Prevalent hypovitaminosis D in Crohn's disease correlates highly with mediators of osteoimmunology.

PURPOSE: In the context of osteoimmunology in Crohn's disease, an association was hypothesized among vitamin D and members of the TNF-α family, known as the RANK (receptor-activator of nuclear factor- κB)-RANK ligand-osteoprotegerin pathway. METHODS: This was a cross-sectional study of 95 patients with Crohn's disease (80 with long-standing disease and 15 newly diagnosed, never treated) and two control groups (healthy volunteers, n=30; and ulcerative colitis patients, n=30). Spine and hip bone mineral density was measured by dual-energy x-ray absorptiometry. Serum 25-hydroxyvitamin-D3, TNF-α, IL-6, sRANKL, osteoprotegerin levels and biochemical markers of bone turnover were analyzed. RESULTS: The precursor metabolite, 25(OH)D3, was measured in 95 young adult CD patients (47 men, 48 women; median age 30 years). A suboptimal 25(OH)D3 level was observed in 90% of CD patients, of whom 40% had a serious deficiency. There was no significant difference in 25(OH)D3 levels between CD patients and those with ulcerative colitis. Analysis revealed an association between 25(OH)D3 deficiency and the increased biogenesis of osteoclastically-active sRANKL (p=0.014) and the proinflammatory cytokines TNF-α (p=0.015) and IL-6 (p=0.029) . CD patients with low bone mineral density had a mean 25(OH)D3 (35±18 nmol/l) in the range of serious deficiency to insufficiency, whereas mean 25(OH)D3 was higher (49±28 nmol/l) in patients with healthy bone status, although levels were still inadequate (p=0.004). The logistic model reported low levels of 25(OH)D3 to be a significant predictor of bone disease [odds ratio=2.66(6.8), p < 0.009]. In the multivariable analysis, adjusted for several confounding factors, 25(OH)D3, sRANKL, IL-6 and TNF-α were independently associated with a likelihood of bone disease [odds ratio (range): 1.02(2.75); 1.09(3.71); 1.27(6.95) respectively, p=0.001]. CONCLUSION: The presented findings suggest that a 25(OH)D3 deficiency accompanying an inflammatory state in CD is a high risk condition for metabolic bone disease.

Relationship of methylglyoxal-adduct biogenesis to LDL and triglyceride levels in diabetics.

AIMS: Protein glycation leading to advanced glycation-endproducts (AGE) is enhanced in diabetes by increased blood glucose and collateral endogenous production of reactive α-dicarbonyls. Among AGE precursors, methylglyoxal (MG) is considered as one of the key intermediates. We hypothesized it to be a common product of both carbonyl and oxidative stress, and investigated its biogenesis in relation to glycemic and lipid status in diabetic patients. METHODS: Serum and urine MG-adducts were measured by competitive immunofluorometric assay in 83 diabetic and 20 healthy subjects. KEY FINDINGS: A significant association of MG-adducts serum level with LDL (r=0.31;p=0.003) was observed. A correlation between LDL-c, HDL-C and PPG as independent variables and serum MG-adducts as a dependent variable was found (p<0.014) using multiple stepwise regression, whereas urine albumin/creatinine ratio was independently associated with urine MG-adducts. LDL cut-off >3.0mmol/l discriminated patients with higher serum MG-adducts (p=0.0052), although there was no between-subgroup difference in glycemic control. Patients on statin therapy had a lower MG-adduct level. The positive relationship between LDL-c and MG-adducts (r=0.38;p=0.042) was noted in patients free of statin treatment, whereas an inverse tendency was found in the statin-treated subgroup. SIGNIFICANCE: Significant relationship between LDL and MG-adduct production, as well as tight correlation between triglycerides and urinary MG-adduct excretion suggest that the lipoxidation and glyceraldehyde-3-phosphate route, along with the glycolytic pathway, might be an important source of MG generation. The glycotoxin methylglyoxal seems to be a common factor linking hyperglycemia and intensive lipolysis, two dominant metabolic changes in diabetes.

Proinflammatory cytokines and receptor activator of nuclear factor kappaB-ligand/osteoprotegerin associated with bone deterioration in patients with Crohn's disease.

