Plasma advanced glycosylation end-products in maintenance haemodialysis patients

BACKGROUND.: Pentosidine is a useful marker of advanced glycation end-products(AGE) which form cross-links between proteins and have beenfound elevated in plasma and tissues of uraemic and haemo-dialysedsubjects. The origin and fate of these molecules are not clearlyunderstood, but they might play a role in the cardiovascularcomplications of end stage renal failure. The aim of this studywas to evaluate the effect of different types of substitutivetherapy on the removal of pentosidine. METHODS.: Pentosidine was measured by a two-step HPLC methodology. Itsconcentration was evaluated in plasma before and after dialysissession, in 24-h urine, and in dialysate of subjects treatedwith three types of chronic substitutive therapy: bicarbonatehaemodialysis, acetate-free biofiltration, and haemofiltration.Pentosidine levels were compared among the three therapy modalitiesand correlated with clinical and biochemical parameters. RESULTS.: Plasma pentosidine level was extremely high (23.7±2.0pmol/mg protein) in the patients treated with the differentdialysis modalities. The dialysis session had no significanteffect on its plasma concentration, but haemofiltration seemedto be the most efficient method (300–2000 nmol of pentosidineremoved per session versus 250–700 nmol per session withthe two other approaches). An interesting correlation was foundbetween pentosidine and blood urea nitrogen (r=0.58, P<0.01)and pentosidine with uric acid (r=0.48, P<0.05). CONCLUSIONS.: These results suggest that none of the methodology showed agood removal of pentosidine, but among them haemofiltrationhas the best efficiency. The statistical relationships betweenpentosidine and urea and uric acid respectively might provideinsight into the origin of pentosidine. The accumulation ofreactive AGE in uraemic patients may be implicated in the organand tissue damage observed in uraemia.     

Tight relations between coronary calcification and atherosclerotic lesions in the carotid artery in chronic dialysis patients

Aim: Both vascular calcification and atherosclerosis are highly prevalent in patients with end‐stage renal disease (ESRD) and have been associated with increased cardiovascular morbidity. Because those two phenomena might be only coincidentally related in chronic haemodialysis (HD) patients, in this study, coronary artery calcification (CAC), common carotid artery intima media thickness (CCA‐IMT) and thickness of atherosclerotic plaques in the carotid artery were simultaneously measured. Methods: In a cross‐sectional study of 47 HD patients (31 male, mean age 56.8 ± 11.4 years, and 16 female, mean age 56.0 ± 7.5 years) without history of major cardiovascular complications. CCA‐IMT and presence and thickness of atherosclerotic plaques were measured with ultrasound and CAC with multidetector computed tomography. Results: The CAC were present in 70.2% of patients. The mean CAC was 1055 ± 232, the mean CCA‐IMT was 0.96 ± 0.21. The atherosclerotic plaques in the common carotid arteries were visualized in 38 patients (80.1%), the mean thickness of the atherosclerotic plaque was 1.61 ± 0.8 mm. We found a significant positive correlation between CAC and CCA‐IMT (r = 0.70, P < 0.001). The thickness of atherosclerosis plaque positively correlated with CAC as well as with CCA‐IMT (r = 0.60, P < 0.001 and r = 0.7, P < 0.003, respectively). Conclusion: The study revealed close relationships between CAC, intima media thickness and the thickness of atherosclerotic plaques in dialysis patients. It may indicate that both vascular calcification and atherosclerotic lesions frequently coexist in patients with ESRD and that the intima media thickness could serve as a surrogate marker of vascular calcification.     

Plasma pentosidine and total homocysteine levels in relation to change in common carotid intima-media area in the first year of dialysis therapy

BACKGROUND: Homocysteine and advanced glycation end-products (AGEs), which accumulate in chronic kidney disease (CKD), are recently proposed cardiovascular risk factors. In this study, we evaluated the association between changes in calculated intima media (cIM) area of the common carotid artery during the first year of dialysis therapy and plasma total homocysteine (tHcy) level as well as circulating AGEs such as plasma pentosidine level. METHODS: We studied 63 CKD patients (38 males) aged 52 +/- 12 years at a time-point close to start of dialysis treatment and after 12 months of dialysis treatment (41 on peritoneal and 22 on hemodialysis). The tHcy and plasma pentosidine levels were measured by HPLC. Change in cIM area was evaluated by non-invasive B mode ultrasonography. Malnutrition was assessed by subjective global assessment (SGA). RESULTS: At basal, 70% of the patients had carotid plaques, 32% had symptomatic CVD, 38% had malnutrition, 30% had inflammation (CRP > or = 1 mg/dl) and 23% had diabetes mellitus, respectively. At baseline, the mean plasma pentosidine levels were similar in the patients with and without carotid plaques (36 +/- 21 vs 36 +/- 19 pmol/mg albumin, respectively), whereas the median plasma tHcy was significantly lower in the patients with carotid plaques than in the patients without carotid plaques (32 +/- 21 vs 52 +/- 42 pmol/l, p < 0.01, respectively). The prevalence of hyperhomocysteinemia (tHcy level > 13.7 micromol/l) was 95%. In univariate analysis, the change in cIM area during the first year of dialysis was significantly correlated with basal plasma pentosidine level (p = 0.31, p = 0.01), but not with basal tHcy (p = -0.11). However, neither pentosidine nor tHcy levels were correlated with cIM area at basal or at 12 months. In a stepwise multiple regression model, age and plasma pentosidine content, but not the tHcy level, associated with changes in the cIM area. CONCLUSION: Progression of atherosclerosis, as indicated by changes in carotid intima-media area during the course of dialysis treatment, was associated with pentosidine, but not with tHcy, levels at baseline in these CKD patients. This suggests that the accumulation of AGEs in CKD patients may have a role in the pathogenesis of CVD in these patients. Since almost all CKD patients have hyperhomocysteinemia, this finding, however, does not exclude a role ofhomocysteine as a risk factor for CVD in CKD patients.     

