Slot Blot- and Electrospray Ionization-Mass Spectrometry/Matrix-Assisted Laser Desorption/Ionization-Mass Spectrometry-Based Novel Analysis Methods for the Identification and Quantification of Advanced Glycation End-Products in the Urine.

Proteins, saccharides, and low molecular organic compounds in the blood, urine, and saliva could potentially serve as biomarkers for diseases related to diet, lifestyle, and the use of illegal drugs. Lifestyle-related diseases (LSRDs) such as diabetes mellitus (DM), non-alcoholic steatohepatitis, cardiovascular disease, hypertension, kidney disease, and osteoporosis could develop into life-threatening conditions. Therefore, there is an urgent need to develop biomarkers for their early diagnosis. Advanced glycation end-products (AGEs) are associated with LSRDs and may induce/promote LSRDs. The presence of AGEs in body fluids could represent a biomarker of LSRDs. Urine samples could potentially be used for detecting AGEs, as urine collection is convenient and non-invasive. However, the detection and identification of AGE-modified proteins in the urine could be challenging, as their concentrations in the urine might be extremely low. To address this issue, we propose a new analytical approach. This strategy employs a method previously introduced by us, which combines slot blotting, our unique lysis buffer named Takata's lysis buffer, and a polyvinylidene difluoride membrane, in conjunction with electrospray ionization-mass spectrometry (ESI)/matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS). This novel strategy could be used to detect AGE-modified proteins, AGE-modified peptides, and free-type AGEs in urine samples.

Association of four differently processed diets with plasma and urine advanced glycation end products and serum soluble receptor for advanced glycation end products concentration in healthy dogs.

Advanced glycation end products (AGEs), formed via the Maillard reaction (MR) during processing of foods, have been implicated in inflammatory and degenerative diseases in human beings. Cellular damage is primarily caused by AGE binding with the receptor for AGEs (RAGE) on cell membranes. An isoform of RAGE, soluble RAGE (sRAGE), acts as a decoy receptor binding circulating AGEs preventing cellular activation. Pet food manufacturing involves processing methods similar to human food processing that may increase dietary AGEs (dAGEs). We hypothesized that diet, plasma and urine AGEs, and serum sRAGE concentrations would differ between thermally processed diets. This study examined the association of four differently processed diets: ultra-processed canned wet food (WF); ultra-processed dry food (DF); moderately processed air-dried food (ADF) and minimally processed mildly cooked food (MF) on total plasma levels of the AGEs, carboxymethyllysine (CML), carboxyethyllysine (CEL), methylglyoxal hydroimidazolone-1, glyoxal hydroimidazolone-1, argpyrimidine, urine CML, CEL and lysinoalanine, and serum sRAGE concentration. Ultra-high-performance liquid chromatography-tandem mass spectrometry was used to measure AGEs. sRAGE concentration was measured using a commercial canine-specific enzyme-linked immunosorbent assay kit. Total dAGEs (mg/100 kcal as fed) were higher in WF than in other diets. Plasma total AGEs (nM/50 µL) were significantly higher with WF, with no difference found between DF, ADF, and MF; however, ADF was significantly higher than MF. Urine CML (nmol AGEs/mmol creatinine) was significantly higher with DF than with WF and MF. There were no significant differences in total urine AGEs or serum sRAGE concentration between diets. In conclusion, different methods of processing pet foods are associated with varied quantities of AGEs influencing total plasma AGE concentration in healthy dogs. Serum sRAGE concentration did not vary across diets but differences in total AGE/sRAGE ratio were observed between MF and WF and, ADF and DF.

Urinary excretion of amino acids and their advanced glycation end-products (AGEs) in adult kidney transplant recipients with emphasis on lysine: furosine excretion is associated with cardiovascular and all-cause mortality.

