Role of N-acetylcysteine in protecting against 2,5-hexanedione neurotoxicity in a rat model: changes in urinary pyrroles levels and motor activity performance.

The interference of N-acetylcysteine (NAC) on 2,5-hexanedione (2,5-HD) neurotoxicity was evaluated through behavioral assays and the analysis of urinary 2,5-HD, dimethylpyrrole norleucine (DMPN), and cysteine-pyrrole conjugate (DMPN NAC), by ESI-LC-MS/MS, in rats exposed to 2,5-HD and co-exposed to 2,5-HD and NAC. Wistar rats were treated with 4 doses of: 400mg 2,5-HD/kg bw (group I), 400mg 2,5-HD/kg bw+200mg NAC/kg bw (group II), 200mg NAC/kg bw (group III) and with saline (group IV). The results show a significant decrease (p<0.01) in urinary DMPN and free 2,5-HD, a significant increase (p<0.01) in DMPN NAC excretion, and a significant recovery (p<0.01) on motor activity in rats co-exposed to 2,5-HD+NAC, as compared with rats exposed to 2,5-HD alone. Taken together, our findings suggest that at the studied conditions NAC protects against 2,5-HD neurotoxicity and DMPN may be proposed as a new sensitive and specific biomarker of 2,5-HD neurotoxicity in animals treated with a toxic amount of 2,5-hexanedione.

Transport of the advanced glycation end products alanylpyrraline and pyrralylalanine by the human proton-coupled peptide transporter hPEPT1.

The glycation compound pyrraline, which originates from the advanced Maillard reaction, appears in urine after consumption of pyrraline-containing food. We hypothesized that the absorption of pyrraline occurs in the form of dipeptides rather than the free amino acid. The human intestinal peptide transporter hPEPT1 was transiently expressed in HeLa cells. In hPEPT1-transfected cells but not in cells transfected with empty vector, the uptake of [(14)C]glycylsarcosine was strongly inhibited by alanylpyrraline (Ala-Pyrr) and pyrralylalanine (Pyrr-Ala). Free pyrraline did not inhibit peptide uptake. In Xenopus laevis oocytes expressing human PEPT1, both Ala-Pyrr and Pyrr-Ala generated significant inward directed currents. In a third approach, uptake of the dipeptides into hPEPT1-transfected HeLa cells was analyzed by HPLC. Ala-Pyrr and Pyrr-Ala were taken up by hPEPT1-expressing cells at a 4- to 7-fold higher rate than by HeLa cells transfected with the empty vector. We conclude that pyrraline containing dipeptides are transported by hPEPT1 in an electrogenic manner into intestinal cells.

Glycation in food and metabolic transit of dietary AGEs (advanced glycation end-products): studies on the urinary excretion of pyrraline.

Pyrraline [epsilon-(2'-formyl-5'-hydroxymethyl-pyrrolyl)-L-norleucin] belongs to the group of AGEs (advanced glycation end-products) formed in the final stage of the Maillard reaction in foods and in vivo. As it is generally accepted that AGEs are pathophysiologically relevant in aging and in diseases such as diabetes and uraemia, physiological consequences resulting from the ingestion of dietary AGEs are discussed, but balance studies for well defined AGEs are still lacking. The aim of our study was to investigate the influence of nutrition on the urinary excretion of pyrraline. After the first day without dietary restrictions, seven healthy volunteers were asked, starting on the morning of day 2, to ingest a diet virtually free of Maillard compounds (i.e. no cooked or roasted foods, no bakery products, no coffee, etc.). Dietary control was stopped on the morning of day 5. We collected 24 h urine samples for these 5 days, which were analysed for free pyrraline by reverse-phase HPLC with UV detection at 297 nm. We found that urinary excretion of free pyrraline was directly affected by the composition of the diet, decreasing from 4.8+/-1.1 mg/day on day 1 to levels of 1.6, 0.4 and 0.3 mg/day on days 2, 3 and 4 respectively, followed by a significant increase to 3.2+/-1.4 mg/day on the 5th day. The results of this work prove, for the first time, that urinary excretion of pyrraline is strongly dependent on its dietary intake. Thus the influence of nutrition should be taken into consideration in studies directed to the physiological role of glycation compounds.

