Plasma Pentosidine Research Articles

Association between pentosidine and arteriosclerosis in patients receiving hemodialysis.

It has been suggested that advanced glycation endproducts (AGEs) accumulate in arteriosclerotic lesions, playing and important role in the development and progression of arteriosclerosis. A chemical quantification method using high-performance liquid chromatography (HPLC) has been established to determine pentosidine levels in these products. Some studies reported that the abdominal aorta calcification index (ACI), obtained by computed tomography (CT), was useful for noninvasively diagnosing arteriosclerosis and determining its severity. In the present study, we measured the ACI and plasma pentosidine in patients receiving maintenance hemodialysis, and investigated the association between arteriosclerosis and pentosidine. In 73 patients receiving maintenance hemodialysis (43 men; 30 women), we determined the ACI, and investigated the association of the ACI with plasma total pentosidine, total cholesterol, triglyceride, high-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol, serum creatinine, and parathyroid hormone (PTH), as well as the product of serum calcium and serum phosphorus, duration of dialysis, and age. The ACI did not correlate with total cholesterol, triglyceride, HDL cholesterol, LDL cholesterol, serum creatinine, PTH, or the product of serum calcium and serum phosphorus. Age, duration of dialysis, and plasma total pentosidine correlated with the ACI: (y = -33.12 + 0.913x; r = 0.407; P < 0.01), (y = 13.94 + 0.403x; r = 0.488; P < 0.01), and (y = 14.13 + 0.630x; r = 0.365; P < 0.01), respectively. It is suggested that pentosidine may be associated with arteriosclerotic development in hemodialysis patients. It has been suggested that advanced glycation endproducts (AGEs) accumulate in arteriosclerotic lesions, playing an important role in the development and progression of arteriosclerosis. A chemical quantification method using high-performance liquid chromatography (HPLC) has been established to determine pentosidine levels in these products. Some studies reported that the abdominal aorta calcification index (ACI), obtained by computed tomography (CT), was useful for noninvasively diagnosing arteriosclerosis and determining its severity. In the present study, we measured the ACI and plasma pentosidine in patients receiving maintenance hemodialysis, and investigated the association between arteriosclerosis and pentosidine.

Advanced glycation end products and oxidative stress are increased in chronic allograft nephropathy

Background: The histologic picture of chronic allograft nephropathy (CAN) resembles early arteriosclerotic lesion. Oxidative stress and advanced glycation end products (AGES) have been implicated in the pathogenesis of atherosclerosis and progression of renal disease. Methods: The authors serially measured the plasma malonyldialdehyde (MDA), carbonyl protein (CP), pentosidine, and argpyramidine levels in 11 postrenal transplant patients with normal renal function (KPT) and 10 patients with biopsy proven CAN at 1, 3, 6, 9 through 12, and 18 through 24 months posttransplant. Data were also obtained in 16 controls and 13 patients with chronic renal failure (CRF). Results: Although serum creatinine, MDA, CP, pentosidine, and argpyrimidine levels decreased during follow-up in KPT, it progressively increased in patients with CAN. The mean serum creatinine level was higher in patients with CRF (3.3 ± 0.8 mg/dL [291.7 ± 70.7 μmol/L]) and CAN (2.4 ± 1.1 mg/dL [212.1 ± 96.6 μmol/L]) than in controls (1.2 ± 0.3 mg/dL [105.8 ± 26.7 μmol/L]) and KPT patients (1.2 ± 0.2 mg/dL [109.7 ± 17.7 μmol/L]; P < 0.001). Markers of oxidative stress and AGEs measured at 18 to 24 months posttransplant in patients with CAN were higher than in KPT, controls, and CRF patients. MDA (nmol/mL) was significantly higher in patients with CAN (1.30 ± 0.30) compared with controls (0.53 ± 0.12), KPT (0.52 ± 0.15), and CRF (0.74 ± 0.27) groups (P < 0.001). Plasma CP (nmol/mg protein) in patients with CAN (4.3 ± 1.00) was higher than in controls (1.90 ± 0.69) and KPT (2.62 ± 1.00) groups at the same time-point (P < 0.001), but comparable with CRF (2.69 ± 1.20). Plasma pentosidine (pmol/μmol protein) level in patients with CAN (19.69 ± 5.05) was higher compared with controls (2.49 ± 0.86), CRF (13.10 ± 3.68), and KPT (14.32 ± 6.28) groups (P < 0.001). Plasma argpyrimidine (pmol/10 μmol protein) was higher in patients with CAN (123.8 ± 17.9) compared with controls (4.81 ± 1.91), CRF (56.92 ± 29.67), and KPT (31.1 ± 11.1; P < 0.001) groups. Conclusion: Oxidative stress and AGEs are increased in patients with CAN, which cannot be explained by the decline in renal function alone. Oxidative stress and AGEs may be one among the nonimmune mediators of CAN.