Arginine (Arg) and lysine (Lys) moieties of proteins undergo various post-translational modifications (PTM) including enzymatic NG- and Nε-methylation and non-enzymatic NG- and Nε-glycation. In a large cohort of stable kidney transplant recipients (KTR, n = 686), high plasma and low urinary concentrations of asymmetric dimethylarginine (ADMA), an abundant PTM metabolite of Arg, were associated with cardiovascular and all-cause mortality. Thus, the prediction of the same biomarker regarding mortality may depend on the biological sample. In another large cohort of stable KTR (n = 555), higher plasma concentrations of Nε-carboxymethyl-lysine (CML) and Nε-carboxyethyl-lysine (CEL), two advanced glycation end-products (AGEs) of Lys, were associated with higher cardiovascular mortality. Yet, the associations of urinary AGEs with mortality are unknown. In the present study, we measured 24 h urinary excretion of Lys, CML, and furosine in 630 KTR and 41 healthy kidney donors before and after donation. Our result indicate that lower urinary CML and lower furosine excretion rates are associated with higher mortality in KTR, thus resembling the associations of ADMA. Lower furosine excretion rates were also associated with higher cardiovascular mortality. The 24 h urinary excretion rate of amino acids and their metabolites decreased post-donation (varying as little as - 24% for CEL, and as much as - 62% for ADMA). For most amino acids, the excretion rate was lower in KTR than in donors pre-donation [except for S-(1-carboxyethyl)-L-cysteine (CEC) and NG-carboxyethylarginine (CEA)]. Simultaneous GC-MS measurement of free amino acids, their PTM metabolites and AGEs in urine is a non-invasive approach in kidney transplantation.

Urinary excretion of advanced glycation end products in dogs and cats.

The present study was conducted with privately owned dogs and cats to investigate whether a relationship exists between the dietary AGEs and the urinary excretion of AGEs, as indication of possible effective absorption of those compounds in the intestinal tract of pet carnivores. For this purpose, data were collected from both raw fed and dry processed food (DPF) fed to dogs and cats, through spot urine sampling and questionnaires. Raw pet food (RF, low in AGE diets) was fed as a primary food source to 29 dogs and DPF to 28 dogs. Cats were categorized into 3 groups, which were RF (n = 15), DPF (n = 14) and dry and wet processed pet food (DWF, n = 25). Urinary-free carboxymethyllysine (CML), carboxyethyllysine (CEL) and lysinoalanine (LAL) were analysed using ultrahigh-performance liquid chromatography (UHPLC)-mass spectrometry, and were standardized for variable urine concentration by expressing the AGE concentrations as a ratio to urine creatinine (Ucr) concentration (µg/µmol Ucr). Urinary excretion of CML, CEL and LAL in dogs fed with DPF was 2.03, 2.14 and 3 times higher compared to dogs fed with RF (p < .005). Similar to the dogs, a significant difference in CML:Ucr, CEL:Ucr and LAL:Ucr between the three diet groups was observed in cats (p-overall < 0.005, ANOVA), in which the RF fed group excreted less AGEs than the other groups. Linear regression coefficients and SE of CML:Ucr, CEL:Ucr and LAL:Ucr showed that body weight and neuter status were significantly correlated with CML and CEL excretion, but not to LAL excretion. Our results revealed a significant correlation between dietary AGEs and urinary excretion of free CML, CEL and LAL, and also showed that endogenous formation of these AGEs occurs in both dogs and cats under physiological conditions.

Therapy with atorvastatin versus rosuvastatin reduces urinary podocytes, podocyte-associated molecules, and proximal tubule dysfunction biomarkers in patients with type 2 diabetes mellitus: a pilot study.

BACKGROUND: Diabetic nephropathy is a severe complication of Type 2 diabetes. Tubular lesions may play an important role in its early stages. The aim of our study was to determine if atorvastatin protects the podocytes and the proximal tubule in patients with Type 2 diabetes. METHODS: A total of 63 patients with Type 2 diabetes completed this 6-months prospective pilot study. They were randomized to continue rosuvastatin therapy (control group) or to be administered an equipotent dose of atorvastatin (intervention group), and were assessed regarding urinary podocytes, podocyte-associated molecules, and biomarkers of proximal tubule dysfunction. RESULTS: The patients from the intervention group presented a significant reduction in podocyturia (from 7.0 to 4.0 cells/ml, p < .05), urinary nephrin (from 1.7 to 1.3 mg/g, p < .001), urinary vascular endothelial growth factor (from 262.8 to 256.9, p < .01), urinary alpha1-microglobulin (from 10.0 to 8.3 mg/g, p < .01), urinary kidney injury molecule-1 (from 139.5 to 136.3 ng/g, p < .001), and urinary advanced glycation end-products (from 112.6 to 101.3 pg/ml, p < .001). Podocyturia correlated directly with the podocyte damage biomarkers, proximal tubule dysfunction biomarkers, albumin to creatinine ratio, and advanced glycation end-products, and inversely with the glomerular filtration rate. CONCLUSIONS: In patients with Type 2 diabetes, atorvastatin exerts favorable effects on the kidney. There is a correlation between the evolution of the podocytes and of the proximal tubule biomarkers, supporting the hypothesis that the glomerular changes parallel proximal tubule dysfunction in the early stages of diabetic nephropathy.

