Homeostatic imbalance of purine catabolism in first-episode neuroleptic-naïve patients with schizophrenia.

BACKGROUND: Purine catabolism may be an unappreciated, but important component of the homeostatic response of mitochondria to oxidant stress. Accumulating evidence suggests a pivotal role of oxidative stress in schizophrenia pathology. METHODOLOGY/PRINCIPAL FINDINGS: Using high-pressure liquid chromatography coupled with a coulometric multi-electrode array system, we compared 6 purine metabolites simultaneously in plasma between first-episode neuroleptic-naïve patients with schizophrenia (FENNS, n = 25) and healthy controls (HC, n = 30), as well as between FENNS at baseline (BL) and 4 weeks (4w) after antipsychotic treatment. Significantly higher levels of xanthosine (Xant) and lower levels of guanine (G) were seen in both patient groups compared to HC subjects. Moreover, the ratios of G/guanosine (Gr), uric acid (UA)/Gr, and UA/Xant were significantly lower, whereas the ratio of Xant/G was significantly higher in FENNS-BL than in HC. Such changes remained in FENNS-4w with exception that the ratio of UA/Gr was normalized. All 3 groups had significant correlations between G and UA, and Xan and hypoxanthine (Hx). By contrast, correlations of UA with each of Xan and Hx, and the correlation of Xan with Gr were all quite significant for the HC but not for the FENNS. Finally, correlations of Gr with each of UA and G were significant for both HC and FENNS-BL but not for the FENNS-4w. CONCLUSIONS/SIGNIFICANCE: During purine catabolism, both conversions of Gr to G and of Xant to Xan are reversible. Decreased ratios of product to precursor suggested a shift favorable to Xant production from Xan, resulting in decreased UA levels in the FENNS. Specifically, the reduced UA/Gr ratio was nearly normalized after 4 weeks of antipsychotic treatment. In addition, there are tightly correlated precursor and product relationships within purine pathways; although some of these correlations persist across disease or medication status, others appear to be lost among FENNS. Taken together, these results suggest that the potential for steady formation of antioxidant UA from purine catabolism is altered early in the course of illness.

Environmental impacts of the coal ash spill in Kingston, Tennessee: an 18-month survey.

An 18 month investigation of the environmental impacts of the Tennessee Valley Authority (TVA) coal ash spill in Kingston, Tennessee combined with leaching experiments on the spilled TVA coal ash have revealed that leachable coal ash contaminants (LCACs), particularly arsenic, selenium, boron, strontium, and barium, have different effects on the quality of impacted environments. While LCACs levels in the downstream river water are relatively low and below the EPA drinking water and ecological thresholds, elevated levels were found in surface water with restricted water exchange and in pore water extracted from the river sediments downstream from the spill. The high concentration of arsenic (up to 2000 μg/L) is associated with some degree of anoxic conditions and predominance of the reduced arsenic species (arsenite) in the pore waters. Laboratory leaching simulations show that the pH and ash/water ratio control the LCACs' abundance and geochemical composition of the impacted water. These results have important implications for the prediction of the fate and migration of LCACs in the environment, particularly for the storage of coal combustion residues (CCRs) in holding ponds and landfills, and any potential CCRs effluents leakage into lakes, rivers, and other aquatic systems.

Expression of liver-specific member of the 3β-hydroxysteroid dehydrogenase family, an isoform possessing an almost exclusive 3-ketosteroid reductase activity

We have recently characterized two types of rat 3β-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase (3β-HSD) isoenzymes expressed in adrenals and gonads. In addition, we have cloned a third type of cDNA encoding a predicted type III 3β-HSD protein specifically expressed in the male rat liver which shares 80% similarity with the two other isoenzymes. Transient expression in human HeLa cells of the cDNAs reveals that the type III 3β-HSD protein does not display oxidative activity for the classical substrates of 3β-HSD, in contrast to the type I 3β-HSD isoenzyme. However, in the presence of NADH, type III isoenzyme, in common with the type I isoform, converts 5α-androstane-3,17-dione (A-dione) and 5α-dihydrotestosterone (DHT) to the corresponding 3β-hydroxysteroids. In fact, the type I and the type III isoenzymes have the same affinity for DHT with K(m) values of 5.05 and 6.16 μM, respectively. When NADPH is used as cofactor, the affinity for DHT of the type III isoform becomes higher than that of the type I isoform with K(m) values of 0.12 and 1.18 μM, respectively. The type III isoform is thus a 3-ketoreductase using NADPH as preferred cofactor which is responsible for the conversion of 3-keto-saturated steroids such as DHT and A-dione into less active steroids.

