The Structure of Mammalian Prions and Their Aggregates.

Prion diseases, such as Creutzfeldt-Jakob disease in humans, bovine spongiform encephalopathy in cattle, chronic wasting disease in cervids (i.e., deer, elk, moose, and reindeer), and sheep scrapie, are caused by the misfolding of the cellular prion protein (PrPC) into a disease-causing conformer (PrPSc). PrPC is a normal, GPI-anchored protein that is expressed on the surface of neurons and other cell types. The structure of PrPC is well understood, based on studies of recombinant PrP, which closely mimics the structure of native PrPC. In contrast, PrPSc is prone to aggregate into a variety of quaternary structures, such as oligomers, amorphous aggregates, and amyloid fibrils. The propensity of PrPSc to assemble into these diverse forms of aggregates is also responsible for our limited knowledge about its structure. Then again, the repeating nature of certain regular PrPSc aggregates has allowed (lower resolution) insights into the structure of the infectious conformer, establishing a four-rung ß-solenoid structure as a key element of its architecture.

Lesión oxidativa de proteínas de hígado y corazón de rata durante el proceso de envejecimiento / Protein oxidative damage in rat liver and heart during the ageing process

Objetivos: el proceso de envejecimiento afecta a todos los organismos y es de suponer que sus mecanismos básicos esten conservados entre especies. La oxidación de proteínas se ha propuesto como uno de estos mecanismos básicos enlazando los radicales derivados del oxígeno con el proceso básico de envejecimiento. Así, si la lesión oxidativa de proteínas esta implicada en el envejecimiento, los animales con edades avanzadas deberían presentar valores basales elevados de lesión proteínica en comparación con animales más jóvenes. Sin embargo, la información disponible sobre esta predicción es limitada considerando la utilización de marcadores químicos específicos. Material y método: en este estudio, la concentración basal de diferentes marcadores de lesión proteínica (oxidación [semialdehídos glutámico y aminoadípico], glucoxidación [carboxietil-lisina], mixto de gluco y lipoxidación [carboximetil-lisina] y lipoxidación [malondialdehído-lisina]), así como el perfil de ácidos grasos de membrana, han sido determinados en el hígado y corazón de ratas macho adultas jóvenes (8 meses) y edad avanzada (30 meses). Resultados: los resultados mostraron incrementos significativos de los marcadores de oxidación y glucoxidación en el grupo de animales de 30 meses tanto en hígado como en corazón. Con relación a la lesión lipoxidativa, también se detectó un aumento significativo de ésta en el grupo de edad avanzada, probablemente asociado al incremento del índice de dobles enlaces y peroxidabilidad mostrados por las membranas celulares del hígado y corazón. Comparativamente, el corazón mostró grados de lesión oxidativa y lipoxidativa significativamente superiores a los del hígado. Los niveles de lesión glucoxidativa fueron superiores en hígado que en corazón. Asimismo, el corazón mostró mayor sensibilidad al envejecimiento, como lo demuestran los mayores incrementos porcentuales en lesión oxidativa, gluco y lipoxidativa. Conclusiones: estos resultados, en el contexto de la teoría del envejecimiento basada en el estrés oxidativo, además de poner de manifiesto las diferencias existentes entre órganos, refuerzan el papel del grado de insaturación de membrana, así como de los mecanismos homeostáticos que regulan el valor basal de modificación oxidativa en dicho proceso biológico