A functional variant of the myeloperoxidase gene is associated with cardiovascular disease in end-stage renal disease patients

Cardiovascular disease (CVD) is the leading cause of mortality in end-stage renal disease (ESRD) patients and there is emerging evidence that genetic factors may contribute to the development of atherosclerosis. Myeloperoxidase (MPO) is an abundant enzyme involved in the production of free radicals. A functional G-->A single nucleotide polymorphism (SNP) has been identified at position -463, where the A allele is associated with lower MPO expression. To analyze the association between this SNP and inflammation, oxidative stress, and CVD, we studied a cohort of 155 ESRD patients (52 +/- 1 years, 62% males, 22% diabetics) shortly before the initiation of dialysis treatment. CVD was defined by medical history criteria; plasma interleukin-6 (IL-6) was used as a marker of inflammation, and plasma pentosidine as an estimation of oxidative protein damage. DNA from leukocytes was used for genotyping, performed by the pyrosequencing reaction. Only five patients (3%) had the genotype AA at the -463 position, whereas 38 (25%) had the GA and 112 (72%) had the GG genotype. No differences were noted in plasma IL-6 levels between the genotype groups, whereas the pentosidine levels were higher in the GG group (28.4 pmol/mg albumin [range, 8.5 to 123 pmol/mg albumin]) compared to the other two groups (21.4 pmol/mg albumin [range, 7.6 to 384 pmol/mg albumin; P < 0.05]). Patients with the GG genotype had a higher prevalence of positive serology for Chlamydia pneumoniae (51%) when compared to the carriers of the A allele (24%) (P < 0.05). The prevalence of CVD was lower in the AA (0%) and GA genotypes (18%), compared to the GG genotype (35%). The GG genotype was still associated with CVD after correction for age, diabetes, smoking, malnutrition, and inflammation. Our findings suggest that the -463 G-->A SNP, which supposedly results in lower MPO activity, is associated with a lower prevalence of CVD in ESRD patients. It could be speculated that this effect is mediated by a decreased oxidative stress due to lower production of free radicals.

Does Very Low Dialysis Endtoxin Level Influence the Plasma Pentosidine Levels in Dialysis Patients?

In order to confirm whether or not very low endtoxin level in dialysate influences the formation of advanced glycation end products (AGEs) in dialysis patients, plasma pentosidine were determined in the dialysis patients before and after the switch to new water-supply system. Method: Plasma pentosidine were measured by high-performance liquid chromatography in 84 patients on long-term hemodialysis before 3 month, 6 months after the switch of dialysate endtoxin level from 0.020–0.025 to 0.001 EU/ml. Endtoxin measurement was done by the Wellreader SK603 (Seikagaku Kogyo Co. Tokyo) by which detection limit is less than 0.001 EU/ml. The plasma pentosidine levels fell from 1.55 ± 0.61 nmol/ml to 1.38 ± 0.52 nmol/ml (3 month after, p < 0.0001) and 1.31 ± 0.50 nmol/ml (6 month after, p < 0.0001). The fall in plasma pentosidine levels were equally observed in patients given dialysis with high-flux polysulfone, PMMA, and cellulose acetate membranes. Unexpectedly, the plasma triglycerides levels fell from 150 ± 116 mg/dl to 124 ± 79 mg/dl (3 month after, p < 0.01) and 119 ± 75 mg/dl (6 month after, P < 0.01). The levels of total cholesterol, c-reactive protein, β 2-microglobulin did not change during this study. Conclusion: Water purity of dialysis fluid has an impact on AGE formation and improve a status of lipid metabolism in hemodialysis patients even if endtoxin level was very low.

