LC-MS/MS analysis of carboxymethylated and carboxyethylated phosphatidylethanolamines in human erythrocytes and blood plasma

An amino group of phosphatidylethanolamine (PE) is considered as a target for nonenzymatic glycation, and the potential involvement of lipid glycation in the pathogenesis of diabetic complications has generated interest. However, unlike an early glycation product of PE (Amadori-PE), the occurrence and roles of advanced glycation end products of PE (AGE-PE) in vivo have been unclear. Here, we developed an LC-MS/MS method for the analysis of AGE-PE [carboxymethyl-PE (CM-PE) and carboxyethyl-PE (CE-PE)]. Collision-induced dissociation of CM-PE and CE-PE produced characteristic ions, permitting neutral loss scanning (NLS) and multiple reaction monitoring (MRM) of AGE-PE. By NLS analysis, a series of AGE-PE molecular species was detected in human erythrocytes and blood plasma. In LC-MS/MS analysis, MRM enabled the separation and determination of the predominant AGE-PE species. Between healthy subjects and diabetic patients, no significant differences were observed in AGE-PE concentrations in erythrocytes and plasma, whereas Amadori-PE concentrations were higher in diabetic patients. These results provide direct evidence for the presence of AGE-PE in human blood, and indicated that, compared with Amadori-PE, AGE-PE is less likely to be accumulated in diabetic blood. The presently developed LC-MS/MS method appears to be a powerful tool for understanding in vivo lipid glycation and its pathophysiological consequence.     

Aminophospholipid glycation and its inhibitor screening system: a new role of pyridoxal 5'-phosphate as the inhibitor

Peroxidized phospholipid-mediated cytotoxity is involved in the pathophysiology of a number of diseases [i.e., the abnormal increase of phosphatidylcholine hydroperoxide (PCOOH) found in the plasma of type 2 diabetic patients]. The PCOOH accumulation may relate to Amadori-glycated phosphatidylethanolamine (deoxy-D-fructosyl PE, or Amadori-PE), because Amadori-PE causes oxidative stress. However, lipid glycation inhibitor has not been discovered yet because of the lack of a lipid glycation model useful for inhibitor screening. We optimized and developed a lipid glycation model considering various reaction conditions (glucose concentration, temperature, buffer type, and pH) between PE and glucose. Using the developed model, various protein glycation inhibitors (aminoguanidine, pyridoxamine, and carnosine), antioxidants (ascorbic acid, alpha-tocopherol, quercetin, and rutin), and other food compounds (L-lysine, L-cysteine, pyridoxine, pyridoxal, and pyridoxal 5'-phosphate) were evaluated for their antiglycative properties. Pyridoxal 5'-phosphate and pyridoxal (vitamin B(6) derivatives) were the most effective antiglycative compounds. These pyridoxals could easily be condensed with PE before the glucose/PE reaction occurred. Because PE-pyridoxal 5'-phosphate adduct was detectable in human red blood cells and the increased plasma Amadori-PE concentration in streptozotocin-induced diabetic rats was decreased by dietary supplementation of pyridoxal 5'-phosphate, it is likely that pyridoxal 5'-phosphate acts as a lipid glycation inhibitor in vivo, which possibly contributes to diabetes prevention.     

Lipid glycation and protein glycation in diabetes and atherosclerosis

Recent instrumental analyses using a hybrid quadrupole/linear ion trap spectrometer in LC-MS/MS have demonstrated that the Maillard reaction progresses not only on proteins but also on amino residues of membrane lipids such as phosphatidylethanolamine (PE), thus forming Amadori-PE (deoxy-d-fructosyl PE) as the principal products. The plasma Amadori-PE level is 0.08 mol% of the total PE in healthy subjects and 0.15–0.29 mol% in diabetic patients. Pyridoxal 5′-phosphate and pyridoxal are the most effective lipid glycation inhibitors, and the PE-pyridoxal 5′-phosphate adduct is detectable in human red blood cells. These findings are beneficial for developing a potential clinical marker for glycemic control as well as potential compounds to prevent the pathogenesis of diabetic complications and atherosclerosis. Glucose and other aldehydes, such as glyoxal, methylglyoxal, and glycolaldehyde, react with the amino residues of proteins to form Amadori products and Heynes rearrangement products. Because several advanced glycation end-product (AGE) inhibitors such as pyridoxamine and benfotiamine inhibit the development of retinopathy and neuropathy in streptozotocin (STZ)-induced diabetic rats, AGEs may play a role in the development of diabetic complications. In the present review, we describe the recent progress and future applications of the Maillard reaction research regarding lipid and protein modifications in diabetes and atherosclerosis.     