Influence of hemodialysis membrane type on pentosidine plasma level, a marker of "carbonyl stress".

Influence of hemodialysis membrane type on pentosidine plasma level, a marker of "carbonyl stress." BACKGROUND: The accumulation of advanced glycation end products (AGEs) in uremia has been ascribed to the retention of carbonyl precursors of AGEs. Pentosidine plasma level has been identified as a surrogate marker of carbonyl precursors ("carbonyl stress"). The influence of hemodialysis (HD) membrane type and residual diuresis on carbonyl stress has not been studied. METHODS: We measured protein-linked and free plasma pentosidine (a surrogate marker of carbonyl stress) by high-performance liquid chromatography in patients on HD with low-flux cellulose (N = 29), high-flux polysulfone (PS; N = 57), polymethylmethacrylate (PMMA) (N = 25), and AN69 (N = 15). RESULTS: Both protein-linked and free pentosidine were similar on low-flux cellulose, high-flux PMMA, and AN69, but were lower (P < 0.01) on high-flux PS. Pentosidine levels were virtually identical on Fresenius and Asahi PS in Japanese and Belgian patients. By multivariate analysis, only the type of HD membrane and residual diuresis proved to be independent determinants (P < 0.001) of pentosidine levels. During a single HD session, the clearance of free pentosidine was similar with all membranes. In three patients who were switched from AN69 to PS, the protein-linked pentosidine level dropped to the control level after resumption of the AN69 membrane. CONCLUSIONS: Both HD membrane type and residual diuresis are independent determinants of pentosidine plasma level, which is a marker of carbonyl stress.     

Advanced glycated end-products (AGE) during haemodialysis treatment: discrepant results with different methodologies reflecting the heterogeneity of AGE compounds.

BACKGROUND: There has been much recent interest in accumulation of advanced glycation end-products (AGE) in uraemic patients. Analysis of AGE has been difficult, because commonly used methodologies, i.e. immunodetection assays or fluorescence measurements, reflect group reactivity and are not specific for chemically defined substances. Some investigators measured individual AGE compounds, e.g. pentosidine, carboxymethyllysine, pyrraline or imidazolone, but a systematic assessment of known compounds using specific HPLC methods in diabetic and non-diabetic end-stage renal disease (ESRD) patients during treatment has not been performed. METHODS: For the present study, the concentrations of early and late products of the Maillard reaction in plasma and ultrafiltrate were monitored during high-flux dialysis sessions in diabetic and non-diabetic patients. AGE were analysed by fluorescence spectroscopy and size exclusion chromatography with fluorescence detection. Specific HPLC methods were used to quantify the Amadori product fructoselysine and the AGE compounds pentosidine and pyrraline in acid or enzymatic hydrolysates. RESULTS: Using size exclusion chromatography, we confirmed a similar fluorescent peak distribution for diabetic and non-diabetic ESRD patients. Main fractions were found at approximately 70, approximately 14 and <2 kDa, confirming results obtained by other authors. In diabetic patients, the fluorescence intensity of the low molecular weight fraction was higher. Uraemic patients differed from controls mainly by the fluorescence of the low molecular weight fraction. The peak spectrum in ultrafiltrates was similar to that in plasma regarding low molecular weight fractions and the 14 kDa peak, but no protein-bound fluorescence was found at 70 kDa. HPLC analysis revealed a significant reduction of plasma pentosidine during high-flux dialysis in non-diabetic (from 9.1+/-5.1 to 8.5+/-4.7 pmol/mg protein; P<0.05) and diabetic patients (from 10.0+/-9.1 to 6.8+/-4.0 pmol/mg protein; P<0.05). In contrast, plasma fructoselysine showed only a non-significant trend to decrease in diabetic (from 3.24+/-0.88 to 3.05+/-0.77 nmol/mg protein) and non-diabetic patients (from 2.69+/-0.52 to 2.56+/-0.50 nmol/mg protein). Pyrraline, a nonfluorescent late AGE product derived from reaction of 3-deoxyglucosone with lysine, could not be detected (detection limit approximately 40 pmol/mg protein). Comparing HPLC and size exclusion analysis, it was found that pentosidine accumulated in the range of low molecular weight substances and was removed by high-flux dialysis. CONCLUSIONS: High-flux dialysis reduces the plasma concentration of fluorescent AGE compounds, i.e. pentosidine, but the Amadori product fructoselysine is not removed, indicating that this compound is protein associated.     