Short-term effects of dietary advanced glycation end products in rats.

Dietary advanced glycation end products (AGE) formed during heating of food have gained interest as potential nutritional toxins with adverse effects on inflammation and glucose metabolism. In the present study, we investigated the short-term effects of high and low molecular weight (HMW and LMW) dietary AGE on insulin sensitivity, expression of the receptor for AGE (RAGE), the AGE receptor 1 (AGER1) and TNF-α, F2-isoprostaglandins, body composition and food intake. For 2 weeks, thirty-six Sprague-Dawley rats were fed a diet containing 20% milk powder with different proportions of this being given as heated milk powder (0, 40 or 100%), either native (HMW) or hydrolysed (LMW). Gene expression of RAGE and AGER1 in whole blood increased in the group receiving a high AGE LMW diet, which also had the highest urinary excretion of the AGE, methylglyoxal-derived hydroimidazolone 1 (MG-H1). Urinary excretion of N ε-carboxymethyl-lysine increased with increasing proportion of heat-treated milk powder in the HMW and LMW diets but was unrelated to gene expression. There was no difference in insulin sensitivity, F2-isoprostaglandins, food intake, water intake, body weight or body composition between the groups. In conclusion, RAGE and AGER1 expression can be influenced by a high AGE diet after only 2 weeks in proportion to MG-H1 excretion. No other short-term effects were observed.

Detection of diabetic nephropathy from advanced glycation endproducts (AGEs) differs in plasma and urine, and is dependent on the method of preparation.

Increased advanced glycation endproducts (AGEs) and oxidation products (OPs) have been proposed as pathogenic for diabetic nephropathy (DN). We investigated the relationship between AGEs and OPs measured in different plasma and urine preparations, and progression of DN in 103 young, normoalbuminuric, normotensive participants with type 1 diabetes in the Natural History of Diabetic Nephropathy Study. The primary endpoint was electron microscopy-measured change in glomerular basement membrane (GBM) width from baseline to 5 years; change in mesangial fractional volume was a secondary endpoint. Fast progressors (FP) were defined as the upper quartile (n = 24) of rate of GBM thickening; slow progressors (SP) were the remainder (n = 79). Four AGEs [3-deoxyglucosone and methylglyoxal hydroimidazolones (DG3H1, MGH1) and carboxymethyl and ethyl lysine (CML, CEL)], and two oxidation products methionine sulfoxide and aminoadipic acid were measured by liquid chromatography, triple quadrupole mass spectrometry. Measurements were done on 10 K plasma filtrates and plasma proteolytic digests (PPD) at year 5, and at four time points over 5 years for urinary 10 K filtrates. Urinary filtrate CEL levels were significantly higher in FP, but not after adjustment for HbA1c, sex, and duration of diabetes. MGHI, CEL, and CML plasma filtrate levels were significantly higher in FP relative to SP (p < 0.05). In PPD, only MGHI showed borderline significantly higher levels in FP relative to SP (p = 0.067), while no other product showed correlation. AGE and OP measurements were not correlated with mesangial expansion. In plasma filtrates, HbA1c at year 5 accounted for 4.7 % of the variation in GBM width. The proportion of variation in GBM width was increased to 11.6 % when MGHI, CEL, and CML were added to the model (6.9 % increase).

Urinary IL-33 and galectin-3 increase in patients with interstitial cystitis/bladder pain syndrome (review).