Formation of pentosidine-like AGE structure from creatine

It is reported that plasma pentosidine levels could reflect the severity of diabetic nephropathy and chronic renal failure. Furthermore, plasma pentosidine and creatine levels are known to increase in diabetic nephropathy, suggesting that pentosidine generated in vivo plays an important role in the pathogenesis of diabetic complications especially in renal diseases. Pentosidine is usually formed by the reaction of ribose with lysine and arginine. In the present study, we tested whether pentosidine is also generated from the reaction of ribose with lysine and creatine. Lysine and ribose were incubated with creatine and it was analyzed by HPLC. A novel fluorescent peak (λ ex/em=335/385 nm) was detected in retention time at 9 min, under which the authentic pentosidine was eluted at a retention time of 12 min. Upon FAB-MS analysis, this novel fluorescent peak was shown to possess the ion peak identical with pentosidine except that an arginine residue was replaced by creatine. Furthermore, anti-pentosidine antibody significantly reacted with the novel fluorescent peak named C-pentosidine. Our results indicate that ribose reacts with creatine at its guanidino group to form C-pentosidine, suggesting that C-pentosidine generated from creatine in vivo may play some role in diabetic nephropathy.

Low parathyroid hormone and pentosidine in hemodialysis patients

Background: Accumulation of advanced glycation end products (AGEs) has been linked to the severity of osteoarticular and cardiovascular damage in patients with end-stage renal disease. Methods: We studied the relationship between plasma pentosidine and parathyroid hormone (PTH) levels and bone turnover in a group of hemodialysis patients (n = 85) with minimal aluminum exposure. Results: Plasma pentosidine levels were greater than the upper limit of normal range (cutoff value, 2.46 pmol/mg protein) in all dialysis patients. When patients were divided into three tertiles according to plasma pentosidine levels, serum PTH levels were approximately six times lower in patients in the third pentosidine tertile than in those in the first tertile (P = 0.008), and a similar association (P = 0.009) was found between pentosidine and bone alkaline phosphatase levels. Multivariate analysis confirmed that these relationships were independent of established risk factors for low bone turnover. Forty patients (47%) had serum PTH levels less than 125 pg/mL (13.2 pmol/L). Of note, in a multiple logistic regression model, the relative risk for low PTH level was 4.02 (95% confidence interval, 1.30 to 12.40; P = 0.02) times greater in patients in the third pentosidine tertile than in the first tertile. Conclusion: Pentosidine, a reliable indicator of AGEs, is related inversely to circulating PTH and bone alkaline phosphatase levels. These associations are in agreement with recent experimental data indicating that AGE accumulation may be a factor involved in low bone turnover in dialysis patients. © 2002 by the National Kidney Foundation, Inc.

Renal Proximal Tubular Metabolism of Protein-Linked Pentosidine, an Advanced Glycation End Product

Background: Pentosidine, an advanced glycation end product, accumulates in plasma proteins of uremic patients. Its fate is, however, yet to be fully understood. Methods: Three cell lines, JTC-12 (proximal tubular cells), MDCK (distal tubular cells), and BALB3T3 (nonrenal cells), were cultured in a double chamber system and were exposed to uremic serum, and the contents of protein-linked pentosidine derived from uremic sera were determined in each medium by HPLC assay. The presence of pentosidine in the cytoplasm of these cells was assessed by immunoperoxidase staining. Results: When the apical cell membrane was exposed to uremic serum (fortified in the upper chamber), the contents of protein-linked pentosidine in the upper medium decreased by up to 30% after 24- and 48-hour incubations of JTC-12 cells but not of other cells. On the other hand, the contents of protein-linked pentosidine in the lower medium did not change. By contrast, exposure of the basolateral cell membrane of the three cell lines to uremic serum (fortified in the lower chamber) did not change the contents of protein-linked pentosidine both in the upper and lower medium after a 24-hour incubation. Pentosidine was detected immunohistochemically in the cytoplasm of JTC-12 cells, but not of BALB3T3 and MDCK cells, the apical membranes of which were exposed to uremic sera for 8 h. The immunoreaction disappeared 48 h after exposure. Pentosidine was not detected in the cytoplasm of JTC-12 cells, the basolateral membranes of which were exposed to uremic sera. The relevance of the in vitro results to humans was demonstrated by immunohistochemical studies in normal human kidney tissues showing that pentosidine was identified in the proximal renal tubules. Conclusion: These results suggest that the proximal tubular cells play a role in the disposal of plasma pentosidine.