Pretreatment Effect on Pt/CeO2 Catalyst in the Selective Hydrodechlorination of Trichloroethylene

Pt-ceria catalysts present different surface chemistries depending on the preparation method and the pretreatment. The catalytic behavior of Pt/CeO2 catalysts in the hydrodechlorination of trichloroethylene (TCE) to ethylene was examined as a function of the pretreatment conditions and the noble metal precursor salts. Using FTIR and X-ray photoelectron spectroscopy, significant differences were observed in the surface properties of Pt/CeO2 prepared from the H2PtCl6 precursor after different pretreatment procedures (i.e.. reduction or oxidation-reduction). These surface changes are related to chloride residues from the synthesis. Strong changes were observed in the selectivity of the catalysts to ethylene depending on the pretreatment conditions. The 0.5%Pt/CeO2 catalyst showed a 13% selectivity toward ethylene after reduction, whereas alter oxidation, followed by reduction, the selectivity increased up to 85% at the same conversion level. This effect was only observed when a chloride-containing precursor was used in the preparation. In this way, it is demonstrated that the use of a Cl-containing Pt precursor and an air treatment prior to reduction strongly improves the ethylene selectivity of Pt-CeO2 dechlorination catalysts. This can be explained by formation or a CeOCl phase during the synthesis that decomposes upon air tempering, producing oxygen vacancies on the ceria support. We propose that these oxygen vacancies are active for cleaving off Cl from the TCE. Pt then supplies II to clean-off Cl as HCl. Reaction of TCE on Pt produces rather ethane, so Pt may be partly Cl-poisoned for the hydrodechlorination reaction but not for II, dissociation or CO adsorption.

New Cu-Based Catalysts Supported on TiO2 Films for Ullmann SNAr-Type C-O Coupling Reactions

New routes for the preparation of highly active TiO2-supported Cu and CuZn catalysts have been developed for C-O coupling reactions. Slurries of a titania precursor were dip-coated onto glass beads to obtain either structured mesoporous or non-porous titania thin films. The Cu and CuZn nanoparticles, synthesized using a reduction by solvent method, were deposited onto calcined films to obtain a Cu loading of 2 wt%. The catalysts were characterized by inductively coupled plasma (ICP) spectroscopy, temperature-programmed oxidation/reduction (TPO/TPR) techniques, Cu-63 nuclear magnetic resonance (NMR) spectroscopy, X-ray diffraction (XRD), scanning and transmission electron microscopy (S/TEM-EDX) and X-ray photo-electron spectroscopy (XPS). The activity and stability of the catalysts obtained have been studied in the C-O Ullmann coupling of 4-chloropyridine and potassium phenolate. The titania-supported nanoparticles retained catalyst activity for up to 12 h. However, catalyst deactivation was observed for longer operation times due to oxidation of the Cu nanoparticles. The oxidation rate could be significantly reduced over the CuZn/TiO2 catalytic films due to the presence of Zn. The 4-phenoxypyridine yield was 64% on the Cu/nonporous TiO2 at 120 degrees C. The highest product yield of 84% was obtained on the Cu/mesoporous TiO2 at 140 degrees C, corresponding to an initial reaction rate of 104 mmol g(cat)(-1) s(-1). The activation energy on the Cu/mesoporous TiO2 catalyst was found to be (144 +/- 5) kJ mol(-1), which is close to the value obtained for the reaction over unsupported CuZn nanoparticles (123 +/- 3 kJ mol(-1)) and almost twice the value observed over the catalysts deposited onto the non-porous TiO2 support (75 +/- 2 kJ mol(-1)).