15N chemically induced dynamic nuclear polarization during reaction of N-acetyl-L-tyrosine with the nitrating systems nitrite/hydrogen peroxide/horseradish peroxidase and nitrite/hypochloric acid

While the Maillard reaction of amino acids and proteins as well as its consequences in vivo has been thoroughly investigated, little attention has so far been paid to the glycation of aminophospholipids such as phosphatidylethanolamine (PE) or phosphatidylserine (PS), which are essential for structure and functionality of biological membranes. PE-derived glucosylamines (Schiff-PEs) and aminoketoses (Amadori-PEs) have now for the first time been simultaneously identified and quantified in erythrocytes from diabetics and healthy individuals by liquid chromatography-electrospray mass spectrometry (LC-(ESI)MS). The amounts of glycated PE (gPE) were significantly higher in diabetics (0.18-34.2 mol% Schiff-PE and 0.047-0.375 mol% Amadori-PE) than in controls (0.12-3.99 mol% Schiff-PE and 0.018-0.055 mol% Amadori-PE). A positive correlation between fructosylated hemoglobin (HbA(1c)) and the gPE levels was established. No advanced glycation endproducts (AGEs) like 5-hydroxymethylpyrrole-2-carbaldehyde (pyrrole-PE), carboxymethyl (CM-PE), or carboxyethyl (CE-PE) derivatives were detected. To investigate the influence of gPE on lipid peroxidation of biological membranes, liposomes consisting of soy-PE and synthetically prepared Amadori-PE (16:0-16:0) were incubated for several days and the formation of oxidation products was monitored. It could be shown that Amadori-PE extensively promotes lipid peroxidation even in the absence of transition metal ions like Cu(2+) and Fe(3+). Oxidative damage to membrane lipids therefore is supposed to be at least partially caused by the glycation of aminophospholipids.     

Synthetically prepared Aamadori-glycated phosphatidylethanolaminecan trigger lipid peroxidation via free radical reactions

This study for the first time confirmed the peroxidative role of the Amadori product derived from the glycation of phosphatidylethanolamine (PE), namely Amadori-PE. The product was synthesized from the reaction of dioleoyl PE with D-glucose, and then purified by a solid-phase extraction procedure, which was a key step in the next HPLC technique for the isolation of essentially pure Amadori-PE. When the synthetically prepared Amadori-PE was incubated with linoleic acid in the presence of Fe(3+) in micellar system, a remarkable formation of thiobarbituric acid reactive substances was observed together with increases in lipid hydroperoxides. In addition, the lipid peroxidation caused by Amadori-PE was effectively inhibited by superoxide dismutase, mannitol, catalase and metal chelator. These results indicated that Amadori-PE triggers oxidative modification of lipids via the generation of superoxide, and implied the involvement of 'lipid glycation' along with membrane lipid peroxidation in the pathogenesis of diabetes and aging.     

UV analysis of Amadori-glycated phosphatidylethanolamine in foods and biological samples

Maillard reactions are among the most important of the chemical and oxidative changes occurring in food and biological samples that contribute to food deterioration and to the pathophysiology of human disease. Although the association of lipid glycation with this process has recently been shown, the number of lipid glycation products in food and biological materials has not been clear. In this study, we synthesized the Amadori products derived from the glycation of phosphatidylethanolamine (PE), i.e., Amadori-PEs. Dioleoyl PE was incubated with glucose and lactose for 15 days, and the resultant Amadori-PEs were purified and isolated using solid phase extraction followed by HPLC. With this procedure, essentially pure (>98% purity) Amadori-PEs glycated with glucose (Glc-PE) and with lactose (Lac-PE) were obtained and used as standards in the subsequent studies.To determine the presence of Amadori-PEs in food and biological samples, the carbonyl group of Amadori-PEs was ultraviolet (UV)-labeled with 3-methyl-2-benzothiazolinone hydrazone, and the labeled Amadori-PEs were analyzed with normal phase HPLC-UV (318 nm). The detection limit was 4.5 ng (5 pmol) for Glc-PE and 5.3 ng (5 pmol) for Lac-PE. Among the several food samples examined, infant formula and chocolate contained a high amount of both Glc-PE and Lac-PE over wide concentration ranges, such as 1.5-112 microg/g. Testing biological materials showed Amadori-PE (Glc-PE) was detectable in rat plasma.     