Interstitial cystitis/bladder pain syndrome (IC/BPS) is an enigmatic chronic disorder characterized by vague bladder pain of variable severity accompanied by urinary symptoms. The pathogenesis and etiology of IC/BPS remain incompletely defined. However, there is an emerging consensus about the central role of epithelial dysfunction, bladder sensory nerve up-regulation, and mast cell activation in the genesis of IC/BPS. Accumulating evidences have suggested that tissue damage is recognized at the cell level via receptor-mediated detection of intracellular proteins (so-called alarmins) released by the necrotic cells. Among these proteins IL-33, galectin-3 (Gal-3) and advanced glycation end products (AGE), may have an important role because they can be participated as cellular components that stimulate the immune system. We determined IL-33, Gal-3, and AGE in 24-hour urine specimens from patients with IC/BPS and healthy subjects. Study participants included 43 woman with IC/BPS and 29 female volunteers. Urinary IL-33, EGF and Gal-3 levels were measured using an enzyme-linked immunosorbent assay, whereas the content of AGE was quantified by natural AGE-specific fluorescence (Ex. 370 nm, Em. 440 nm). Urinary IL-33, and Gal-3 levels were normalized by urinary creatinine (Cr) levels and compared among subgroups. We have found that the levels of IL-33 and Gal-3 were significantly increased in IC/BPS. The level of the IL-33 in the urine of healthy women was equal to 0.32, while the level of IL-33 in IC/BPS patients increases up to 0.58 (p<0.05). Further, the amounts of urine Gal-3 were also elevated in IC/BPS compared to healthy subjects (0.16 versus 0.07; p>0.01) and AGE-specific fluorescence in urine was increased up to 140% in IC/BPS patients. These data suggest on the participation of IL-33, Gal-3 and AGE in the pathogenesis of IC/BPS.

Dietary Advanced Glycation End Products (AGEs) and Urinary Fluorescent AGEs in Children and Adolescents: Findings from the Italian I.Family Project.

Advanced glycation end products (AGEs) have been implicated in chronic diseases in adults, but their role in paediatric populations remains uncertain. This study, conducted on the Italian sample of the I.Family project, aimed to investigate the relationship between dietary and urinary fluorescent AGEs in children and adolescents. The secondary objective was to investigate the sources of dietary AGEs (dAGEs) and their association with dietary composition and anthropometric parameters. Dietary data were collected from 1048 participants via 24 h dietary recall in 2013/2014 to estimate dAGEs intake, while urinary fluorescent AGE levels were measured in 544 individuals. Participants were stratified based on dAGEs intake and compared with respect to urinary fluorescent AGE levels, anthropometric measurements, and dietary intake. The results showed no significant correlation between dietary and urinary fluorescent AGE levels, nor between dAGEs and anthropometric parameters. Notably, higher dAGEs were associated with a diet richer in protein (especially from meat sources) and fat and lower in carbohydrates. In addition, the consumption of ultra-processed foods was lower in participants with a higher DAGE intake. This study highlights the lack of a clear association between dietary and urinary fluorescent AGEs in children, but suggests a distinctive dietary pattern associated with increased dAGEs intake. Further investigation is warranted to elucidate the potential health implications of dAGEs in paediatric populations.

In end stage osteoarthritis, cartilage tissue pentosidine levels are inversely related to parameters of cartilage damage.

OBJECTIVES: Age is the most prominent predisposition for development of osteoarthritis (OA). Age-related changes of articular cartilage are likely to play a role. Advanced glycation endproducts (AGEs) accumulate in cartilage matrix with increasing age and adversely affect the biomechanical properties of the cartilage matrix and influence chondrocyte activity. In clinical studies AGEing of cartilage and its relation to actual cartilage damage can only be measured by surrogate markers (e.g., serum, skin or urine AGE levels and imaging or biochemical markers of cartilage damage). In this study actual cartilage AGE levels were directly related to actual cartilage damage by use of cartilage obtained at joint replacement surgery. METHODS: Cartilage and urine samples were obtained from 69 patients undergoing total knee replacement. Samples were analyzed for pentosidine as marker of AGE. Cartilage damage was evaluated macroscopically, histologically, and biochemically. RESULTS: Cartilage and urine pentosidine both increased with increasing age. The higher the macroscopic, histological, and biochemical cartilage damage the lower the cartilage pentosidine levels were. In multiple regression analysis age is not found to be a confounder. CONCLUSION: There is an inverse relation between cartilage AGEs and actual cartilage damage in end-stage OA. This is likely due to ongoing (ineffective) increased turnover of cartilage matrix proteins even in end stage disease.