Objectives: ageing affects all organisms and its basic mechanisms can be expected to be conserved across species. Protein oxidation has been proposed as one of the basic mechanisms linking oxygen radicals with the basic ageing process. If oxidative damage to proteins is involved in ageing, aged animals should show higher steady-state levels of specific markers of this kind of damage than younger adult animals. However, the evidence available on this prediction is limited when the use of specific chemical markers is considered. Material and method: we measured steady-state levels of markers of different kinds of protein damage-oxidation (glutamic and aminoadipic semialdehydes), glycoxidation (carboxyethyl-lysine), mixed glyco- and lipoxidation (carboxymethyl-lysine), and lipoxidation (malondialdehydelysine)-, as well as fatty acid profile in the livers and hearts of young adult (8 months old) and aged (30 months old) male rats. Results: oxidative and glycoxidative markers were significantly increased in both the liver and heart of aged rats. Lipoxidation damage was also significantly increased in aged rats. This result was probably associated with the significantly increased double bond and peroxidizability indexes found in liver and heart cell membranes. Steady-state levels of oxidation and lipoxidation damage were significantly higher in the heart than in the liver. In contrast, glycoxidation damage was greater in the liver than in the heart. Accumulation rates of protein damage were higher in the heart than in the liver during aging. Conclusions: in the context of the oxidative stress theory of ageing, these results reveal differences between organs and reinforce the role of membrane unsaturation as well as the homeostatic mechanisms that maintain the steady-state level of protein damage in this biological process

Recent advances in the analysis of oxidized proteins.

Glutamic semialdehyde is a product of oxidation of arginine and proline, and aminoadipic semialdehyde, of oxidation of lysine. These two carbonyl-containing compounds are the main carbonyl products of metal-catalyzed oxidation of proteins, accounting for 55-100% of the total carbonyl value. Accordingly, they are quantitatively very important contributors to the total value of protein carbonyls in tissues as measured by the classic spectophotometric assay. Sensitive gas chromatography-mass spectrometry based analytical methods allow their quantitation in a variety of biological samples, including tissue protein, cell cultures and lipoproteins. These measurements provide specific information on the oxidative status of proteins that is complementary to that afforded by protein carbonyls, and will be useful tools in the ongoing effort to define and assess the role of protein oxidation in pathology and aging.

Protein modification by advanced Maillard adducts can be modulated by dietary polyunsaturated fatty acids.

Advanced Maillard adducts, such as N epsilon-(carboxymethyl)lysine and N epsilon-(carboxyethyl)lysine, can be formed efficiently in vitro from both peroxidation of polyunsaturated fatty acids and glycolysis intermediates. In an attempt to differentiate the in vivo influence of the two pathways in these modifications, Wistar rats were chronically fed with specially designed diets rich in saturated or unsaturated fats. The degree of fatty acid unsaturation of all analysed organs (liver, kidney, brain) was altered by these dietary stresses. Protein glycoxidative and lipoxidative modifications were measured by GC/MS. In accordance with fatty acid profiles, concentrations of N epsilon-(malondialdehyde)lysine in these tissues were significantly increased in animals fed the unsaturated fat diet. In contrast, N epsilon-(carboxymethyl)lysine and N epsilon-(carboxyethyl)lysine concentrations were strongly dependent on the tissue analysed; although the unsaturated fat diet increased their levels significantly in brain, levels were unchanged in kidney and decreased in liver. These later results could be interpreted on the basis that polyunsaturated fatty acids decrease the expression of several glycolytic enzymes in liver. Globally, these data suggest that tissue-specific metabolic characteristics play a key role in the degree of cellular protein modification by Maillard reactions, e.g. by modulation of the concentration of glycolysis intermediates or via specific defensive systems in these organs.

Copper-catalyzed oxidation of the recombinant SHa(29-231) prion protein.

Metal-catalyzed oxidation may result in structural damage to proteins and has been implicated in aging and disease, including neurological disorders such as Alzheimer's disease and amyotrophic lateral sclerosis. The selective modification of specific amino acid residues with high metal ion affinity leads to subtle structural changes that are not easy to detect but may have dramatic consequences on physical and functional properties of the oxidized protein molecules. PrP contains a histidine-rich octarepeat domain that binds copper. Because copper-binding histidine residues are particularly prone to metal-catalyzed oxidation, we investigated the effect of this reaction on the recombinant prion protein SHaPrP(29-231). Using Cu2+/ascorbate, we oxidized SHaPrP(29-231) in vitro. Oxidation was demonstrated by liquid chromatography/mass spectrometry, which showed the appearance of protein species of higher mass, including increases in multiples of 16, characteristic of oxygen incorporation. Digestion studies using Lys C indicate that the 29-101 region, which includes the histidine-containing octarepeats, is particularly affected by oxidation. Oxidation was time- and copper concentration-dependent and was evident with copper concentrations as low as 1 microM. Concomitant with oxidation, SHaPrP(29-231) suffered aggregation and precipitation, which was nearly complete after 15 min, when the prion protein was incubated at 37 degrees C with a 6-fold molar excess of Cu2+. These findings indicate that PrP, a copper-binding protein, may be particularly susceptible to metal-catalyzed oxidation and that oxidation triggers an extensive structural transition leading to aggregation.