Plasma phosphatidylcholine hydroperoxide as a new marker of oxidative stress in alcoholic patients

Quantitative analysis of plasma phosphatidylcholine hydroperoxide (PCOOH) is an important step in evaluating the biochemical processes leading to oxidative injury. However, secondary products of lipid peroxidation are now used as indices. One hundred nine alcoholic patients, aged 22-81 years (mean +/- SEM, 52.0 +/- 1.3 years), and 21 healthy volunteers, aged 41-79 years (51.2 +/- 2.2 years), participated in this study. Plasma PCOOH was measured by HPLC with chemiluminescence detection. Plasma PCOOH concentration was significantly higher in alcoholic patients (46.1 +/- 4.1 pmol/ml) than in controls (15.6 +/- 1.8 pmol/ml). It was significantly higher in patients with blood alcohol (88.0 +/- 10.5 pmol/ml) than in those without alcohol (32.6 +/- 3.1 pmol/ml). The patients with high levels of aspartate aminotransferase, alanine aminotransferase, gamma-glutamyl transpeptidase (gamma-GTP), and triglyceride (TG) showed significantly higher PCOOH concentrations than did patients with normal levels. The PCOOH level was positively correlated with levels of gamma-GTP, HDL, blood alcohol concentration, and TG. Plasma PCOOH levels in 29 alcoholic patients after a 6 week abstinence were decreased significantly (22.8 +/- 11.1 pmol/ml), which was associated with improvement on liver function tests. This is the first measurement of plasma PCOOH in alcoholic patients. These results suggest the involvement of lipid peroxidation in alcohol-induced liver damage and confirm that the PCOOH plasma concentration is a new marker of alcohol consumption as well as oxidative stress in alcoholic patients.     

Amadori-glycated phosphatidylethanolamine induces angiogenic differentiations in cultured human umbilical vein endothelial cells

Glycation has been implicated in the endothelial dysfunction that contributes to both diabetes- and aging-associated vascular complications. The aim of the present study was to determine whether Amadori-glycated phosphatidylethanolamine (Amadori-PE), a lipid-linked glycation compound that is formed at an increased rate in hyperglycemic states, affected proliferation, migration and tube formation of cultured human umbilical vein endothelial cells (HUVEC). Amadori-PE at a low concentration of less than 5 microM significantly enhanced these three factors involved in angiogenesis. Furthermore, stimulation of HUVEC with Amadori-PE resulted in secretion of matrix metalloproteinase 2 (MMP-2), a pivotal enzyme in the initial step of angiogenesis. Our results demonstrated for the first time that Amadori-PE may be an important compound that promotes vascular disease as a result of its angiogenic activity on endothelial cells. We also demonstrated that MMP-2 is a primary mediator of Amadori-PE-driven angiogenesis.     

Amadori-glycated phosphatidylethanolamine up-regulates telomerase activity in PANC-1 human pancreatic carcinoma cells

Several lines of experimental data have highlighted a key role of Amadori-glycated phosphatidylethanolamine (Amadori-PE) in the development of diabetic complications. Recent epidemiological studies suggest that diabetes mellitus could be a risk factor for some cancers. A characteristic of cancer cells is their immortal phenotype, and the enzyme telomerase contributes to the infinite replicative potential of cancer cells. The purpose of this study was to obtain new information about the effect of Amadori-PE on the regulation of telomerase in PANC-1 human pancreatic carcinoma cells. Amadori-PE enhanced cellular telomerase in a time- and dose-dependent manner by up-regulating hTERT expression through induction of c-myc. These results provide experimental evidence for a novel role of Amadori-PE in linking diabetes and cancer.     