OBJECTIVES: The high incidence of bone disease and the increasing evidence of Crohn's disease (CD) bone decline in corticosteroid users and nonusers suggest that bone metabolism is affected by inflammatory process. The aim of the study was to compare serum levels of proinflammatory cytokines, markers of bone turnover and regulatory molecules of osteoclast biogenesis, receptor activator of nuclear factor kappaB-ligand (RANKL) and osteoprotegerin (OPG), between naïve and long-standing CD patients. METHODS: The study included 95 CD patients, 15 of them with newly diagnosed and previously untreated CD. The spine and hip bone mineral density was measured by dual-energy X-ray absorptiometry. Biochemical markers were determined by immunoassay. RESULTS: Osteopenia was recorded at diagnosis in 53% of naïve patients and osteoporosis was found in 26% of long-standing CD patients. The newly diagnosed patients showed correlation between TNF-alpha and soluble RANKL (sRANKL) (r=0.5; P=0.04), and this positive relationship characterized the study population as a whole (r=0.3; P=0.003). Analysis of the OPG and sRANKL relationship showed absence of correlation in patients with healthy skeleton, whereas an inverse correlation was found in those with osteopenia (r=-0.31; P=0.033) and osteoporosis (r=-0.48; P=0.028). In naïve patients with reduced T score, the correlation between sRANKL and OPG was highly inverse (r=-0.8; P=0.02) and these patients were characterized by lower BMI, significantly higher level of proinflammatory cytokines, elevated C-reactive protein, and increased activity of free sRANKL and OPG. CONCLUSION: Bone disease that accompanies CD at diagnosis suggests that bone metabolism is affected by the underlying inflammatory process per se, as probably confirmed by our finding of the central proinflammatory cytokine TNF-alpha being strongly associated with the osteoclastogenic mediator RANKL, and inversely with bone density.

Methylglyoxal-derivative advanced glycation endproducts: detection by competitive immunofluorometric assay and quantifying in serum and urine.

BACKGROUND: Advanced glycation endproducts (AGE) are a family of heterogeneous chemical structures formed on the host protein in the conditions of carbonyl or oxidative stress. Among AGE precursors, methylglyoxal (MG) is considered one of the key intermediates. METHODS: In the current study, we describe and evaluate a solid phase time-resolved fluoroimmunoassay (DELFIA) based on the competitive reaction between MG-AGE antibody and competitive antigen for detecting MG-adducts in serum and urine. The fluorometry assay was validated by comparison with previously established competitive ELISA. RESULTS: The concentration of MG-adducts assayed by competitive DELFIA in the sera of diabetic patients (DM, n=66) were higher in comparison to non-diabetic controls (C, n=28); DM median=294 mgEq/L (10th and 90th percentile 158-564) vs. C median=224 mgEq/L (10th and 90th percentile 124-290) (p=0.0022). In diabetic subjects, urinary excretion of MG-adducts exceeded the mean level measured in controls; DM median=36.0 mgEq/L (10th and 90th percentile 1-210) vs. C median=11.5 mgEq/L (10th and 90th percentile 1-49) p=0.0036. MG-adducts in urine were low and undetectable in 8 of 28 control subjects and 2 of 66 diabetics. The percentage recovery of MG-adduct added in the same concentration to control and diabetic pool sera showed under-recovery in the latter. Comparison of total AGE level and the amount of MG-adducts revealed MG-derivatives to account for 37% of the heterogeneous structure commonly termed AGE. CONCLUSIONS: The fluoroimmunoassay for MG-derivative AGE evaluated can be utilized on biomonitoring of MG-adducts in serum and its urinary excretion. The competitive DELFIA assay was found to be substantially more sensitive than the standard ELISA having a wider dynamic range, while sharing similar quenching attributes.

Advanced glycated human serum albumin as AGE-carrier protein in enzyme-linked immunosorbent assay.

Competitive ELISAs for advanced glycation end product (AGE) measurements are based on anti-AGE-antibody and in vitro prepared AGE-carrier competitive antigen. AGE-human serum albumin (HSA) prepared by non-enzymatic reaction between protein and glucose is often used as a competitive antigen. The aim of the study was to examine the impact of pH on AGE-HSA preparation. The sets of AGE-HSA analyzed by gel filtration chromatography, IEF and UV/VIS showed significant modifications of native albumin. A competitive ELISA was developed by using polyclonal-AGE-antibody and in vitro prepared AGE-HSA at pH 4.5-8.0. AGE-HSA preparations showed an impact on the ELISA ability to recognize AGE-immunoreactivity of serum proteins. The highest AGE-immunoreactivity was recorded with the antigen prepared at pH 4.5; detection limit declined by approximately 50% with antigens prepared at pH 6.5, 7.5 or 8.0. Study results confirmed the impact of pH on the glycation adduct formation, thus significantly modifying the results of competitive ELISA measurements.