Renal accumulation of pentosidine in non-diabetic proteinuria-induced renal damage in rats.

BACKGROUND: Advanced glycation end-products (AGEs) contribute to the pathogenesis of diabetic glomerulopathy. The role of AGEs in non-diabetic renal damage is not well characterized. First, we studied whether renal AGE accumulation occurs in non-diabetic proteinuria-induced renal damage and whether this is ameliorated by renoprotective treatment. Secondly, we investigated whether renal AGE accumulation was due to intrarenal effects of local protein trafficking. METHODS: Pentosidine was measured (by high-performance liquid chromatography) in rats with chronic bilateral adriamycin nephropathy (AN), untreated and treated with lisinopril. Age-matched healthy rats served as negative controls. Secondly, we compared renal pentosidine in mild proteinuric and non-proteinuric kidneys of unilateral AN and in age-matched controls at 12 and 30 weeks. Intrarenal localization of pentosidine was studied by immunohistochemistry. RESULTS: Renal pentosidine was elevated in untreated AN (0.14+/-0.04 micromol/mol valine) vs healthy controls (0.04+/-0.01 micromol/mol valine, P<0.01). In lisinopril-treated AN, pentosidine was lower (0.09+/-0.02 micromol/mol valine) than in untreated AN (P<0.05). In unilateral proteinuria, pentosidine was similar in non-proteinuric and proteinuric kidneys. After 30 weeks of unilateral proteinuria, pentosidine was increased in both kidneys (0.26+/-0.10 micromol/mol valine) compared with controls (0.18+/-0.06 micromol/mol valine, P<0.05). Pentosidine (AN, week 30) was also increased compared with AN at week 12 (0.16+/-0.06 micromol/mol valine, P<0.01). In control and diseased kidneys, pentosidine was present in the collecting ducts. In proteinuric kidneys, in addition, pentosidine was present in the brush border and cytoplasm of dilated tubular structures, i.e. at sites of proteinuria-induced tubular damage. CONCLUSION: Pentosidine accumulates in non-diabetic proteinuric kidneys in damaged tubules, and renoprotective treatment by angiotensin-converting enzyme (ACE) inhibitors inhibits AGE accumulation, supporting a relationship between abnormal renal protein trafficking, proteinuria-induced tubular damage and tubular pentosidine accumulation. Future studies, applying specific AGE inhibitors, should be conducted to provide insight into the pathophysiological significance of renal AGEs in non-diabetic renal disease.     

Plasma pentosidine is associated with inflammation and malnutrition in end-stage renal disease patients starting on dialysis therapy

Pentosidine is an advanced glycation end-product (AGE), formed by glycosylation and oxidation, that accumulates markedly in end-stage renal disease (ESRD). It has been speculated that AGE and carbonyl stress contributes to long-term complications such as cardiovascular disease (CVD) in ESRD patients. This study determined plasma levels of pentosidine as well as the presence of inflammation (CRP > or = 10 mg/L), clinical CVD (CVD(clin)), and malnutrition (subjective global assessment [SGA] > 1) in a cohort of 191 ESRD patients, median age of 55 yr (range, 23 to 70 yr) and median GFR = 7 ml/min (range, 2 to 17 ml/min), close to start of renal replacement therapy. Fifty-one elderly subjects, median age of 82 yr (range, 71 to 110 yr), with mild renal impairment, median GFR = 67 ml/min (range, 38 to 113 ml/min), were also studied for comparative analysis of plasma pentosidine. The plasma pentosidine content was elevated in all patients compared with the levels in the elderly subjects and were negatively correlated with GFR both in the ESRD patients (Rho = -0.24; P < 0.01; n = 159) and in the elderly subjects (Rho = -0.31; P < 0.05). Moreover, the plasma pentosidine content was correlated with age in the ESRD patients (Rho = 0.26; P < 0.001) and in the elderly subjects (Rho = 0.44; P < 0.001). The 63 malnourished ESRD patients (35%) had a significantly higher (P < 0.05) median plasma pentosidine than the well-nourished patients (39 versus 27 pmol/mg albumin). Similarly, 73 inflamed patients (38%) had a significantly higher (P < 0.001) median pentosidine content compared with 118 non-inflamed patients (37 versus 24 pmol/mg albumin). Also, the plasma pentosidine content showed weak but significant positive correlations with CRP (Rho = 0.28; P < 0.0001), fibrinogen (Rho = 0.23; P < 0.01; n = 126), IL-6 (Rho = 0.22; P < 0.01; n = 169), and soluble vascular cellular adhesion molecule-1 (Rho = 0.38; P < 0.001; n = 74). On the other hand, no significant differences in plasma pentosidine content were noted between the patients with and those without CVD(clin) (32 versus 27 pmol/mg albumin, respectively). Analyses of all-cause mortality, by Kaplan-Meier, showed that mortality was not linked to the plasma pentosidine content. Moreover, survival analysis by the Cox regression model showed that age (P < 0.001), diabetes mellitus (P < 0.01), malnutrition (P < 0.01), and CVD(clin) (P < 0.01) independently predicted poor outcome, whereas an elevated plasma pentosidine content did not. The present study shows that an elevated plasma pentosidine content in ESRD patients is significantly associated with both inflammation and malnutrition and confirms that low residual renal function and high age further contribute to an increased plasma pentosidine content. However, in this small cohort, the plasma pentosidine content did not predict outcome. Thus, accumulation of plasma pentosidine is unlikely to be an appropriate clinically useful marker to predict mortality in ESRD patients.     