Liquid chromatography–tandem mass spectrometry determination of human plasma 1-palmitoyl-2-hydroperoxyoctadecadienoyl-phosphatidylcholine isomers via promotion of sodium adduct formation

Accumulation of phosphatidylcholine hydroperoxide (PCOOH), a primary oxidation product of phosphatidylcholine, in blood plasma has been observed in various pathological conditions, including atherosclerosis. In this study, we investigated the use of liquid chromatography–tandem mass spectrometry (LC–MS/MS) to develop a method for accurate quantification of PCOOH (1-palmitoyl-2-hydroperoxyoctadecadienoyl-sn-glycero-3-phosphocholine, 16:0/HpODE PC), focusing on isomers such as 16:0/13-HpODE PC and 16:0/9-HpODE PC. Sodiated PCOOH ([M+Na]⁺, m/z 812) provided not only a known product ion (m/z 147) but also characteristic product ions (m/z 541 for 16:0/13-HpODE PC and m/z 388 for 16:0/9-HpODE PC). Thus, three multiple reaction monitorings (MRMs) could be performed. MRM (812/147) enabled determination of 16:0/HpODE PC, and MRM (812/541) and MRM (812/388) allowed specific measurement of 16:0/13-HpODE PC and 16:0/9-HpODE PC, respectively. By using this method, we could determine plasma PCOOH concentrations in healthy subjects and patients with angiographically significant stenosis. In healthy subject and patient plasma, the concentration of 16:0/HpODE PC was close to the sum of the concentrations of 16:0/13-HpODE PC and 16:0/9-HpODE PC. This finding shows that radical and/or enzymatic oxidation, rather than singlet oxygen oxidation, is recognized to cause peroxidation of PC. The newly developed LC–MS/MS method appears to be a powerful tool for developing a better understanding of in vivo lipid peroxidation and its involvement in human diseases.     

Overexpression of glyoxalase-I reduces hyperglycemia-induced levels of advanced glycation end products and oxidative stress in diabetic rats

The reactive advanced glycation end product (AGE) precursor methylglyoxal (MGO) and MGO-derived AGEs are associated with diabetic vascular complications and also with an increase in oxidative stress. Glyoxalase-I (GLO-I) transgenic rats were used to explore whether overexpression of this MGO detoxifying enzyme reduces levels of AGEs and oxidative stress in a rat model of diabetes. Rats were made diabetic with streptozotocin, and after 12 weeks, plasma and multiple tissues were isolated for analysis of AGEs, carbonyl stress, and oxidative stress. GLO-I activity was significantly elevated in multiple tissues of all transgenic rats compared with wild-type (WT) littermates. Streptozotocin treatment resulted in a 5-fold increase in blood glucose concentrations irrespective of GLO-I overexpression. Levels of MGO, glyoxal, 3-deoxyglucosone, AGEs, and oxidative stress markers nitrotyrosine, malondialdehyde, and F2-isoprostane were elevated in the diabetic WT rats. In diabetic GLO-I rats, glyoxal and MGO composite scores were significantly decreased by 81%, and plasma AGEs and oxidative stress markers scores were significantly decreased by ～50%. Hyperglycemia induced a decrease in protein levels of the mitochondrial oxidative phosphorylation complex in the gastrocnemius muscle, which was accompanied by an increase in the lipid peroxidation product 4-hydroxy-2-nonenal, and this was counteracted by GLO-I overexpression. This study shows for the first time in an in vivo model of diabetes that GLO-I overexpression reduces hyperglycemia-induced levels of carbonyl stress, AGEs, and oxidative stress. The reduction of oxidative stress by GLO-I overexpression directly demonstrates the link between glycation and oxidative stress.     