Effect of Irbesartan treatment on plasma and urinary markers of protein damage in patients with type 2 diabetes and microalbuminuria.

The aim of this study was to assess the effect of the angiotensin II receptor blocker Irbesartan on protein damage by glycation, oxidation and nitration in patients with type 2 diabetes and microalbuminuria. In a double-masked randomised crossover trial of 52 hypertensive type 2 diabetic patients, antihypertensive treatment was replaced with bendroflumethiazide. After 2-months wash-out, patients were treated randomly with Irbesartan 300, 600, and 900 mg o.d., each dose for 2 months in a three-way crossover study. Glycation, oxidation and nitration adduct residues in plasma protein and related urinary free adducts were determined by stable isotopic dilution analysis liquid chromatography-tandem mass spectrometry. Treatment with Irbesartan decreased urinary excretion of advanced glycation endproducts (AGEs)--methylglyoxal- and glyoxal-derived hydroimidazolones, MG-H1 and G-H1. Urinary AGEs were decreased by 30-32%. In plasma protein, treatment with Irbesartan increased content of glycation adducts Nε-fructosyl-lysine, AGEs Nε-carboxymethyl-lysine, Nε-carboxyethyl-lysine and pentosidine, and also increased content of oxidation markers N-formylkynurenine and dityrosine. This was attributed to decreased clearance of plasma protein modified by Nε-fructosyl-lysine and oxidative markers through the glomerular filter tightened by Irbesartan treatment. Treatment of patients with type 2 diabetes with Irbesartan decreased urinary excretion of MG-H1, G-H1 and 3-NT, which may result from decreased exposure to these AGEs. This is likely achieved by blocking angiotensin II signalling and related down-regulation of glyoxalase 1 and may contribute to health benefits of Irbesartan therapy.

Development of a method for the determination of advanced glycation end products precursors by liquid chromatography and its application in human urine samples.

A liquid chromatographic method with fluorimetric detection has been developed to determine the most abundant α-dicarbonyl compounds, generated as intermediates in the Maillard's reaction, previous derivatization to high fluorescent pteridinic derivatives. Hence, the biomarkers D-glucosone, 3-deoxyglucosone, glyoxal, methylglyoxal, diacetyl, 2,3-pentanedione, and phenylglyoxal were quantified using a gradient elution mode. The experimental conditions of the derivatization reaction and mobile phase composition were optimized. Linearity ranges (peak area versus α-dicarbonyl compound concentration) from 1.0 to 100.0 ng mL(-1) were obtained. Detection limits were comprised between 0.3 and 11.0 ng mL(-1). The high sensitivity of the method allows the determination of α-dicarbonyl compounds present in human urine, such as D-glucosone, 3-deoxyglucosone, glyoxal, and methylglyoxal, that are used as biomarkers, in order to investigate their roles in several diseases, with special emphasis in diabetes mellitus. With the aim of avoiding the interferences due to pteridinic compounds present in urine, a cleanup step with an ISOLUTE ENV+ cartridge was carried out. The concentrations of these urinary biomarkers have been reported as a normalized ratio to urinary creatinine, and determined in healthy and in diabetic volunteers, of different ages and sex. In all urine samples, standard addition and external calibration procedures were applied and compared.

Association between Urinary Advanced Glycation End Products and Subclinical Inflammation in Children and Adolescents: Results from the Italian I.Family Cohort.