A sensitive and specific ELISA for plasma pentosidine.

BACKGROUND: Advanced glycation end products are formed by non-enzymatic glycation and oxidation reaction. Pentosidine is a well-known and characterized structure among them, and has been implicated in the pathogenesis of complications associated with chronic renal failure and long-term dialysis, such as dialysis-related amyloidosis and atherosclerosis. METHODS: We established a highly sensitive and specific competitive enzyme-linked immunosorbent assay (ELISA) for plasma pentosidine and applied it to large numbers of plasma samples including haemodialysis (HD) and continuous ambulatory peritoneal dialysis (CAPD) patients. We compared their plasma pentosidine levels determined by the competitive ELISA with those determined by high-performance liquid chromatographic (HPLC) assay currently used. RESULTS: The plasma pentosidine levels determined by the ELISA were correlated well with those determined by sophisticated instrumental HPLC assay, both in non-diabetic and diabetic dialysis patients. Both analyses yielded comparable results, with over 8-fold higher plasma pentosidine levels in HD and CAPD patients, irrespective of the presence or absence of diabetes, as compared to normal subjects and non-uraemic diabetic patients. CONCLUSIONS: The competitive ELISA will provide a rapid and convenient determination of plasma pentosidine content and thus be useful to assess the carbonyl stress in uraemic patients.     

Influence of dialysis modalities on serum AGE levels in end-stage renal disease patients.

BACKGROUND: The accumulation of advanced glycation end-products (AGEs) in end-stage renal disease (ESRD) influenced by dialysis modalities is of current interest. Highly permeable membranes in haemodialysis or haemofiltration should be able to eliminate circulating AGEs as well as their AGE precursors more efficiently. METHODS: In our study, 10 non-diabetic and 10 diabetic ESRD patients were on haemodialysis with low-flux membranes (LF) followed by a cross-over haemodialysis with high-flux or super-flux polysulfone membranes (HF, SF) for 6 months each. We measured the protein-bound pentosidine and free pentosidine serum levels by high-performance liquid chromatography (HPLC) as well as the serum AGE peptide, AGE-beta(2)-microglobulin and beta(2)-microglobulin concentrations, using ELISA assays. RESULTS: All parameters investigated were significantly higher in dialysis patients than in healthy subjects. The reduction rates during a single dialysis session were found to be higher using the SF than those obtained with the HF (free pentosidine 82.4+/-7.3 vs 76.6+/- 8.7%; AGE peptides 79.7+/-7.7 vs 62.3+/-14.7%; AGE-beta(2)-microglobulin 64.0+/-16.5 vs 45.4+/-17.7%; beta(2)-microglobulin 70.5+/-5.6 vs 58.2+/-6.0%). The protein-bound pentosidine levels remained constant over the respective dialysis sessions. In the 6-month treatment period with the SF, decreased pre-dialysis serum levels of protein-bound pentosidine, free pentosidine and AGE peptides were observed in non-diabetics and diabetics as compared with values obtained with the LF. The respective pre-dialysis AGE-beta(2)-microglobulin concentrations decreased insignificantly, whereas those of beta(2)-microglobulin were significantly lower. Using the HF dialyser, only moderate changes of the parameters measured were noted. CONCLUSION: Treatment with the biocompatible polysulfone SF dialyser seems to be better suited to lower serum AGE levels and to eliminate their precursors.     