基于液质联用的莱茵衣藻极性甘油酯组定性定量分析

以模式藻株莱茵衣藻(Chlamydomonas reinhardtii)为材料, 基于液质联用技术对其极性甘油酯组进行定性定量分析。通过综合利用UPLC-ESI-Q-Trap/MS的一级质谱扫描(中性丢失或母离子扫描)及UPLC-ESI-Orbitrap/MS²的二级碎片信息扫描, 共鉴定出109种极性甘油酯分子; 再通过外标法利用UPLC-ESI-Q-Trap/MS在多级反应监测模式下对各分子进行靶向定量分析。结果表明, 莱茵衣藻的极性脂以糖脂MGDG、DGDG及甜菜碱脂DGTS为主, 所有极性脂的分子组成表明, DGDG、SQDG、DGTS及PI是C18脂肪酸的去饱和载体。该研究利用液质联用技术建立了莱茵衣藻极性甘油酯组的结构图谱及定量分析技术平台, 为微藻极性脂生物学功能及脂质代谢研究奠定了基础。     

Ion-trap tandem mass spectrometric analysis of squalene monohydroperoxide isomers in sunlight-exposed human skin

We previously discovered that squalene monohydroperoxide (SQ-OOH) was produced on human forehead skin and suggested that skin squalene (SQ) may be the principal target lipid for oxidative stress (e.g., sunlight exposure). Because of its six double bonds, SQ peroxidation can yield various positional hydroperoxide isomers. However, the structural characterization of skin SQ-OOH isomers has never been reported. Here, we prepared pure SQ-OOH isomers and developed an analytical method for SQ-OOH isomers using a quadrupole/linear ion-trap mass spectrometer (QTRAP) MS/MS system. Collision-induced dissociation produced specific fragment ions for each SQ-OOH isomer, which permitted discrimination between SQ-OOH isomers by multiple reaction monitoring (MRM). When lipid extract from human forehead skin was subjected to LC-MS/MS with MRM, individual SQ-OOH isomers could be separated and detected with a sensitivity of 0.05 ng/injection. The total concentration of SQ-OOH isomers in forehead skin was approximately 956 microg/g skin lipids, but it increased up to 2,760 microg/g skin lipids after 3 h of sunlight exposure. The LC-MS/MS method was useful for investigating the peroxidation mechanisms of SQ as well as SQ-OOH-mediated skin disorders.     

Direct-injection screening for acidic drugs in plasma and neutral drugs in equine urine by differential-gradient LC-LC coupled MS/MS

Direct-injection LC-LC hybrid tandem MS methods have been developed for undertaking broad-based screening for acidic drugs in protein-precipitated plasma and neutral doping agents in equine urine. In both analyses, analytes present in the matrix were trapped using a HLB extraction column before being refocused and separated on a Chromolith RP-18e monolithic analytical column using a controlled differential gradient generated by proportional dilution of the first column's eluent with water. Each method has been optimised by the adoption of a mobile phase and gradient that was tailored to enhance ionisation in the MS source while maintaining good chromatographic behaviour for the majority of the target drugs. The analytical column eluent was fed into the heated nebulizer (HN) part of the Duospray interface attached to a 4000 QTRAP mass spectrometer. Information dependent acquisition (IDA) with dynamic background subtraction (DBS) was configured to trigger a sensitive enhanced product ion (EPI) scan when a multiple reaction monitoring (MRM) survey scan signal exceeded the defined criteria. Ninety-one percent of acidic drugs in protein-precipitated plasma and 80% of the neutral compounds in equine urine were detected when spiked at 10 ng/ml.     

Advanced glycation end products (AGEs), free radicals and diabetes

Nonenzymatic glycation, i.e. binding of monosaccharides to amino groups of proteins, gives rise to complex components called "advanced glycation end-products" (AGEs), which alter protein structure and functions, and participate in diabetic long-term complications. Glycation and oxidative stress are closely linked, and are often referred to as "glycoxidation" processes. Experimental data support these interactions. a) All glycation steps generate oxygen free radicals, some of these steps being common with these of lipid peroxidation. b) AGEs bind to membrane receptors such as RAGE, and induce an oxidative stress and a pro-inflammatory status. c) Glycated proteins modulate cellular oxidative functions: glycated collagens induce an inappropriate oxidative response in PMNs. d) Products of lipid peroxidation (MDA) bind to proteins and amplify glycoxidation-induced damages. Glycoxydation intensity increases in diabetes mellitus, ageing, renal failure and other pathological states with oxidative stress. Therapies aiming at limiting glycoxidation take into account its oxidative part.     