Methylglyoxal in food and living organisms.

Methylglyoxal (MG) is a highly reactive alpha-oxoaldehyde formed endogenously in numerous enzymatic and nonenzymatic reactions. It modifies arginine and lysine residues in proteins forming advanced glycation end-products such as N(delta)-(5-methyl-4-imidazolon-2-yl)-L-ornithine (MG-H1), 2-amino-5-(2-amino-5-hydro-5-methyl-4-imidazolon-1-yl)pentanoic acid (MG-H2), 2-amino-5-(2-amino-4-hydro-4-methyl-5-imidazolon-1-yl)pentanoic acid (MG-H3), argpyrimidine, N(delta)-(4-carboxy-4,6-dimethyl-5,6-dihydroxy-1,4,5,6-tetrahydropyrimidine-2-yl)-L-ornithine (THP), N(epsilon)-(1-carboxyethyl)lysine (CEL), MG-derived lysine dimer (MOLD), and 2-ammonio-6-({2-[4-ammonio-5-oxido-5-oxopently)amino]-4-methyl-4,5-dihydro-1H-imidazol-5-ylidene}amino)hexanoate (MODIC), which have been identified in vivo and are associated with complications of diabetes and some neurodegenerative diseases. In foodstuffs and beverages, MG is formed during processing, cooking, and prolonged storage. Fasting and metabolic disorders and/or defects in MG detoxification processes cause accumulation of this reactive dicarbonyl in vivo. In addition, the intake of low doses of MG over a prolonged period of time can cause degenerative changes in different tissues, and can also exert anticancer activity. MG in biological samples can be quantified by HPLC or GC methods with preliminary derivatization into more stable chromophores and/or fluorophores, or derivatives suitable for determination by MS by use of diamino derivatives of benzene and naphthalene, 6-hydroxy-2,4,5-triaminopyrimidine, cysteamine, and o-(2,3,4,5,6-pentafluorobenzyl) hydroxylamine. The methods include three basic steps: deproteinization, incubation with derivatization agent, and chromatographic analysis with or without preliminary extraction of the formed products.

Humoral methylglyoxal level reflects glycemic fluctuation.

OBJECTIVES: According to the nonenzymatic glycation hypothesis, excessive production of toxic alpha-oxoaldehydes is associated with diabetes tissue damage. The aim of this study was to examine the hypothesis that methylglyoxal overproduction is affected by glycemic fluctuation. DESIGN AND METHODS: Methylglyoxal was measured by HPLC in 41 patients with diabetes, and correlated with 9-point daily glucose profiles, fasting glucose level, as well as early (HbA1c) and advanced glycation products. The 24-h glycemia variability was expressed as the M value, a quantitative index of diurnal glucose fluctuation. RESULTS: Methylglyoxal was, in parallel, analyzed in the whole blood and the plasma of the same individual. Significantly higher concentrations were measured in plasma samples of both, patients with diabetes (n=41) (742+/-141 vs. 409+/-131 nmol/L; P=0.000001) and normoglycemic controls (n=10) (520+/-42 vs. 338+/-62 nmol/L; P=0.0002). Difference between plasma and whole blood methylglyoxal (DeltaMG) in the same individual showed higher DeltaMG values in patients with diabetes (346+/-165 vs. 167+/-86 nmol/L; P=0.0027). Elevated methylglyoxal production was observed in patients with M values>20, yielding a significant correlation between M values and methylglyoxal levels. A significant negative correlation between methylglyoxal and creatinine clearance was observed (r=-0.38, P=0.019). CONCLUSIONS: Methylglyoxal was demonstrated to be a parameter characterized by high sensitivity to glycemic fluctuation. The difference between plasma and whole blood concentrations, in the diabetic population versus control subjects might be associated with increased biogenesis, less efficient endogenous detoxification and/or decreased elimination.

Humoral SPARC/osteonectin protein in plasma cell dyscrasias.