Pentosidine Is Associated With Cortical Bone Geometry and Insulin Resistance in Otherwise Healthy Children

Pentosidine is an advanced glycation end product (AGE) associated with fracture in adults with diabetes. AGE accumulation in bone collagen contributes to bone fragility but might also adversely influence bone turnover and, consequently, bone geometry. The relationships between AGEs and bone health have yet to be studied in children. Thus, the objective of this study was to assess relationships between pentosidine and cortical bone volumetric density, geometry, and estimated strength in children. Participants were otherwise healthy black and white boys and girls, ages 9 to 13 years, who were at sexual maturation stage 2 or 3 (N = 160). Tibia and radius cortical bone and muscle area (66% site) were assessed via pQCT. In fasting sera, insulin, glucose, and pentosidine were measured. The Quantitative Insulin Sensitivity Check Index (QUICKI), a measure of insulin sensitivity, was calculated. While controlling for race, sex, maturation, and height, pentosidine negatively correlated with QUICKI (P < 0.05). In unadjusted analyses, pentosidine was associated with lower radius and tibia cortical volumetric bone mineral density, bone mineral content (Ct.BMC), area (Ct.Ar), and thickness (Ct.Th); a larger radius endosteal circumference (Endo.Circ); and lower tibia polar strength strain index (all P < 0.05). While controlling for race, sex, maturation, height, and muscle area, pentosidine was negatively associated with tibia Ct.BMC, Ct.Ar, and Ct.Th but positively associated with Endo.Circ (all P < 0.05). Linear regression revealed a significant interaction between pentosidine and QUICKI in relation to tibia Ct.Th (p_interaction = 0.049), indicating that the negative relationship between pentosidine and Ct.Th was stronger in those with lower QUICKI (ie, greater insulin resistance). This is the first study to report evidence of a potentially adverse influence of AGEs on bone strength in otherwise healthy children. This relationship was strongest in children with the greatest insulin resistance, supporting further work in youth with chronic metabolic health conditions. © 2019 American Society for Bone and Mineral Research.     

Involvement of oxidative stress in the accelerated formation of pentosidine in patients with end-stage renal failure

SUMMARY: Advanced glycation end products (AGEs) have been found to accumulate in the amyloid deposits, skin and plasma of haemodialysis patients (HD), implicating the possible involvement of AGE-modified protein in pathogenesis in dialysis-related amyloidosis. Pentosidine, an AGE cross-link, is a specific marker for AGEs. Plasma pentosidine levels in HD patients were increased dramatically. In the present study, plasma pentosidine, fructoselysine, advanced oxidation protein products (AOPP) and glutathione peroxidase (GSHPx) levels were measured to elucidate the role of oxidative stress in pentosidine formation in nondiabetic HD patients. Plasma pentosidine did not correlate with fructoselysine; plasma AOPP levels were significantly higher than those in normal subjects (201.45 ± 57.93 vs. 55.91 ± 6.57 μmol/L, P <0.001) and correlated positively with plasma pentosidine in HD patients ( r =0.52, P <0.005); plasma GSHPx levels were significantly lower than those in normal subjects (168.40 ± 65.08 vs. 348.87 ± 86.10 U/I, P <0.001) and correlated negatively with plasma pentosidine ( r =0.54, P <0.001) in HD patients. Decreased GSHPx levels may lead to the accumulation of hydrogen peroxide. These findings implicate the involvement of oxidative stress in the accelerated formation of pentosidine in uraemia and suggest that pentosidine could be considered as an oxidative stress biomarker to estimate the degree of oxidative-stress-mediated protein damage.     

The influence of gender, weight, height and BMI on pentosidine concentrations in plasma of hemodialyzed patients

BACKGROUND/AIMS: Advanced glycation end-products (AGEs) such as pentosidine play an important role in complications associated with chronic renal failure (CRF) and hemodialysis (HD). This study was undertaken to determine the influence of anthropometric parameters and inflammation on plasma pentosidine concentrations. METHODS: We measured total and free pentosidine in the plasma of 49 patients on chronic HD. Acid hydrolysis of plasma and protein precipitation with trichloroacetic acid was done in the case of total and free pentosidine, respectively. Pentosidine was measured by high performance liquid chromatography (HPLC). C-reactive protein (CRP) was measured by the nephelometric method. RESULTS: A strong negative correlation between dry weight and mean concentration of total pentosidine before and after HD was found (R = -0.47, p < 0.001). This correlation was stronger in males (R = -0.47, p = 0.017) than females (R = -0.34, p = 0.10). Even stronger correlations were noted between body mass index (BMI) and total (R = -0.55, p < 0.001), as well as free (R = -0.39, p = 0.01) pentosidine. Multivariate analysis demonstrated that BMI and time on HD were two independent factors influencing total pentosidine concentrations. CRP did not correlate with pentosidine or BMI. CONCLUSIONS: Lower BMI values are associated with significantly higher plasma pentosidine concentrations in patients on HD. Presumably this relationship is mediated by hypercatabolism observed in these patients. Catabolism produces weight loss and reduces BMI concurrently with the induction of oxidative and carbonyl stresses that stimulate the generation of pentosidine and other harmful AGEs in dialyzed patients.     