Thioredoxin converts the Syrian hamster (29-231) recombinant prion protein to an insoluble form.

The prion protein (PrP) is an essential, and probably the only, component of the infectious agent responsible for the transmissible spongiform encephalopathies. In its cellular (PrP(C)) form, it is a soluble, alpha-helix-rich protein of yet unknown function attached to the outer membrane of neurons through a glycosylphosphatidyl inositol anchor. In its pathogenic, "scrapie" form (PrP(Sc)), it appears as an aggregate showing no detectable covalent modifications but displaying a profoundly altered conformation enriched in beta-sheet structure. Reduction of the single disulfide bridge in the prion protein with millimolar concentrations of dithiothreitol results in transformation of the alpha-helix-rich to the beta-sheet-rich conformation, with concomitant decrease in solubility. We report here that thioredoxin coupled with thioredoxin reductase and NADPH efficiently reduces recombinant Syrian hamster (29-231) prion protein under physiologically relevant conditions. The reduced prion protein immediately becomes insoluble and precipitates, although it does not gain significant resistance to proteinase K. The thioredoxin/thioredoxin reductase system is approximately 7000 times more efficient than dithiothreitol.

Glutamic and aminoadipic semialdehydes are the main carbonyl products of metal-catalyzed oxidation of proteins.

Metal-catalyzed oxidation results in loss of function and structural alteration of proteins. The oxidative process affects a variety of side amino acid groups, some of which are converted to carbonyl compounds. Spectrophotometric measurement of these moieties, after their reaction with 2,4-dinitrophenylhydrazine, is a simple, accurate technique that has been widely used to reveal increased levels of protein carbonyls in aging and disease. We have initiated studies aimed at elucidating the chemical nature of protein carbonyls. Methods based on gas chromatography/mass spectrometry with isotopic dilution were developed for the quantitation of glutamic and aminoadipic semialdehydes after their reduction to hydroxyaminovaleric and hydroxyaminocaproic acids. Analysis of model proteins oxidized in vitro by Cu2+/ascorbate revealed that these two compounds constitute the majority of protein carbonyls generated. Glutamic and aminoadipic semialdehydes were also detected in rat liver proteins, where they constitute approximately 60% of the total protein carbonyl value. Aminoadipic semialdehyde was also measured in protein extracts from HeLa cells, and its level increased as a consequence of oxidative stress to cell cultures. These results indicate that glutamic and aminoadipic semialdehydes are the main carbonyl products of metal-catalyzed oxidation of proteins, and that this reaction is a major route leading to the generation of protein carbonyls in biological samples.

Low fatty acid unsaturation: a mechanism for lowered lipoperoxidative modification of tissue proteins in mammalian species with long life spans.

Carbonyl compounds generated by the nonenzymatic oxidation of polyunsaturated fatty acids react with nucleophilic groups in proteins, leading to their modification. It has not been tested whether fatty acid unsaturation is related to steady-state levels of lipoxidation-derived protein modification in vivo. A low fatty acid unsaturation, hence a low protein lipoxidation, in tissues of longevous animals would be consistent with the free radical theory of aging, because membrane lipids increase their sensitivity to oxidative damage as a function of their degree of unsaturation. To evaluate the relationship between fatty acid composition, protein lipoxidation, and maximum life span (MLSP), we analyzed liver fatty acids and proteins from seven mammalian species, ranging in MLSP from 3.5 to 46 years. The results show that the peroxidizability index of fatty acids and the sensitivity to in vitro lipid peroxidation are negatively correlated with the MLSP. Based on gas chromatography and mass spectroscopy analyses, liver proteins of all these species contain malondialdehyde-lysine and Nepsilon-carboxymethyllysine adducts, two biomarkers of protein lipoxidation. The steady-state levels of malondialdehyde-lysine and Nepsilon-carboxymethyl lysine are directly related to the peroxidizability index and inversely related to the MLSP. We propose that a low degree of fatty acid unsaturation may have been selected in longevous mammals to protect their tissue lipids and proteins against oxidative damage while maintaining an appropriate environment for membrane function.