High levels of urinary pentosidine, an advanced glycation end product, in children with acute exacerbation of atopic dermatitis: relationship with oxidative stress.

Pentosidine is an advanced glycation end product formed by sequential glycation and oxidation. The formation of pentosidine is increased in diseases associated with oxidative stress, such as inflammatory conditions. The aim of the present study was to determine the urinary concentration of pentosidine in atopic dermatitis (AD) and its relationship to the inflammatory status of AD. Urine samples of 32 children with AD and 30 age-matched healthy control subjects were assayed for pentosidine, pyrraline (another advanced glycation end product formed by nonoxidative glycation), and 8-hydroxy-2'-deoxyguanosine (8-OHdG) (an established marker of oxidative stress). Of these 3 markers, urinary concentrations of pentosidine were significantly higher in patients with acute exacerbation of AD than in healthy controls and patients with stable AD. Urinary concentrations of 8-OHdG were significantly higher in AD patients with and without acute exacerbation than in healthy controls. Urinary pentosidine levels correlated significantly with those of 8-OHdG when all data of healthy controls and AD patients were plotted. In patients with acute exacerbation of AD, both urinary pentosidine and 8-OHdG significantly decreased after 7 to 9 days of treatment. Our findings in patients with acute exacerbation of AD suggest that pentosidine levels are partly determined by the prevailing oxidative stress in these patients.

Formation of advanced glycosylation end products and oxidative stress in young patients with type 1 diabetes.

Increased production of advanced glycosylation end products (AGEs) and augmented oxidative stress may contribute to vascular complications in diabetes. Little is known about the formation and accumulation of AGEs in young patients with type 1 diabetes. The aim of the present study was to investigate whether AGE production and oxidative stress are augmented in young patients with type 1 diabetes at early clinical stages of the disease. Urine samples of 38 patients with type 1 diabetes [mean age (+/-SD), 12.8 +/- 4.5 y; diabetes duration, 5.7 +/- 4.3 y; HbA1c, 8.0 +/- 1.6%; urinary albumin excretion, 12.6 +/- 14.4 mg/g creatinine (Cr)] and those of 60 age-matched healthy control subjects were assayed for AGEs, pentosidine and pyrraline, and markers of oxidative stress, 8-hydroxy-2'-deoxyguanosine (8-OHdG) and acrolein-lysine. Of these four markers, urinary concentrations of pentosidine, 8-OHdG, and acrolein-lysine were significantly higher in the patients with diabetes than in the healthy control subjects. For the patient group, pentosidine correlated significantly with 8-OHdG and acrolein-lysine, and pyrraline correlated significantly with acrolein-lysine. Urinary pentosidine, 8-OHdG, and acrolein-lysine but not pyrraline correlated significantly with urinary albumin excretion. Patients with microalbuminuria (> or =15 mg/g Cr) showed significantly higher levels of all four markers than did normoalbuminuric patients and control subjects. The present study indicates that accumulation of AGEs, whose formation is closely linked to oxidative stress, and resultant endothelial dysfunction may start early in the course of type 1 diabetes. This means that the risk of vascular complications may be present at an early age and that the best possible glycemic control should be emphasized from the diagnosis of diabetes.

Increased urinary levels of pentosidine, pyrraline and acrolein adduct in type 2 diabetes.