Mechanism of the inhibitory effect of OPB-9195 [(+/-)-2-isopropylidenehydrazono-4-oxo-thiazolidin-5-yla cetanilide] on advanced glycation end product and advanced lipoxidation end product formation

The accumulation in uremic plasma of reactive carbonyl compounds (RCO) derived from both carbohydrates and lipids ("carbonyl stress") contributes to uremic toxicity by accelerating the advanced glycation and lipoxidation of proteins. It was previously demonstrated that OPB-9195 [(+/-)-2-isopropylidenehydrazono-4-oxo- thiazolidin-5-ylacetanilide] inhibited the in vitro formation of advanced glycation end products (AGE) in uremic plasma. This study was designed to elucidate the mechanism of action of OPB-9195 by further delineating the AGE and advanced lipoxidation end product (ALE) precursors targeted by this drug. The inhibitory effects of OPB-9195 on the formation of two AGE (N:epsilon-carboxymethyllysine and pentosidine) on bovine serum albumin incubated with various AGE precursors were examined. Inhibition of N:epsilon-carboxymethyllysine and pentosidine formation with OPB-9195 was more efficient than with aminoguanidine. OPB-9195 also proved effective in blocking the carbonyl amine chemical processes involved in the formation of two ALE (malondialdehyde-lysine and 4-hydroxynonenal-protein adduct). The efficiency of OPB-9195 was similar to that of aminoguanidine. When glucose-based peritoneal dialysis fluid was incubated in the presence of OPB-9195, a similar inhibition of AGE formation was observed. The direct effect of OPB-9195 on major glucose-derived RCO in peritoneal dialysis fluids was then evaluated. The effects of OPB-9195 could be accounted for by its ability to trap RCO. The concentrations of three major glucose-derived RCO (glyoxal, methylglyoxal, and 3-deoxy-glucosone) were significantly lower in the presence of OPB-9195 than in its absence. Aminoguanidine had a similar effect. In conclusion, OPB-9195 inhibits both AGE and ALE formation, probably through its ability to trap RCO. OPB-9195 might prove to be a useful tool to inhibit some of the effects of RCO-related uremic toxicity.     

Nitric oxide inhibits the formation of advanced glycation end products

BACKGROUND: Advanced glycation end products (AGEs) are elevated in renal failure and have been implicated in the pathogenesis of several uremic complications. Their formation is closely associated with oxidative stress. The recent observation that nitric oxide (NO) has an antioxidant effect led us to examine the possible role of NO in the generation of AGEs. METHODS: We examined the effect of NO donors, 2, 2'-(hydroxynitrosohydrazono)bis-ethanamine (NOC18) and S-nitroso-N-acetyl-DL-penicillamine (SNAP), on the in vitro formation of pentosidine, which was used as a surrogate marker for AGEs. Bovine serum albumin was incubated under air at 37 degrees C in a medium containing either several AGE precursors or uremic plasma. To elucidate further the mechanism of the NO effect on AGE formation, we examined the generation of free radicals and carbonyls in pentose-driven pentosidine formation. RESULTS: NO donors significantly inhibit the formation of pentosidine in a dose-dependent manner. The effect is abolished by the addition of a NO scavenging agent, 2-(4-carboxyphenyl)-4,4,5, 5-tetramethylimidazoline-1-oxyl 3-oxide (carboxy-PTIO). The inhibitory effect results from NO but not from the NO donor molecule. It is best explained by the ability of NO to scavenge carbon-centered radicals, hydroxyl radical, and carbonyl compounds. CONCLUSIONS: NO inhibits pentosidine formation by scavenging free radicals and by inhibiting carbonyl compound formation. NO might be implicated in the atherogenic and inflammatory effects of AGEs: Reduced NO production and increased oxidative stress associated with atherosclerotic lesions may accelerate AGE formation and, thus, exacerbate endothelial dysfunction and accelerate the development of atherosclerosis in uremia.     

Effect of dwell time on carbonyl stress using icodextrin and amino acid peritoneal dialysis fluids

BACKGROUND: Deterioration of the peritoneal membrane limits the technical survival of peritoneal dialysis (PD). Advanced glycation of the membrane has been incriminated in this evolution. Advanced glycation end products (AGEs) develop under the influence of glucose and of its degradation products, mainly reactive carbonyl compounds (RCOs) such as glyoxal (GO), methylglyoxal (MGO), and 3-deoxyglucosone (3-DG). The present study was undertaken to evaluate the impact of recently developed glucose-free PD fluids on AGE generation. METHODS: Recently developed glucose-free PD fluids containing either icodextrin or amino acids were investigated. GO, MGO, and 3-DG [high-performance liquid chromatography (HPLC)] and total RCOs (spectrophotometry) were measured in fresh solutions and in effluents after various dwell duration. The AGE formation potential of PD fluids and effluents was assessed by incubation at 37 degrees C, for one week, with bovine serum albumin and by the eventual measurement of pentosidine (HPLC) and Nepsilon-carboxymethyllysine (CML; gas chromatography/mass spectrometry). RESULTS: GO, MGO, and 3-DG (P < 0. 001) as well as total RCOs levels (P < 0.01) were significantly lower in icodextrin and amino acid PD fluid than in commercial, heat-sterilized, 1.36% glucose PD fluid. Pentosidine and CML generation were also significantly lower (P < 0.001) in icodextrin and amino acid PD fluid than in conventional 1.36% glucose PD fluid. The levels of total RCOs, however, increased in icodextrin and amino acid PD fluid effluents with dwell time. AGE formation potential rose accordingly, as demonstrated by a parallel increase in the generation of pentosidine and CML during incubation of PD effluents. CONCLUSION: The present data demonstrate lower RCO contents and AGE formation potential in fresh icodextrin and amino acid PD fluids than in fresh heat-sterilized glucose PD fluids. However, this difference decreases progressively during dwell time, mainly as a result of the influx of total RCOs.     