Plasma Pentosidine Levels Measured by a Newly Developed Method Using ELISA in Patients with Chronic Renal Failure

The plasma pentosidine levels in patients with renal disease were measured by a simple method which was established for plasma and urinary pentosidine determinations. The method, which can be completed within a few hours, involves pretreating plasma with proteolytic enzyme (pronase) and measuring the concentration of pentosidine in the sample by ELISA using antipentosidine antibodies. The prepared antibodies showed no cross-reaction with the raw materials for pentosidine synthesis or with compounds having similar structures. SDS-PAGE indicated that the antibodies had a high purity. The reaction of the antibodies and keyhole limpet hemocyanin-pentosidine in the competitive ELISA system was inhibited by free pentosidine. Excellent standard curves for pentosidine determination were obtained. In actual measurements of clinical samples from patients, a good correlation (r = 0.9356) was obtained between the values measured by ELISA and HPLC. The plasma pentosidine level in patients with renal disease correlated significantly with plasma creatinine, urea nitrogen, β₂-microglobulin, and creatinine clearance, indicating its usefulness in evaluating the severity of renal disease. A significant elevation in plasma pentosidine levels was observed in mild renal dysfunction, whereas no significant increases in creatinine and urea nitrogen levels were detected, suggesting that the plasma pentosidine level is useful in the early diagnosis of beginning renal failure. In patients with chronic renal failure, no difference in plasma pentosidine levels was observed between diabetic nephropathy and chronic glomerulonephritis, while a significant correlation was observed with phosphatidylcholine hydroperoxide, suggesting the possibility that the plasma pentosidine level reflects injury due to oxidation. From these results, the quantitative measurement method developed by us is judged to be a superior innovation for measuring pentosidine in body fluids. The plasma pentosidine level may be useful for the early diagnosis of mild renal failure and to estimate the degree of the severity of renal diseases.

'Carbonyl stress' and dialysis-related amyloidosis.

Advanced glycation end products (AGEs) are formed by nonenzymatic glycation and oxidation (glycoxidation) reactions. As AGE formation is related to hyperglycaemia, they have been implicated in the pathogenesis of diabetic complications. They also increase in normoglycaemic uraemic patients: AGEs, such as pentosidine and carboxymethyllysine (CML), are elevated in both the plasma proteins and skin collagen of uraemic patients, being several times greater than in normal subjects and nonuraemic diabetic patients. However, AGE concentrations do not differ between diabetics and non-diabetics in uraemia. AGE accumulation in uraemia, therefore, cannot be attributed to hyperglycaemia, or simply to the decreased removal by glomerular filtration of AGE-modified proteins as over 90% of plasma pentosidine and CML are linked to albumin. Recently, evidence has suggested that, in uraemia, the increased carbonyl compounds, derived from both carbohydrates and lipids, modify proteins both by the glycoxidation reaction (leading to augmented AGE production) and also by the lipoxidation reaction (leading to the augmentation of the advanced lipoxidation end product, ALE, production). Thus, uraemia might be a state of carbonyl overload with potentially damaging proteins ('carbonyl stress'). Carbonyl stress in uraemia appears to be relevant to long-term complications associated with chronic renal failure and dialysis, such as dialysis-related amyloidosis. Immunohistochemical studies, with specific antibodies to AGEs and ALEs, identified carbonyl stress in long-lived beta2-microglobulin amyloid deposits. Furthermore, proteins modified with carbonyl stress exhibit several biological activities through interactions with several types of cell, e.g. monocytes/macrophages, synovial cells and osteoclasts/osteoblasts, which might partially account for dialysis arthropathies.

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

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. 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. 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. 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.

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." 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. 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). 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. Both HD membrane type and residual diuresis are independent determinants of pentosidine plasma level, which is a marker of carbonyl stress.

A Sensitive and specific ELISA for plasma pentosidine

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. 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. 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. 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.

