therapeutic approaches.

A specific sequence of the noncollagenous domain of the alpha3(IV) chain of type IV collagen inhibits expression and activation of matrix metalloproteinases by tumor cells.

The invasive properties of melanoma cells correlate with the expression of matrix metalloproteinases (MMPs) and their physiological modulators (tissue inhibitors of metalloproteinase and membrane-type MMPs) and with that of the alphaVbeta3 integrin. We investigated the effect of anterior lens capsule type IV collagen and of the alpha3(IV) collagen chain on the invasive properties of various tumor cell lines (HT-144 melanoma cells, HT-1080 fibrosarcoma cells). We demonstrated that anterior lens capsule type IV collagen or specifically the synthetic peptide alpha3(IV) 185-203 inhibited both the migration of melanoma or fibrosarcoma cells as well as the activation of membrane-bound MMP-2 by decreasing the expressions of MT1-MMP and the beta3 integrin subunit.

A glucose-containing fraction extracted from the young erythrocyte membrane is capable of transferring glucose to hemoglobin in vitro.

Red blood cell (RBC) membranes are rich in a glycoconjugate that is extractable in chloroform/methanol solutions (2/1, v/v) and contains several hexoses, such as glucose. Old and young RBC are separated and their respective glycoconjugates are prepared. HbA0 is purified by column chromatography and incubated with solutions of this conjugate. After 24-h incubation, Hb is dialyzed and the amount of glycosylated Hb is measured by a method of column chromatography adapted from Trivelli. A very significant amount of HBAlc is formed when young RBC extracts are incubated: 3.6% of total Hb becomes HBAlc with the extracts, versus 3.2% with free glucose, and only 2.5% for controls. No increase in HbAlc is obtained when extracts of old RBC are incubated. Another difference between the action of the glycoconjugate and free glucose is that the former induces the increase of only the HBAlc fraction, whereas glucose induces the increase of all the minor Hb fractions. The evaluation of glucose contained in the conjugate before and after the glycosylation reaction demonstrates that it is due to an exchange of glucose units from the conjugate to Hb. The reaction is stereospecifically inhibited by p-nitrophenyl-beta-D-glucoside. The nature of the formed HbAlc is demonstrated by isoelectric focusing. A slight increase of HbAlc observed in the incubated controls may be due to an internal migration of some residues of glucose primitively bound to lysyl residues in an unstable form and also to some degree of denaturation during the incubation.

[Multicenter evaluation of four homogenous LDL-cholesterol assays]. / Evaluation multicentrique de quatre méthodes de dosage direct du cholestérol-LDL.

International guidelines emphasize the importance of LDL cholesterol (LDL-C) assay in the care and follow-up of patients with cardiovascular risk. Most studies and common practice use Friedewald's formula for LDL-C calculation. The accuracy of the result depends closely on the precision of the input parameters (total cholesterol, triglycerides (TG) and HDL cholesterol), and discrepancies between calculated LDL-C and measurement by reference methods appear when TG exceed 4.5 mmol/L, or in the presence of abnormal lipoproteins. These restrictions and uncertainties in calculations have prompted the recent development of direct and homogeneous methods that fit all analyzers. A multicenter evaluation of four direct assays of LDL-C (Daiichi, Denka Seiken, Kyowa, Wako) was carried out on 45 serum samples (TG below 3.1 mmol/L) in eight laboratories using different analyzers. For three methods (Daiichi, Kyowa, Wako), the interlaboratory reproducibility was markedly improved relative to that of calculation. A strong correlation was found for all new methods when compared with a beta-quantification assay. Average bias in Denka Seiken assays was greater than Kyowa's and Daiichi's (although less dispersed for the latter) and for Wako all bias were positive. The relationship between bias variations and the lipid parameters of the samples was studied. Three methods, Daiichi, Kyowa and Wako, revealed a significant positive correlation between bias and serum VLDL-C/TG ratio, clearly indicating that cholesterol enrichment of VLDL was a source of variability in these assays. Specificity of the four methods was tested in situation of dyslipidemia by spiking isolated lipoproteins (chylomicrons, VLDL and HDL). This experiment revealed differences in behavior, most evidently upon addition of VLDL. No method was truly specific, but up to 8 mmol/L of TG the variations were acceptable. In the presence of type III hyperlipoproteinemia, however, only the Denka Seiken method was reliable. Linearity up to 20 mmol/L (Daiichi, Denka Seiken) or 14 mmol/L (Kyowa, Wako) of LDL-C allows these tests to be used in main routine cases. New direct assays are an obvious technological advance in terms of analytical performance and conveniency. Their use for the diagnosis and follow-up of hyperlipidemic patients offers an alternative that overcomes the limitations of the Friedewald calculation.