Studies on structural changes of the carotid arteries and the heart in asymptomatic renal transplant recipients.

BACKGROUND: The present study was designed to characterize early structural changes of large arteries in renal transplant recipients with no clinical evidence of cardiovascular disease and normal blood pressure values, and to analyse the relationship between arterial alterations and those of the heart. METHODS: Intima media thickness and atherosclerotic plaques of the carotid arteries as well as left ventricular geometry and function were examined in 35 asymtomatic renal transplant recipients and 29 age- and sex-matched healthy controls by high resolution B-mode ultrasound and by echocardiography. RESULTS: Intima-media thickness of the carotid arteries was significantly higher in renal transplant recipients (1.21+/-0.08 mm) than in healthy controls (0.74+/-0.04 mm) (P<0.001). Atherosclerotic plaques were found in the majority of renal transplant recipients (71% vs 14% in healthy controls, P<0.001). Left ventricular mass index was significantly increased in the group of renal transplant recipients (264+/-13 g, 146+/-7 g/m2) when compared with healthy controls (155+/-8 g, 83+/-4 g/m2) (P<0.001). Multiple regression analysis in renal transplant recipients showed that intima media thickness of the carotid arteries was significantly related to left ventricular mass index (P<0.02), but not to age, blood pressure, body mass index, serum creatinine, cholesterol and lipoprotein (a) levels. In the group of healthy controls, intima-media thickness of the carotid artery was related to age (P<0.002), but not to left ventricular mass index or the other independent variables. CONCLUSIONS: The present study documents pronounced intima-media thickening in asymptomatic renal transplant recipients. Atherosclerotic lesions are present in most renal transplant recipients with no clinical evidence of cardiovascular disease. We observed a parallelism between arterial wall thickening and left ventricular hypertrophy, although blood pressure levels were normal during haemodialysis therapy and after renal transplantation.     

Association of Serum Pentosidine With Arterial Stiffness in Hemodialysis Patients

Pentosidine is an advanced glycation end product (AGE). The present study was undertaken to investigate the association of serum pentosidine with carotid distensibility as a measure of arterial stiffness in hemodialysis patients. One hundred and three patients on maintenance hemodialysis were recruited. The distensibility coefficient of the common carotid artery was evaluated by an ultrasonic phase‐locked echo‐tracking system. Serum pentosidine was measured by competitive enzyme‐linked immunosorbent assay. Serum albumin, lipid profile, calcium, phosphorus, intact parathyroid hormone (iPTH), high‐sensitivity C‐reactive protein (hs‐CRP), and oxidized low‐density lipoprotein (ox‐LDL) levels were also measured. Correlation was determined by linear and multiple stepwise regression analysis. Serum pentosidine level studied in hemodialysis patients was 0.54 ± 0.13 µg/mL. No significant difference in serum pentosidine level was noted between patients with and without diabetes (0.59 ± 0.10 µg/mL vs. 0.53 ± 0.13 µg/mL, P = 0.062) as well as between patients with and without prior cardiovascular disease (CVD) history (0.56 ± 0.14 µg/mL vs. 0.53 ± 0.12 µg/mL, P = 0.206). In multivariate regression analysis, only age (β = 0.363, P < 0.001) and ox‐LDL (β = 0.262, P = 0.004) were identified as independent determinants for serum pentosidine. Serum pentosidine was significantly correlated with carotid distensibility (r = ?0.387, P < 0.001), as well as age, ox‐LDL, and hs‐CRP. After adjustment for age, blood pressure, history of diabetes, prior CVD history, lipid profile, calcium, phosphorus, iPTH, hs‐CRP, and ox‐LDL, serum pentosidine was still negatively correlated with distensibility (β = ?0.175, P = 0.044). Serum pentosidine was independently associated with carotid distensibility in hemodialysis patients. This finding suggested that the accumulation of AGE might be an important pathway in the development of arterial stiffness in end‐stage renal disease.     