Cell-associated pentosidine as a marker of aging in human diploid cells in vitro and in vivo

Cellular aging is characterized by alterations at both the morphological and molecular levels, some of which are decreased mitotic rate, increased cytoplasmic vacuolization, and changes in intrinsic cellular constituents (Stanulis-Praeger, 1987. Mech. Ageing Dev. 38, 1–48). In the present investigation, glycoxidation is studied as a marker for cellular aging by measuring cell-associated pentosidine levels in human skin fibroblasts as a function of replicative life span and in human peripheral blood T lymphocytes as a function of chronological age. Fibroblasts were isolated from culture by detachment/centrifugation while lymphocytes were isolated from blood by a Ficoll-Paque/Lympho-Kwik T-Cell Prep technique. Pentosidine levels were measured in acid-hydrolyzed cell pellet suspensions by high-pressure liquid chromatography. Results show that pentosidine was detected in early and late cultured reticular and papillary fibroblasts. Pentosidine, expressed as either protein, DNA, or cell number, significantly ( P<0.0006) increased with in vitro passage and was significantly ( P<0.01) related to cell proliferation as measured by cell density and cell doublings per day during culture. Cell-associated pentosidine was measured in T lymphocytes isolated from healthy, diabetic, and uremic individuals. In healthy controls, levels significantly ( P<0.0003) increased with age. In uremic individuals, a large variation was observed with many values above the 95% confidence intervals determined for controls. Since a previous study showed that plasma pentosidine in healthy subjects does not increase with age, these results suggest that cellular turnover perhaps coupled to a deterioration in cellular anti-glycoxidation defensive mechanisms play a substantial role in explaining increased pentosidine concentrations during cellular aging.

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&lt;0.001) and correlated positively with plasma pentosidine in HD patients (r=0.52, P&lt;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&lt;0.001) and correlated negatively with plasma pentosidine (r=0.54, P&lt;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.

Skeletal muscle protein oxidation and oxidative stress in exercise, immobilization and aging: Possible mechanisms

Pentosidine is an advanced glycation end product (AGE) formed during Maillard or browning reaction by non-enzymatic glycation and oxidation (glycoxidation). Recent studies demonstrated the increased plasma pentosidine levels not only in diabetic patients with hyperglycemia but also in normoglycemic uremic patients. The mechanism of increased glycoxidation reaction in uremia, however, remains unknown. As superoxide dismutases (SODs) and glutathione peroxidase (GPx) are antioxidant enzymes involved in the metabolism of H2O2which accelerates the glycoxidation reaction, we measured their activities by enzymatic assays in the plasma of normal and non-diabetic hemodialysis patients and examined a link between redox regulation by antioxidant enzymes and glycoxidation reaction. The activities of GPx were significantly lower in the plasma of hemodialysis patients than in normal subjects, whereas those of SODs were higher in the former than in the latter. As plasma SODs comprise three isozymes,i.e.,Cu/Zn-SOD, Mn-SOD, and extracellular (EC)-SOD, we determined the levels of each SOD isozyme by ELISAs. The plasma concentrations of Cu/Zn-SOD and EC-SOD were significantly higher in hemodialysis patients than in normal subjects, whereas those of Mn-SOD did not differ between the two groups. It is of note that GPx activities correlated inversely with pentosidine in the plasma of hemodialysis patients (r2= 0.262,P< 0.01). There was no significant correlation between total SOD activities and pentosidine levels in the plasma of hemodialysis patients, but, among the three SOD isozymes, the plasma EC-SOD levels correlated with the levels of pentosidine in hemodialysis patients (r2= 0.286,P< 0.05). As decreased GPx and increased SOD activities result in the increased H2O2generation, which accelerates the glycoxidation of protein, these data suggest a link of altered redox regulation by antioxidant enzymes to increased glycoxidation reaction in the uremic plasma. This paper provides the first time evidence for the possible involvement of enzymatic redox regulation in the non-enzymatic glycoxidation reactionin vivo.

Implication of Altered Redox Regulation by Antioxidant Enzymes in the Increased Plasma Pentosidine, an Advanced Glycation End Product, in Uremia