Glycation of apolipoprotein E impairs its binding to heparin: identification of the major glycation site.

The increased glycation of plasma apolipoproteins represents a possible major factor for lipid disturbances and accelerated atherogenesis in diabetic patients. The glycation of apolipoprotein E (apoE), a key lipid-transport protein in plasma, was studied both in vivo and in vitro. ApoE was shown to be glycated in plasma very low density lipoproteins of both normal subjects and hyperglycemic, diabetic patients. However, diabetic patients with hyperglycemia showed a 2-3-fold increased level of apoE glycation. ApoE from diabetic plasma showed decreased binding to heparin compared to normal plasma apoE. The rate of Amadori product formation in apoE in vitro was similar to that for albumin and apolipoproteins A-I and A-II. The glycation of apoE in vitro significantly decreased its ability to bind to heparin, a critical process in the sequestration and uptake of apoE-containing lipoproteins by cells. Diethylenetriaminepentaacetic acid, a transition metal chelator, had no effect on the loss of apoE heparin-binding activity, suggesting that glycation rather than glycoxidation is responsible for this effect. In contrast, glycation had no effect on the interaction of apoE with amyloid beta-peptide. ApoE glycation was demonstrated to be isoform-specific. ApoE(2) showed a higher glycation rate and the following order was observed: apoE(2)>apoE(4)>apoE(3). The major glycated site of apoE was found to be Lys-75. These findings suggest that apoE is glycated in an isoform-specific manner and that the glycation, in turn, significantly decreases apoE heparin-binding activity. We propose that apoE glycation impairs lipoprotein-cell interactions, which are mediated via heparan sulfate proteoglycans and may result in the enhancement of lipid abnormalities in hyperglycemic, diabetic patients.     

Effect of aminophospholipid glycation on order parameter and hydration of phospholipid bilayer

The effect of aminophospholipid glycation on lipid order and lipid bilayer hydration was investigated using time-resolved fluorescence spectroscopy. The changes of lipid bilayer hydration were estimated both from its effect on the fluorescence lifetime of The 1-[4-(trimethylammonium)-phenyl]-6-phenylhexa-1,3,5-triene (TMA-DPH) and 1,6-diphenylhexa-1,3,5-triene (DPH) and using solvatochromic shift studies with 1-anilinonaphthalene-8-sulfonic acid. The head-group and acyl chain order were determined from time-resolved fluorescence anisotropy measurements of the TMA-DPH and DPH. The suspensions of small unilamellar vesicles (with phosphatidylethanolamine/phosphatidylcholine molar ratio 1:2.33) were incubated with glyceraldehyde and it was found that aminophospholipids react with glyceraldehyde to form products with the absorbance and the fluorescence properties typical for protein advanced glycation end products. The lipid glycation was accompanied by the progressive oxidative modification of unsaturated fatty acid residues. It was found that aminophospholipid glycation increased the head-group hydration and lipid order in both regions of the membrane. The lipid oxidation accompanying the lipid glycation affected mainly the lipid order, while the effect on the lipid hydration was small. The increase in the lipid order was presumably the result of two effects: (1) the modification of head-groups of phosphatidylethanolamine by glycation; and (2) the degradation of unsaturated fatty acid residues by oxidation.     

A Contrast of the Plasma Membrane Lipid Composition of Oat and Rye Leaves in Relation to Freezing Tolerance