Pyridoxamine improves functional, structural, and biochemical alterations of peritoneal membranes in uremic peritoneal dialysis rats

BACKGROUND: We previously suggested that biochemical alterations of peritoneal membrane associated with long-term peritoneal dialysis might be, at least in part, accounted for by reactive carbonyl compounds overload originating both from uremic circulation and heat sterilization of glucose peritoneal dialysis fluid. In the present study, we utilized a uremic rat model on peritoneal dialysis and evaluated the protective effects of pyridoxamine, a recently developed inhibitor of advanced glycation end product (AGE), on structural, functional, and biochemical alterations of peritoneal membrane. METHODS: Uremic rats were generated by subtotal nephrectomy, some of which were undergone peritoneal dialysis with dialysate and/or given intraperitoneal pyridoxamine. Functional [dialysate/plasma ratio (D/P)(urea, creatinine), D/D(0 glucose)], structural (density of blood vessels in peritoneal membrane tissues), and molecular biochemical [formation of pentosidine, an AGE, by high-performance liquid chromatography (HPLC) assay and expressions of vascular endothelial growth factor (VEGF), and fibroblast growth factor 2 (FGF-2), by semiquantitative polymerase chain reaction (PCR) and/or immunohistochemistry] alterations of peritoneal membrane were assessed. RESULTS: Uremic peritoneal membrane was characterized by an increased functional area of exchange for small solutes between blood and dialysate, vascular proliferation, increased AGE genesis, and up-regulated expressions of angiogenic cytokines. The peritoneal membrane alterations associated with peritoneal dialysis are similar but more severe than those in uremia without peritoneal dialysis. Pyridoxamine given in uremic rats with peritoneal dialysis significantly improved functional and structural alterations. This improvement was accompanied by reduction of AGE accumulation and of angiogenic cytokines expressions. CONCLUSION: Peritoneal carbonyl stress derived from uremia as well as peritoneal dialysis procedure might contribute to the vascular proliferation through induction of bioactive molecules and to an increased functional area, eventually leading to ultrafiltration failure. Pyridoxamine may be beneficial in protection of uremic peritoneal membrane on peritoneal dialysis.     

Advanced glycation end products in human penis: elevation in diabetic tissue, site of deposition, and possible effect through inos or enos

Objectives

We hypothesized that advanced glycation end product (AGE) formation contributes to erectile dysfunction (ED) by quenching nitric oxide. Our first goal was to identify the specific AGE pentosidine in the diabetic human penis. Because AGE-mediated effects may involve inducible nitric oxide synthase (iNOS), we performed immunohistochemical and Western blot analysis of diabetic and nondiabetic human penile tissue for iNOS. Finally, because AGEs may act intracellularly to affect proteins, we set out to identify endothelial NOS (eNOS) in the human penis as an initial step in examining a possible intracellular interaction between eNOS and AGEs.

Methods

We performed high-performance liquid chromatographic analysis of diabetic human penile corpus cavernosum and serum for pentosidine and performed immunohistochemical, electron microscopic (EM), and Western blot analysis of the diabetic and nondiabetic penile corpus cavernosum and tunica for pyrraline, iNOS, and eNOS (and neural NOS [nNOS]for comparative purposes) via standard methods.

Results

We found a significant elevation of pentosidine in the penile tissue but not the serum of diabetic patients (average age 55.6 ± 2.3 years) compared with that of nondiabetic patients (average age 61.8 ± 3.6 years). Pentosidine was 117.06 ± 9.19 pmol/mg collagen in the diabetic tunica versus 77.58 ± 5.5 pmol/mg collagen in the nondiabetic tunica (P < 0.01) and 74.58 ± 8.49 pmol/mg collagen in the diabetic corpus cavernosum versus 46.59 ± 2.53 pmol/mg collagen in the nondiabetic corpus cavernosum (P < 0.01), suggesting a tissue-specific effect of the AGEs. We localized the site of deposition of the specific AGE pyrraline to the human penile tunica and the penile corpus cavernosum collagen. Immunohistochemical and EM analysis localized eNOS and iNOS to the cavernosal endothelium and smooth muscle. Western blot analysis in 6 patients revealed the following: iNOS, but no eNOS, in penile tissue from 1 insulin-dependent diabetic man; eNOS only in 1 man after radical prostatectomy; both eNOS and iNOS in 2 men with Peyronie's disease, as well as in 2 other men with impotence and hypertension. Finally, the specific iNOS inhibitor PNU-19451A significantly augmented relaxation of precontracted human cavernosal tissue, from 64.7% ± 5.58 to 80.03% ± 4.55 at 10 μM acetylcholine and 65.06% ± 2.84 to 86.16% ± 3.96 at 0.1 mM acetylcholine (n = 4, P < 0.002 and P < 0.02, respectively).

Conclusions

AGEs are elevated in diabetic human penile tissue, but not in serum, and are localized to the collagen of the penile tunica and corpus cavernosum. We identified eNOS and iNOS in the human penile cavernosal smooth muscle and endothelium. The augmentation of cavernosal relaxation with a specific iNOS inhibitor, combined with the identification of iNOS protein, but not eNOS, in a patient with severe diabetes and ED, allows for speculation of a pathophysiologic mechanism for AGE-mediated ED via upregulation of iNOS and downregulation of eNOS. These data provide further insight into the mechanisms of advanced glycation end product-mediated ED and provide a foundation for further study. UROLOGY 50: 1016-1026, 1997.