Pentosidine is an advanced glycation end product (AGE) formed during Maillard or browning reaction by non-enzymatic glycation and oxidation (glycoxidation). Recent studies demonstrated the increased plasma pentosidine levels not only in diabetic patients with hyperglycemia but also in normoglycemic uremic patients. The mechanism of increased glycoxidation reaction in uremia, however, remains unknown. As superoxide dismutases (SODs) and glutathione peroxidase (GPx) are antioxidant enzymes involved in the metabolism of H2O2which accelerates the glycoxidation reaction, we measured their activities by enzymatic assays in the plasma of normal and non-diabetic hemodialysis patients and examined a link between redox regulation by antioxidant enzymes and glycoxidation reaction. The activities of GPx were significantly lower in the plasma of hemodialysis patients than in normal subjects, whereas those of SODs were higher in the former than in the latter. As plasma SODs comprise three isozymes,i.e.,Cu/Zn-SOD, Mn-SOD, and extracellular (EC)-SOD, we determined the levels of each SOD isozyme by ELISAs. The plasma concentrations of Cu/Zn-SOD and EC-SOD were significantly higher in hemodialysis patients than in normal subjects, whereas those of Mn-SOD did not differ between the two groups. It is of note that GPx activities correlated inversely with pentosidine in the plasma of hemodialysis patients (r2= 0.262,P< 0.01). There was no significant correlation between total SOD activities and pentosidine levels in the plasma of hemodialysis patients, but, among the three SOD isozymes, the plasma EC-SOD levels correlated with the levels of pentosidine in hemodialysis patients (r2= 0.286,P< 0.05). As decreased GPx and increased SOD activities result in the increased H2O2generation, which accelerates the glycoxidation of protein, these data suggest a link of altered redox regulation by antioxidant enzymes to increased glycoxidation reaction in the uremic plasma. This paper provides the first time evidence for the possible involvement of enzymatic redox regulation in the non-enzymatic glycoxidation reactionin vivo.

Increased Pentosidine, an Advanced Glycation End Product, in Plasma and Synovial Fluid from Patients with Rheumatoid Arthritis and Its Relation with Inflammatory Markers

Pentosidine is an advanced glycation end product (AGE) formed by combined processes of glycation and oxidation (glycoxidation) between carbohydrate-derived carbonyl group and protein amino group. Recent studies demonstrated the increased pentosidine levels not only in diabetic patients with hyperglycemia but also in normoglycemic uremic patients due to increased oxidative stress. Rheumatoid arthritis (RA) is a state of increased oxidative stress associated with chronic inflammation. This suggested an enhanced glycoxidation reaction and increased AGE levels in RA patients. In the present study, we therefore determined, by high performance liquid chromatographic (HPLC) assay, the concentrations of pentosidine in plasma and synovial fluid from 22 patients with rheumatoid arthritis (RA) and compared their levels with those in 17 patients with osteoarthritis (OA), 26 diabetic patients, and 25 normal subjects. The levels of inflammatory markers and markers of tissue destruction, metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs), were also measured in the same samples. Pentosidine levels in plasma and synovial fluid from RA patients were significantly higher than those in OA patients, diabetic patients, and normal subjects. There was a significant correlation between the pentosidine levels in plasma and those in synovial fluid. Among markers of inflammation and matrix destruction, pentosidine levels in plasma from RA patients were correlated with the levels of C-reactive protein (CRP), erythrocyte sedimentation rate, white blood cell count, and platelet count. Multiple stepwise regression analysis reveals an independent influence of CRP on plasma pentosidine levels. In conclusion, pentosidine levels are significantly higher in plasma and synovial fluid from RA patients and may be useful as a biomarker of chronic inflammation in RA patients.

Clearance of pentosidine, an advanced glycation end product, by different modalities of renal replacement therapy