Advanced Glycation End Products (AGEs) have been positively correlated with inflammation in adults, while inconsistent evidence is available in children. We evaluated the association between urinary AGEs, measured by fluorescence spectroscopy, and biomarkers of subclinical inflammation in 676 healthy children/adolescents (age 11.8 ± 1.6 years, M ± SD) from the Italian cohort of the I.Family project. Urinary fluorescent AGEs were used as independent variable and high-sensitivity C-reactive protein (hs-CRP) was the primary outcome, while other biomarkers of inflammation were investigated as secondary outcomes. Participants with urinary AGEs above the median of the study population showed statistically significantly higher hs-CRP levels as compared to those below the median (hs-CRP 0.44 ± 1.1 vs. 0.24 ± 0.6 mg/dL, M ± SD p = 0.002). We found significant positive correlations between urinary AGEs and hs-CRP (p = 0.0001), IL-15 (p = 0.001), IP-10 (p = 0.006), and IL-1Ra (p = 0.001). At multiple regression analysis, urinary AGEs, age, and BMI Z-score were independent variables predicting hs-CRP levels. We demonstrated for the first time, in a large cohort of children and adolescents, that the measurement of fluorescent urinary AGEs may represent a simple, noninvasive, and rapid technique to evaluate the association between AGEs and biomarkers of inflammation. Our data support a role of AGEs as biomarkers of subclinical inflammation in otherwise healthy children and adolescents.

The carbonyl scavenger carnosine ameliorates dyslipidaemia and renal function in Zucker obese rats.

The metabolic syndrome is a risk factor that increases the risk for development of renal and vascular complications. This study addresses the effects of chronic administration of the endogenous dipeptide carnosine (ß-alanyl-L-histidine, L-CAR) and of its enantiomer (ß-alanyl-D-histidine, D-CAR) on hyperlipidaemia, hypertension, advanced glycation end products, advanced lipoxidation end products formation and development of nephropathy in the non-diabetic, Zucker obese rat. The Zucker rats received a daily dose of L-CAR or D-CAR (30 mg/kg in drinking water) for 24 weeks. Systolic blood pressure was recorded monthly. At the end of the treatment, plasma levels of triglycerides, total cholesterol, glucose, insulin, creatinine and urinary levels of total protein, albumin and creatinine were measured. Several indices of oxidative/carbonyl stress were also measured in plasma, urine and renal tissue. We found that both L- and D-CAR greatly reduced obese-related diseases in obese Zucker rat, by significantly restraining the development of dyslipidaemia, hypertension and renal injury, as demonstrated by both urinary parameters and electron microscopy examinations of renal tissue. Because the protective effect elicited by L- and D-CAR was almost superimposable, we conclude that the pharmacological action of L-CAR is not due to a pro-histaminic effect (D-CAR is not a precursor of histidine, since it is stable to peptidic hydrolysis), and prompted us to propose that some of the biological effects can be mediated by a direct carbonyl quenching mechanism.

Advanced glycation urinary protein-bound biomarkers and severity of diabetic nephropathy in man.

BACKGROUND/AIMS: The formation of advanced glycation end products (AGEs) is accelerated in patients with diabetic nephropathy. The aim of this study was to ascertain if the urinary excretion of proteins modified by advanced glycation can be used as biomarkers for albuminuria in individuals with type 1 or type 2 diabetes. METHODS: Community-based patients with type 1 (n = 68) or type 2 diabetes (n = 216) attending a diabetes clinic of a tertiary referral hospital were classified as having normoalbuminuria (Normo, albumin excretion rate (AER) <20 µg/min), microalbuminuria (Micro, AER 20-200 µg/min) or macroalbuminuria (Macro, AER ≥200 µg/min). Serum and urine AGE-modified proteins were measured. RESULTS: In patients with both type 1 diabetes and type 2 diabetes, there was a clear association between the degree of albuminuria and urinary AGE-modified proteins (p < 0.0001). Exclusive to patients with type 1 diabetes, urinary excretion of the AGE carboxymethyllysine correlated with AER, whereas patients with type 2 diabetes and macroalbuminuria had an increase in urinary methylglyoxal, an AGE intermediate. These changes were independent of isotopic glomerular filtration rate levels. Serum concentrations of AGEs or soluble receptor for AGEs were not consistently associated with albuminuria in either type 1 or type 2 diabetes. CONCLUSIONS: Urinary excretion of proteins modified by AGEs may be useful biomarkers of albuminuria in individuals with type 1 and type 2 diabetes, warranting prospective investigation in larger diabetic cohorts.

Temporal increases in urinary carboxymethyllysine correlate with albuminuria development in diabetes.