Measurement of pentosidine in biological samples.

Pentosidine is a highly fluorescent advanced glycation end product (AGE) and crosslink derived from one molecule of arginine and one of lysine bridged in an imidazo-pyridinium structure (Fig. 1). It was first isolated from articular cartilage by Sell and Monnier (1), and has now been detected and quantified in a variety of human and animal tissues, including skin and kidney collagen (2-5), lens crystallins (6, 7), plasma (8, 9), serum (10), urine (11), and synovial fluid (12, 13). Pentosidine is readily prepared from arginine, lysine, and a pentose (hence its name). Dyer et al. (14) have also described its formation from glucose, albeit at a slower rate and probably through oxidation of glucose to arabinose (15). Because its formation from either glucose or ribose requires oxidation, pentosidine is both an AGE and a "glycoxidation" product (16). Fig. 1. Structure of pentosidine.

Conversion of lysine to N(epsilon)-(carboxymethyl)lysine increases susceptibility of proteins to metal-catalyzed oxidation.

Metal-catalyzed oxidation (MCO) of proteins leads to the conversion of some amino acid residues to carbonyl derivatives, and may result in loss of protein function. It is well documented that reactions with oxidation products of sugars, lipids, and amino acids can lead to the conversion of some lysine residues of proteins to N(epsilon)-(carboxymethyl)lysine (CML) derivatives, and that this increases their metal binding capacity. Because post-translational modifications that enhance their metal binding capacity should also increase their susceptibility to MCO, we have investigated the effect of lysine carboxymethylation on the oxidation of bovine serum albumin (BSA) by the Fe(3+)/ascorbate system. Introduction of approximately 10 or more mol CML/mol BSA led to increased formation of carbonyls and of the specific oxidation products glutamic and adipic semialdehydes. These results support the view that the generation of CML derivatives on proteins may contribute to the oxidative damage that is associated with aging and a number of age-related diseases.

Thyroid status modulates glycoxidative and lipoxidative modification of tissue proteins.

Steady state protein modification by carbonyl compounds is related to the rate of carbonyl adduct formation and the half-life of the protein. Thyroid hormones are physiologic modulators of both tissue oxidative stress and protein degradation. The levels of the glycation product N(epsilon)-fructoselysine (FL) and those of the oxidation products, N(epsilon)-(carboxymethyl)lysine (CML) and malondialdehyde-lysine (MDA-lys), identified by GC/MS in liver proteins, decreased significantly in hyperthyroid rats, as well as (less acutely) in hypothyroid animals. Immunoblotting of liver proteins for advanced glycation end-products (AGE) is in agreement with the results obtained by GC/MS. Cytosolic proteolytic activity against carboxymethylated foreign proteins measured in vitro was significantly increased in hypo- and hyperthyroidism. Oxidative damage to DNA, estimated as 8-oxo-7,8-dihydro-2'-deoxyguanosine (8oxodG), did not show significant differences between groups. The results suggests that the steady state levels of these markers depend on the levels of thyroid hormones, presumably through their combined effects on the rates of protein degradation and oxidative stress, whereas DNA is more protected from oxidative damage.

The myeloperoxidase system of human phagocytes generates Nepsilon-(carboxymethyl)lysine on proteins: a mechanism for producing advanced glycation end products at sites of inflammation.