We recently demonstrated that pentosidine, an advanced glycation end product, accumulates markedly as albumin-linked form (Palb) and in free-form (Pfree) in the plasma of patients with end-stage renal failure. The present study was undertaken to examine the clearance of Palb and Pfree by different modalities of renal replacement therapy, that is, hemodialysis (HD), continuous ambulatory peritoneal dialysis (CAPD), and renal transplantation. HD cleared Pfree (9.4 +/- 4.3 nmol/kg/HD) but not Palb, by diffusion but not by membrane adsorption, whereas CAPD cleared both Palb (4.03 +/- 2.01 nmol/kg/day) and Pfree (2.43 +/- 1.24 nmol/kg/day). Plasma total pentosidine levels were significantly (P < 0.05) lower in CAPD (0.97 +/- 0.41 nmol/ml) than in HD (1.19 +/- 0.41 nmol/ml), as the result of a lower serum albumin level in the former patients. Indeed, Palb expressed per mg albumin was virtually identical in HD and CAPD. By contrast, Pfree was significantly lower in CAPD than in HD. Palb levels were significantly correlated with plasma Pfree levels in both HD and CAPD patients, but not in the CAPD dialysate. Pentosidine transport across the peritoneum occurs mainly by diffusion, both as Palb and Pfree. Interestingly, peritoneal Palb clearance (0.17 +/- 0.07 ml/min) significantly (P < 0.00001) exceeded albumin clearance (0.11 +/- 0.05 ml/min). Palb levels being significantly higher (P < 0.0005) in the peritoneal fluid (36.28 +/- 18.55 pmol/mg) than in the serum (27.12 +/- 11.71 pmol/mg), thus raises the possibility of a facilitated diffusion of Palb or an active transport mechanism for protein-linked pentosidine into the peritoneal cavity. After renal transplantation, plasma Pfree fell rapidly, remained barely detectable after one month, and returned to normal at six months. By contrast, Palb fell more slowly and remained significantly above normal at six months, but returned eventually to normal levels. These findings demonstrate that: (1) both HD and CAPD remove Pfree; (2) the peritoneal clearance of Palb, might contribute to the lower level of plasma pentosidine in CAPD than in HD patients; and (3) renal transplantation is the best therapeutic modality to normalize both Palb and Pfree levels.

Advanced glycation end-products in diabetic nephropathy.

Advanced glycation end-products (AGEs) are the pigmented and fluorescent adduct formed by a non-enzymatic reaction between sugar and protein. Since AGEs are generated in high glucose milieu, then induce the structural and functional alteration of matnx proteins, and have biological effects on various kinds of cells including mesangial cells, AGEs have been implicated in tissue damage of diabetic nephropathy. To elucidate the factor(s) that determine the AGEs level in diabetic nephropathy, we quantitated the plasma pentosidine level of different status of diabetic nephropathy by HPLC assay. The plasma pentosidine level in diabetic nephropathy was found to be determined by factors such as renal function control of glucose and the patient's age; of these, renal function was the most critical factor. For a better understanding of the pathological role of AGEs in diabetic nephropathy, we then examined renal tissues of diabetic nephropathy immunohistochemically using antibodies specific for AGE proteins. Immunohistochemistry revealed the positive immunostaining for AGEs in the expanded mesangial area of diffuse diabetic glomerulosclerosis. The degree of staining was stronger than that in patients of IgA nephropathy with a similar degree of mesangial expansion. The nodular lesions, characteristic of diabetic nephropathy, were also stained positive for AGEs. These findings suggests a potential link of AGEs accumulation, which may be determined by renal function, control of glucose and age, to renal tissue damage in diabetic nephropathy.

Plasma advanced glycosylation end-products in maintenance haemodialysis patients

Pentosidine is a useful marker of advanced glycation end-products (AGE) which form cross-links between proteins and have been found elevated in plasma and tissues of uraemic and haemodialysed subjects. The origin and fate of these molecules are not clearly understood, but they might play a role in the cardiovascular complications of end stage renal failure. The aim of this study was to evaluate the effect of different types of substitutive therapy on the removal of pentosidine. Pentosidine was measured by a two-step HPLC methodology. Its concentration was evaluated in plasma before and after dialysis session, in 24-h urine, and in dialysate of subjects treated with three types of chronic substitutive therapy: bicarbonate haemodialysis, acetate-free biofiltration, and haemofiltration. Pentosidine levels were compared among the three therapy modalities and correlated with clinical and biochemical parameters. Plasma pentosidine level was extremely high (23.7 +/- 2.0 pmol/mg protein) in the patients treated with the different dialysis modalities. The dialysis session had no significant effect on its plasma concentration, but haemofiltration seemed to be the most efficient method (300-2000 nmol of pentosidine removed per session versus 250-700 nmol per session with the two other approaches). An interesting correlation was found between pentosidine and blood urea nitrogen (r = 0.58, P < 0.01) and pentosidine with uric acid (r = 0.48, P < 0.05). These results suggest that none of the methodology showed a good removal of pentosidine, but among them haemofiltration has the best efficiency. The statistical relationships between pentosidine and urea and uric acid respectively might provide insight into the origin of pentosidine. The accumulation of reactive AGE in uraemic patients may be implicated in the organ and tissue damage observed in uraemia.