This study investigates whether urinary levels of pentosidine, pyrraline and acrolein adduct are increased in type 2 diabetes (DM), and whether these levels are correlated with glycemic control and clinical traits. Urinary levels of pentosidine, pyrraline and acrolein adduct in DM patients (n = 100) recruited from the outpatient clinic of our university hospital were compared with those of age- and sex-matched non-diabetic subjects (n = 50). The correlation of these urinary levels with the glycemic control and the clinical traits were examined. Furthermore, the influence of smoking habit on the levels of acrolein adduct was examined. Urinary levels of pentosidine, pyrraline and acrolein adduct were all significantly (p<0.001) higher in the DM group than in the non-DM group (pentosidine (log(pmol/mgCr)), 1.579 +/- 0.147 vs 1.427 +/- 0.142; pyrraline (log(nmol/mgCr)), 0.888 +/- 0.402 vs 0.581 +/- 0.336; acrolein adduct (log(nmol/mgCr)), 2.316 +/- 0.221 vs 2.051 +/- 0.201). Glycemic control parameters, such as fasting plasma glucose (FPG) and HbA1c, were significantly correlated with these urinary levels. Age was correlated with the urinary levels of pentosidine but not with those of pyrraline and acrolein adduct. The urinary albumin excretion rate did not correlate with any of these urinary levels. The levels of acrolein adduct were higher in the subjects with smoking habit than in those without the habit in the DM group as well as in the non-DM group (DM, 2.391 +/- 0.230 and 2.212 +/- 0.190, p=0.0004; Non-DM, 2.120 +/- 0.171 and 1.993 +/- 0.206, p=0.0503). The urinary levels of pentosidine, pyrraline and acrolein adduct were increased in DM and were significantly correlated with glycemic control levels. In addition, smoking habit seems to increase the urinary levels of acrolein adduct.

Determination of urinary pyrraline by solid-phase extraction and high performance liquid chromatography.

Pyrraline is one of the advanced glycation end products formed under non-enzymatic and non-oxidative conditions in vivo. In this study, we developed a novel method for determination of urinary pyrraline using solid-phase extraction as a pretreatment procedure prior to determination by high performance liquid chromatography (HPLC). The Oasis HLB solid-phase extraction cartridge was used for pretreatment of urine samples without hydrolysis. The chromatogram obtained clearly revealed the peak for urinary pyrraline owing to prior removal of interfering substances in urine samples. The recovery rate of pyrraline was 97.2+/-3.3% (n=6). The mean excretion level of urinary pyrraline in healthy control (20-77 years old, n = 30) was 1.42+/-0.65 micromol/mmol creatinine, and the daily variation in the excretion level was considered to be insignificant. We propose the above procedure as a simple, rapid, and accurate method for determination of pyrraline levels in urine.

Glycaemic control and in vivo non-oxidative Maillard reaction: urinary excretion of pyrraline in diabetes patients.

The presence of pyrraline in human urine has recently been described. Using reversed-phase high-performance liquid chromatography, we measured urinary pyrraline in 45 insulin-treated diabetic patients with preserved renal function and in 30 age- and sex-matched healthy subjects. The relationship between urinary pyrraline and metabolic control parameters in the diabetic population (glycaemia, fructosamine, haemoglobin A1c, and 1-year mean haemoglobin A1c) was evaluated. The mean urinary level of pyrraline in diabetic patients with poor glycaemic control (HbA1c > 9.5%) was higher than that in healthy subjects (1.12 +/- 0.35 vs. 0.75 +/- 0.2 mumol mmol-1 creatinine, P < 0.04), whereas in patients with good to moderate glycaemic control (HbA1c < 9.5) it was slightly but not significantly higher than in healthy subjects (0.80 +/- 0.3 mumol mmol-1 creatinine vs. 0.75 +/- 0.2 mumol mmol-1 creatinine). There is a significant correlation between urinary pyrraline level and glycaemia (P < 0.008), haemoglobin A1c (P < 0.01) and 1-year mean haemoglobin A1c values (P < 0.007), but not with fructosamine. The results of the present work prove, for the first time, that glycaemic status influences circulating levels of advanced Maillard reaction products.

Urinary pyrraline as a biochemical marker of non-oxidative Maillard reactions in vivo.

The presence of pyrraline, a non-oxidative glucose-derived Maillard reaction product in plasma proteins has been established previously. In this study we have investigated the presence of pyrraline in human urine to determine whether pyrraline-containing proteins are metabolized or selectively retained. Pyrraline was detected by means of HPLC, and its presence was confirmed by UV and electrospray-mass spectrometry. The quantification of pyrraline in urine from healthy individuals showed 1.21 +/- 0.4 micrograms/mg creatinine. In urine from diabetic patients, pyrraline levels varied considerably, although the mean level was higher than in healthy subjects (1.37 +/- 0.6 micrograms/mg creatinine). These data further support the presence of a catabolic pathway for advanced non-oxidative Maillard reaction products in vivo and suggest their role in the pathogenesis of diabetes.