BACKGROUND/AIMS: Advanced glycation end products (AGEs) mediate progressive tissue damage in diabetic nephropathy; however, their utility as a noninvasive reliable biomarker of progressive diabetic nephropathy remains to be determined. In this study, we investigated the temporal accumulation of the AGE carboxymethyllysine (CML) at various sites in a model of experimental diabetic nephropathy. METHODS: Diabetic rats were followed for 1, 4, 8, 16 and 32 weeks. Glomerular filtration rate and urinary albumin excretion were measured. CML was determined in the plasma, urine, renal cortical mitochondria and cytosol by an in-house ELISA. Gene expression of AGE receptors were quantified by real-time PCR and urinary excretion of 8-hydroxy-2'-deoxyguanosine (8-OHdG) was determined by EIA. RESULTS: Four weeks after diabetes induction, urinary CML excretion was increased, which preceded the excretion of urinary albumin and continued to rise progressively until 32 weeks. Circulating, mitochondrial and cytosolic CML content and urinary excretion of 8-OHdG were increased 4 weeks after diabetes induction, but did not increase further with diabetes duration. Renal gene expression of AGE receptors was transiently upregulated at 1 week of diabetes, but this was not a sustained phenomenon. CONCLUSIONS: The most informative marker of progressive renal damage linked to the AGE pathway in experimental diabetic nephropathy is urinary excretion of CML, which now warrants clinical investigation as a potential noninvasive sensitive marker of progressive diabetic nephropathy.

Urinary biomarkers of oxidative/nitrosative stress in healthy smokers.

OBJECTIVE: To evaluate the effect of cigarette smoking on oxidative and nitrosative stress, we assessed urinary levels of 8-hydroxy-2'-deoxyguanosine (8-OHdG), isoprostane 15-F(2t)-IsoP, thiobarbituric acid-reacting substances (TBARS), advanced glycation end-products (AGEs), dityrosine (diTyr), hydrogen peroxide, total nitrite and nitrate and trolox equivalent antioxidant capacity (TEAC) in healthy smokers. METHODS: Fluorimetric and spectrophotometric assays were performed in urine samples of 33 healthy smokers and 58 age-matched controls. RESULTS: Levels of 8-OHdG, 15-F(2t)-IsoP and AGES were found significantly higher in smokers than in controls (10.7 ng/mg Cr vs. 8.3 ng/mg Cr, 1.41 ng/mg Cr vs. 1.01 ng/mg Cr and 189 AFU/mg Cr vs. 143 AFU/mg Cr, respectively; P < 0.05 for all). Positive correlations were found between age and levels of AGEs and diTyr in smokers (r = 0.380, P < 0.035 and r = 0.418, P < 0.019, respectively) and also between age and AGEs, diTyr and TEAC in controls (r = 0.474, P < 0.001, r = 0.463, P < 0.001 and r = 0.576, P < 0.001, respectively), being this correlation negative for 8-OHdG in controls (r = -0.295, P = 0.041). Positive correlation between the number of cigarettes smoked per day and AGEs was also found (r = 0.355, P = 0.044). CONCLUSION: Urinary 8-OHdG, 15-F(2t)-IsoP and AGEs may represent a non-invasive quantitative index of oxidant stress in healthy smokers, being AGEs a possible indicator of tobacco toxin exposure. The increased oxidative stress in healthy smokers observed may be generated because of an excessive production of reactive oxygen species and not by exhaustion of antioxidant defenses.

New Advanced Glycation End Products Observed in Rat Urine by Untargeted Metabolomics after Feeding with Heat-Treated Skimmed Milk Powder.

SCOPE: Milk powder is commonly consumed throughout the world. However, advanced glycation end products (AGEs) will form in milk powder during thermal processing and long-term storage. This study aimed to identify such compounds with potential as new urinary biomarkers of intake of heat-treated skimmed milk powder (HSMP). METHODS AND RESULTS: A parallel study is performed with different dosages of HSMP as well as hydrolyzed HSMP and untreated skimmed milk powder (SMP) in 36 rats. The 24-h urine samples on day 7 or 8 are collected and profiled by untargeted UPLC-Qtof-MS metabolomics. Statistical analysis revealed 25 metabolites differentiating SMP and HSMP; nineteen of these structures are proposed as lysine- and arginine-derived AGEs, and heterocyclic compounds. CONCLUSION: These metabolites may potentially serve as biomarkers of food intake pending further validation to assess intakes of heat-processed dairy foods and thus help to elucidate the effects of HSMP consumption or dietary AGEs on human health.