The matricellular protein SPARC (secreted protein acidic and rich in cysteine)/osteonectin was determined in patients with multiple myeloma and related disease to assess the hypothesized role of SPARC as a possible marker of tumor burden and disease progression. Soluble SPARC was measured by competitive enzyme-linked immunosorbent assay (ELISA) in plasma of 42 patients, including sequential measurements in individual patients, and in 20 healthy controls. SPARC values were heterogeneous in multiple myeloma patients showing a decline from baseline levels recorded in controls (456+/-195 vs 600+/-63 ng/ml, p=0.00023). A SPARC showed a significant positive correlation with platelet count (r=0.72, p=0.000000, n=42), hemoglobin (r=0.52, p=0.00037, n=42), and IgG level (r=0.43, p=0.0085, n=42) and negative correlation with beta(2)-microglobulin (r=-0.46, p=0.0023, n=42), aspartate aminotransferase (AST) (r=-0.42, p=0.0061, n=41), interleukin (IL)-6 (r=-0.41, p=0.008, n=42), lactate dehydrogenase (LDH) (r=-0.36, p=0.02, n=41), and percentage of plasma cells in bone marrow aspirate (r=-0.34, p=0.029, n=42). No correlation was found between SPARC and "M" component or disease stage. Investigations performed during the course of disease, including sequential measurements in individual patients, showed a trend to downregulation by disease progression, with the lowest level recorded in the terminal stage (217+/-107 ng/ml, n=11). Patients with established osteolytic lesions had lower plasma SPARC at diagnosis (309+/-197 vs 581+/-293, p=0.021), which correlated with osteocalcin by disease progression (r=0.31, p=0.026). The results of this pilot study revealed abnormalities in the level of humoral SPARC in multiple myeloma and an overall trend to downregulation in the advanced stage of the disease. The regulation of SPARC seems to be opposite to the markers of tumor burden and of aggressive multiple myeloma phenotype.

Preparation and quantification of methylglyoxal in human plasma using reverse-phase high-performance liquid chromatography.

OBJECTIVES: To detect methylglyoxal (MG), a highly reactive alpha-oxoaldehyde found widespread throughout biological life, in human plasma using reverse-phase high-performance liquid chromatography method (RP HPLC) with UV detection. DESIGN AND METHODS: The processing of human plasma required protein precipitation with trifluoroacetic acid (TFA), incubation of the supernatant (2 h) with 1,2-diamino-4,5-dimethoxybenzene (DDB) to convert MG to 6,7-dimethoxy-2-methylquinoxaline (DMQ), freeze-drying, and RP HPLC analysis using 6,7-dimethoxy-2,3-dimethylquinoxaline (DMDQ) as an internal standard (IS). Simplified methods for the synthesis of MG and DDB are also described. RESULTS: Calibration curves were linear in the range of 200-1000 nM. The limit of detection was 30.6 and 45.9 pmol, at 215 and 352 nm, respectively. The intraday coefficients of variation were 6.9-12.6% for 215 nm and 3.5-12.6% for 352 nm. The interday coefficients of variation were 9.6-12.8% for 215 nm and 7.2-14.7% for 352 nm. Sample storage conditions together with statistical evaluation are also described. CONCLUSIONS: Here we present a rapid and inexpensive method for the determination of methylglyoxal in human plasma using RP HPLC with UV detection. The simplicity of the reported RP HPLC method makes it suitable for the detection of methylglyoxal in many human plasma samples.

Advanced glycation endproducts in human diabetic and non-diabetic cataractous lenses.

BACKGROUND: Advanced glycation endproduct (AGE) formation is thought to contribute to aging and cataract formation in the lens. In this study, we evaluated AGE immunoreactivity in human diabetic (n=14) and nondiabetic (n=31) cataractous lenses in relation to high-molecular-weight (HMW) protein content, which is believed to contribute to the onset of cataract. METHODS: AGE immunoreactivity was detected in alkali-soluble individual lens samples. Competitive ELISA with polyclonal anti-AGE antibody was performed to estimate AGEs. SDS-PAGE was used to detect changes in lens protein composition on the basis of molecular size. RESULTS: Regression analysis of data from nondiabetic lenses showed a significant correlation between lens AGE content and patient age (r=0.665, P<0.001). The curve exhibited exponential regression ( y=0.272.e(0.025x)). The level of nonspecified AGEs measured in diabetic lenses showed an overall increase compared with nondiabetic lenses (4.03+/-1.85 vs 1.78+/-0.71 AU/mg protein, P<0.0078). SDS-PAGE showed the occurrence of HMW proteins in both diabetic and nondiabetic lens samples. However, in diabetic patients, who had a higher level of AGEs, a significantly higher proportion of HMW proteins was also observed. The levels of AGE and percent of HMW aggregates showed a very significant correlation ( r=0.68, P<0.007) in the diabetic group, whereas in nondiabetics the correlation, although positive, did not reach statistical significance. CONCLUSION: The AGE distribution, with a higher proportion in the samples of lenses rich in HMW aggregates, corroborates the hypothesis that the advanced glycation process might have a role in degenerative changes in eye lens, which in diabetic patients occur vigorously and much earlier than in those without diabetes.