The lipid composition of the plasma membrane isolated from leaves of spring oat (Avena sativa L. cv Ogle) was vastly different from that of winter rye (Secale cereale L. cv Puma). The plasma membrane of spring oat contained large proportions of phospholipids (28.8 mol% of the total lipids), cerebrosides (27.2 mol%), and acylated sterylglucosides (27.3 mol%) with lesser proportions of free sterols (8.4 mol%) and sterylglucosides (5.6 mol%). In contrast, the plasma membrane of winter rye contained a greater proportion of phospholipids (36.6 mol%), and there was a lower proportion of cerebrosides (16.4 mol%); free sterols (38.1 mol%) were the predominant sterols, with lesser proportions of sterylglucosides (5.6 mol%) and acylated sterylglucosides (2.9 mol%). Although the relative proportions of individual phospholipids, primarily phosphatidylcholine and phosphatidylethanolamine, and the molecular species of these two phospholipids were similar in oat and rye, the relative proportions of di-unsaturated species of these two phospholipids were substantially lower in oat than in rye. The relative proportions of sterol species in oat were different from those in rye; the molecular species of cerebrosides were similar in oat and rye, with only slight differences in the proportions of the individual species. After 4 weeks of cold acclimation, the proportion of phospholipids increased significantly in both oat (from 28.8 to 36.8 mol%) and rye (from 36.6 to 43.3 mol%) as a result of increases in the proportions of phosphatidylcholine and phosphatidylethanolamine. For both oat and rye, the relative proportions of di-unsaturated species increased after cold acclimation, but the increase was greater in rye than in oat. In both oat and rye, this increase occurred largely during the first week of cold acclimation. During the 4 weeks of cold acclimation, there was a progressive decrease in the proportion of cerebrosides in the plasma membrane of rye (from 16.4 to 10.5 mol%), but there was only a small decrease in oat (from 27.2 to 24.2 mol%). In both oat and rye, there were only small changes in the proportions of free sterols and sterol derivatives during cold acclimation. Consequently, the proportions of both acylated sterylglucosides and cerebrosides remained substantially higher in oat than in rye after cold acclimation. The relationship between these differences in the plasma membrane lipid composition of oat and rye and their freezing tolerance is presented.     

Protective effects of fluvastatin against reactive oxygen species induced DNA damage and mutagenesis

Oxidative stress may be an important factor in the development of diabetic complications. Advanced glycation end-products have drown attention as potential sources of oxidative stress in diabetes. We investigated the protective effects of fluvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, on oxidative DNA damage from reactive oxygen species or advanced glycation end-products in vitro, as well as effects of main fluvastatin metabolites and other inhibitors of the same enzyme, pravastatin and simvastatin. Protective effects were assessed in terms of the DNA breakage rate in a single-stranded phage DNA system in vitro. DNA was exposed to either reactive oxygen species or advanced glycation end-products. Fluvastatin and its metabolites showed a strong protective effect comparable to those seen with thiourea and mannitol, though pravastatin and simvastatin did not exert clear protective effects. Furthermore, fluvastatin reduced the mutagenesis by reactive oxygen species or advanced glycation end-products in Salmonella typhimurium test strains. Both pravastatin and simvastatin still lacked protective activity. Fluvastatin and its metabolites protect against oxidative DNA damage and may reduce risk of consequent diabetic complications.     

Differential carbonylation of proteins as a function of in vivo oxidative stress

This study reports for the first time qualitative and quantitative differences in carbonylated proteins shed into blood as a function of increasing levels of OS. Carbonylated proteins in freshly drawn blood from pairs of diabetic and lean rats were derivatized with biotin hydrazide, dialyzed, and enriched with avidin affinity chromatography. Proteins thus selected were used in several ways. Differences between control and diabetic subjects in relative concentration of proteins was achieved by differential labeling of tryptic digests with iTRAQ reagents followed by reversed phase chromatography (RPC) and tandem mass spectrometry (MS/MS). Identification and characterization of OS induced post-translational modification sites in contrast was achieved by fractionation of affinity selected proteins before proteolysis and RPC-MS/MS. Relative quantification of peptides bearing oxidative modifications was achieved for the first time by selective reaction monitoring (SRM). Approximately 1.7% of the proteins in Zucker diabetic rat plasma were selected by the avidin affinity column as compared to 0.98% in lean animal plasma. Among the 35 proteins identified and quantified, Apo AII, clusterin, hemopexin precursor, and potassium voltage-gated channel subfamily H member 7 showed the most dramatic changes in concentration. Seventeen carbonylation sites were identified and quantified, 11 of which changed more than 2-fold in oxidation state. Three types of carbonylation were identified at these sites: direct oxidative cleavage from reactive oxygen species, glycation and addition of advanced glycation end products, and addition of lipid peroxidation products. Direct oxidation was the dominant form of carbonylation observed while hemoglobin and murinoglobulin 1 homologue were the most heavily oxidized proteins.