Oxidative and endoplasmic reticulum stresses mediate apoptosis induced by modified LDL in human retinal Müller cells

We previously showed that extravasated, modified LDL is implicated in pericyte loss in diabetic retinopathy (DR). Here, we investigate whether modified LDL induces apoptosis in retinal Müller glial cells.

Increased methionine sulfoxide content of apoA-I in type 1 diabetes

Cardiovascular disease is a major cause of morbidity and premature mortality in diabetes. HDL plays an important role in limiting vascular damage by removing cholesterol and cholesteryl ester hydroperoxides from oxidized low density lipoprotein and foam cells. Methionine (Met) residues in apolipoprotein A-I (apoA-I), the major apolipoprotein of HDL, reduce peroxides in HDL lipids, forming methionine sulfoxide [Met(O)]. We examined the extent and sites of Met(O) formation in apoA-I of HDL isolated from plasma of healthy control and type 1 diabetic subjects to assess apoA-I exposure to lipid peroxides and the status of oxidative stress in the vascular compartment in diabetes. Three tryptic peptides of apoA-I contain Met residues: Q(84)-M(86)-K(88), W(108)-M(112)-R(116), and L(144)-M(148)-R(149). These peptides and their Met(O) analogs were identified and quantified by mass spectrometry. Relative to controls, Met(O) formation was significantly increased at all three locations (Met(86), Met(112), and Met(148)) in diabetic patients. The increase in Met(O) in the diabetic group did not correlate with other biomarkers of oxidative stress, such as N(epsilon)-malondialdehyde-lysine or N(epsilon)-(carboxymethyl)lysine, in plasma or lipoproteins. The higher Met(O) content in apoA-I from diabetic patients is consistent with increased levels of lipid peroxidation products in plasma in diabetes. Using the methods developed here, future studies can address the relationship between Met(O) in apoA-I and the risk, development, or progression of the vascular complications of diabetes.

Aminoguanidine and the effects of modified LDL on cultured retinal capillary cells

Compared with normal low density lipoprotein (N-LDL), LDL minimally modified in vitro by glycation, minimal oxidation, or glycoxidation (G-, MO-, GO-LDL) decreases survival of cultured retinal capillary endothelial cells and pericytes. Similar modifications occurring in vivo in diabetes may contribute to retinopathy. The goal of this study was to determine whether low concentrations of aminoguanidine might prevent cytotoxic modification of LDL and/or protect retinal capillary cells from previously modified LDL.

Native and modified LDL activate extracellular signal-regulated kinases in mesangial cells

Glycation and/or oxidation of LDL may promote diabetic nephropathy. The mitogen-activated protein kinase (MAPK) cascade, which includes extracellular signal-regulated protein kinases (ERKs), modulates cell function. Therefore, we examined the effects of LDL on ERK phosphorylation in cultured rat mesangial cells. In cells exposed to 100 microg/ml native LDL or LDL modified by glycation, and/or mild or marked (copper-mediated) oxidation, ERK activation peaked at 5 min. Five minutes of exposure to 10-100 microg/ml native or modified LDL produced a concentration-dependent (up to sevenfold) increase in ERK activity. Also, 10 microg/ml native LDL and mildly modified LDL (glycated and/or mildly oxidized) produced significantly greater ERK activation than that induced by copper-oxidized LDL +/- glycation (P <0.05). Pretreatment of cells with Src kinase and MAPK kinase inhibitors blocked ERK activation by 50-80% (P <0.05). Native and mildly modified LDL, which are recognized by the native LDL receptor, induced a transient spike of intracellular calcium. Copper-oxidized (+/- glycation) LDL, recognized by the scavenger receptor, induced a sustained rise in intracellular calcium. The intracellular calcium chelator (EGTA/AM) further increased ERK activation by native and mildly modified LDL (P <0.05). These findings demonstrate that native and modified LDL activate ERKs 1 and 2, an early mitogenic signal, in mesangial cells and provide evidence for a potential link between modified LDL and the development of glomerular injury in diabetes.