Early urinary markers of diabetic kidney disease: a nested case-control study from the Diabetes Control and Complications Trial (DCCT).

BACKGROUND: Urinary markers were tested as predictors of macroalbuminuria or microalbuminuria in patients with type 1 diabetes. STUDY DESIGN: Nested case-control of participants in the Diabetes Control and Complications Trial (DCCT). SETTING & PARTICIPANTS: 87 cases of microalbuminuria were matched to 174 controls in a 1:2 ratio, while 4 cases were matched to 4 controls in a 1:1 ratio, resulting in 91 cases and 178 controls for microalbuminuria. 55 cases of macroalbuminuria were matched to 110 controls in a 1:2 ratio. Controls were free of micro-/macroalbuminuria when their matching case first developed micro-/macroalbuminuria. PREDICTORS: Urinary N-acetyl-beta-d-glucosaminidase (NAG), pentosidine, advanced glycation end product (AGE) fluorescence, and albumin excretion rate (AER). OUTCOMES: Incident microalbuminuria (2 consecutive annual AERs > 40 but < or = 300 mg/d) or macroalbuminuria (AER > 300 mg/d). MEASUREMENTS: Stored urine samples from DCCT entry and 1-9 years later when macro- or microalbuminuria occurred were measured for the lysosomal enzyme NAG and the AGE pentosidine and AGE fluorescence. AER and adjustor variables were obtained from the DCCT. RESULTS: Submicroalbuminuric AER levels at baseline independently predicted microalbuminuria (adjusted OR, 1.83; P < 0.001) and macroalbuminuria (adjusted OR, 1.82; P < 0.001). Baseline NAG excretion independently predicted macroalbuminuria (adjusted OR, 2.26; P < 0.001) and microalbuminuria (adjusted OR, 1.86; P < 0.001). Baseline pentosidine excretion predicted macroalbuminuria (adjusted OR, 6.89; P = 0.002). Baseline AGE fluorescence predicted microalbuminuria (adjusted OR, 1.68; P = 0.02). However, adjusted for NAG excretion, pentosidine excretion and AGE fluorescence lost the predictive association with macroalbuminuria and microalbuminuria, respectively. LIMITATIONS: Use of angiotensin-converting enzyme inhibitors was not directly ascertained, although their use was proscribed during the DCCT. CONCLUSIONS: Early in type 1 diabetes, repeated measurements of AER and urinary NAG excretion may identify individuals susceptible to future diabetic nephropathy. Combining the 2 markers may yield a better predictive model than either one alone. Renal tubule stress may be more severe, reflecting abnormal renal tubule processing of AGE-modified proteins, in individuals susceptible to diabetic nephropathy.

Modulation of advanced glycation end products by candesartan in patients with diabetic kidney disease--a dose-response relationship study.

Advanced glycation end products (AGEs) are proinflammatory mediators implicated in the pathogenesis of diabetic kidney disease (DKD). In this study, dose-dependent effects of angiotensin receptor blockade on urinary AGEs were evaluated in patients with DKD. Patients with type 2 diabetes and proteinuria ≥500 mg/d (n = 11) were compared with diabetic controls without DKD (n = 10) and normal controls (n = 11). After a 2-week washout period, DKD participants were treated with candesartan doses progressively increasing from 8, 16, 32, to 64 mg/d every 3 weeks for a total of 12 weeks. Other antihypertensive agents were adjusted to maintain stable blood pressure. At baseline and after each dosing period, blood pressure measurements and 24-hour urine collections were obtained. Urinary carboxymethyl lysine, an AGE biomarker, was reduced over the 12-week dose escalation protocol (r = 0.38, P = 0.01) in DKD participants. Creatinine clearance increased slightly, but albuminuria was unaffected by candesartan administration. Baseline urinary transforming growth factor-ß1 excretion was lower in DKD participants than in controls and did not change during the study period. Reducing kidney exposure to AGEs may be a mechanism of protection by angiotensin receptor blockade in DKD. AGEs may also impact the diabetic kidney through mechanisms independent of transforming growth factor-ß1.