Pre-enrichment of modified low density lipoproteins with alpha-tocopherol mitigates adverse effects on cultured retinal capillary cells

We determined whether pre-enrichment of low density lipoproteins (LDL) with alpha-tocopherol mitigates their adverse effects, following in vitro glycation, oxidation or glycoxidation, towards cultured bovine retinal capillary endothelial cells (RCEC) and pericytes.

Carboxymethylethanolamine, a biomarker of phospholipid modification during the maillard reaction in vivo

Nepsilon-(Carboxymethyl)lysine (CML) is a stable chemical modification of proteins formed from both carbohydrates and lipids during autoxidation reactions. We hypothesized that carboxymethyl lipids such as (carboxymethyl)phosphatidylethanolamine (carboxymethyl-PE) would also be formed in these reactions, and we therefore developed a gas chromatography-mass spectrometry assay for quantification of carboxymethylethanolamine (CME) following hydrolysis of phospholipids. In vitro, CME was formed during glycation of dioleoyl-PE under air and from linoleoylpalmitoyl-PE, but not from dioleoyl-PE, in the absence of glucose. In vivo, CME was detected in lipid extracts of red blood cell membranes, approximately 0.14 mmol of CME/mol of ethanolamine, from control and diabetic subjects, (n = 22, p > 0.5). Levels of CML in erythrocyte membrane proteins were approximately 0.2 mmol/mol of lysine for both control and diabetic subjects (p > 0.5). For this group of diabetic subjects there was no indication of increased oxidative modification of either lipid or protein components of red cell membranes. CME was also detected in fasting urine at 2-3 nmol/mg of creatinine in control and diabetic subjects (p = 0.085). CME inhibited detection of advanced glycation end product (AGE)-modified protein in a competitive enzyme-linked immunosorbent assay using an anti-AGE antibody previously shown to recognize CML, suggesting that carboxymethyl-PE may be a component of AGE lipids detected in AGE low density lipoprotein. Measurement of levels of CME in blood, tissues, and urine should be useful for assessing oxidative damage to membrane lipids during aging and in disease.

Quantification of malondialdehyde and 4-hydroxynonenal adducts to lysine residues in native and oxidized human low-density lipoprotein

Malondialdehyde (MDA) and 4-hydroxynonenal (HNE) are major end-products of oxidation of polyunsaturated fatty acids, and are frequently measured as indicators of lipid peroxidation and oxidative stress in vivo. MDA forms Schiff-base adducts with lysine residues and cross-links proteins in vitro; HNE also reacts with lysines, primarily via a Michael addition reaction. We have developed methods using NaBH4 reduction to stabilize these adducts to conditions used for acid hydrolysis of protein, and have prepared reduced forms of lysine-MDA [3-(N epsilon-lysino)propan-1-ol (LM)], the lysine-MDA-lysine iminopropene cross-link [1,3-di(N epsilon-lysino)propane (LML)] and lysine-HNE [3-(N epsilon-lysino)-4-hydroxynonan-l-ol (LHNE)]. Gas chromatography/MS assays have been developed for quantification of the reduced compounds in protein. RNase incubated with MDA or HNE was used as a model for quantification of the adducts by gas chromatography/MS. There was excellent agreement between measurement of MDA bound to RNase as LM and LML, and as thiobarbituric acid-MDA adducts measured by HPLC; these adducts accounted for 70-80% of total lysine loss during the reaction with MDA. LM and LML (0.002-0.12 mmol/ mol of lysine) were also found in freshly isolated low-density lipoprotein (LDL) from healthy subjects. LHNE was measured in RNase treated with HNE, but was not detectable in native LDL. LM, LML and LHNE increased in concert with the formation of conjugated dienes during the copper-catalysed oxidation of LDL, but accounted for modification of <1% of lysine residues in oxidized LDL. These results are the first report of direct chemical measurement of MDA and HNE adducts to lysine residues in LDL. LM, LML and LHNE should be useful as biomarkers of lipid peroxidative modification of protein and of oxidative stress in vitro and in vivo.

The advanced glycation end product, Nepsilon-(carboxymethyl)lysine, is a product of both lipid peroxidation and glycoxidation reactions

Nepsilon-(Carboxymethyl)lysine (CML) is an advanced glycation end product formed on protein by combined nonenzymatic glycation and oxidation (glycoxidation) reactions. We now report that CML is also formed during metal-catalyzed oxidation of polyunsaturated fatty acids in the presence of protein. During copper-catalyzed oxidation in vitro, the CML content of low density lipoprotein increased in concert with conjugated dienes but was independent of the presence of the Amadori compound, fructoselysine, on the protein. CML was also formed in a time-dependent manner in RNase incubated under aerobic conditions in phosphate buffer containing arachidonate or linoleate; only trace amounts of CML were formed from oleate. After 6 days of incubation the yield of CML in RNase from arachidonate was approximately 0.7 mmol/mol lysine compared with only 0.03 mmol/mol lysine for protein incubated under the same conditions with glucose. Glyoxal, a known precursor of CML, was also formed during incubation of RNase with arachidonate. These results suggest that lipid peroxidation, as well as glycoxidation, may be an important source of CML in tissue proteins in vivo and that CML may be a general marker of oxidative stress and long term damage to protein in aging, atherosclerosis, and diabetes.

Glycation, glycoxidation, and cross-linking of collagen by glucose. Kinetics, mechanisms, and inhibition of late stages of the Maillard reaction

The Maillard or browning reaction between sugar and protein contributes to the increased chemical modification and cross-linking of long-lived tissue proteins in diabetes. To evaluate the role of glycation and oxidation in these reactions, we have studied the effects of oxidative and antioxidative conditions and various types of inhibitors on the reaction of glucose with rat tail tendon collagen in phosphate buffer at physiological pH and temperature. The chemical modifications of collagen that were measured included fructoselysine, the glycoxidation products N epsilon-(carboxymethyl)lysine and pentosidine and fluorescence. Collagen cross-linking was evaluated by analysis of cyanogen bromide peptides using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by changes in collagen solubilization on treatment with pepsin or sodium dodecylsulfate. Although glycation was unaffected, formation of glycoxidation products and cross-linking of collagen were inhibited by antioxidative conditions. The kinetics of formation of glycoxidation products proceeded with a short lag phase and were independent of the amount of Amadori adduct on the protein, suggesting that autoxidative degradation of glucose was a major contributor to glycoxidation and cross-linking reactions. Chelators, sulfhydryl compounds, antioxidants, and aminoguanidine also inhibited formation of glycoxidation products, generation of fluorescence, and cross-linking of collagen without significant effect on the extent of glycation of the protein. We conclude that autoxidation of glucose or Amadori compounds on protein plays a major role in the formation of glycoxidation products and cross-liking of collagen by glucose in vitro and that chelators, sulfhydryl compounds, antioxidants, and aminoguanidine act as uncouplers of glycation from subsequent glycoxidation and cross-linking reactions.

Accumulation of Maillard reaction products in skin collagen in diabetes and aging

To investigate the contribution of glycation and oxidation reactions to the modification of insoluble collagen in aging and diabetes, Maillard reaction products were measured in skin collagen from 39 type 1 diabetic patients and 52 nondiabetic control subjects. Compounds studied included fructoselysine (FL), the initial glycation product, and the glycoxidation products, N epsilon-(carboxymethyl) lysine (CML) and pentosidine, formed during later Maillard reactions. Collagen-linked fluorescence was also studied. In nondiabetic subjects, glycation of collagen (FL content) increased only 33% between 20 and 85 yr of age. In contrast, CML, pentosidine and fluorescence increased five-fold, correlating strongly with age. In diabetic patients, collagen FL was increased threefold compared with nondiabetic subjects, correlating strongly with glycated hemoglobin but not with age. Collagen CML, pentosidine and fluorescence were increased up to twofold in diabetic compared with control patients: this could be explained by the increase in glycation alone, without invoking increased oxidative stress. There were strong correlations among CML, pentosidine and fluorescence in both groups, providing evidence for age-dependent chemical modification of collagen via the Maillard reaction, and acceleration of this process in diabetes. These results support the description of diabetes as a disease characterized by accelerated chemical aging of long-lived tissue proteins.