Reactive aldehydes derived from reducing sugars and peroxidation of lipids covalently modify proteins and may contribute to oxidative tissue damage. We recently described another mechanism for generating reactive aldehydes from free alpha-amino acids. The pathway begins with myeloperoxidase, a heme enzyme secreted by activated neutrophils. Conversion of alpha-amino acids to aldehydes requires hypochlorous acid (HOCl), formed from H2O2 and chloride by myeloperoxidase. When L-serine is the substrate, HOCl generates high yields of glycolaldehyde. We now demonstrate that a model protein, ribonuclease A (RNase A), exposed to free L-serine and HOCl exhibits the biochemical hallmarks of advanced glycation end (AGE) products -- browning, increased fluorescence, and cross-linking. Furthermore, Nepsilon-(carboxymethyl)lysine (CML), a chemically well-characterized AGE product, was generated on RNase A when it was exposed to reagent HOCl-serine, the myeloperoxidase-H2O2-chloride system plus L-serine, or activated human neutrophils plus L-serine. CML production by neutrophils was inhibited by the H2O2 scavenger catalase and the heme poison azide, implicating myeloperoxidase in the cell-mediated reaction. CML was also generated on RNase A by a myeloperoxidase-dependent pathway when neutrophils were activated in a mixture of amino acids. Under these conditions, we observed both L-serine-dependent and L-serine-independent pathways of CML formation. The in vivo production of glycolaldehyde and other reactive aldehydes by myeloperoxidase may thus play an important pathogenic role by generating AGE products and damaging tissues at sites of inflammation.

Quantification of N-(glucitol)ethanolamine and N-(carboxymethyl)serine: two products of nonenzymatic modification of aminophospholipids formed in vivo.

Chemical, nonenzymatic modification of protein and lipids by reducing sugars, such as glucose, is thought to contribute to age-related deterioration in tissue protein and cellular membranes and to the pathogenesis of diabetic complications. This report describes the synthesis and quantification of N-(glucitol)ethanolamine (GE) and N-(carboxymethyl)serine (CMS), two products of nonenzymatic modification of aminophospholipids. GE is the product of reduction and hydrolysis of glycated phosphatidylethanolamine (PE), while CMS is formed through reaction of phosphatidylserine (PS) with products of oxidation of either carbohydrate (glycoxidation) or lipids (lipoxidation). Gas chromatography/mass spectrometry procedures for quantification of the N,O-acetyl methyl ester derivatives of the modified head groups were developed. GE and CMS were quantified in samples of PE and PS, respectively, following incubation with glucose in vitro; CMS formation was dependent on the presence of oxygen during the incubation. Both GE and CMS were detected and quantified in lipid extracts of human red blood cell membranes. The content of GE, but not CMS, was increased in the lipids from diabetic compared to nondiabetic subjects. Measurement of these modified lipids should prove useful for assessing the role of carbonyl-amine reactions of aminophospholipids in aging and age-related diseases.

A low degree of fatty acid unsaturation leads to lower lipid peroxidation and lipoxidation-derived protein modification in heart mitochondria of the longevous pigeon than in the short-lived rat.

Birds have a maximum longevity (MLSP) much greater than mammals of similar metabolic rate and body size. Thus, they are ideal models to identify longevity characteristics not linked to low metabolic rates. In this investigation, we show that the fatty acid double bond content of total lipids and phosphatidylcholine, phosphatidylethanolamine and cardiolipin fractions of heart mitochondria is intrinsically lower in pigeons (MLSP = 35 years) than in rats (MLSP = 4 years). This is mainly due to a lower content of the most highly unsaturated docosahexaenoic acid (22:6n-3) and in some fractions arachidonic acid (20:4n-6). The lower double bond content leads to a lower sensitivity to in vitro lipid peroxidation, and is associated with a lower concentration of lipid peroxidation products in vivo, and a lower level of malondialdehyde-lysine protein adducts in heart mitochondria of pigeons than rats. These results, together with those previously obtained in other species or tissues, suggest that a low degree of fatty acid unsaturation is a general characteristic of longevous homeothermic vertebrate animals both when they have low metabolic rates (mammals of large body size) or high metabolic rates (small sized birds). This constitutive trait helps to protect their tissues and mitochondria against lipid peroxidation and oxidative protein modification and can be a factor contributing to their slow rate of aging. The results also show, for the first time in a physiological model, that lipid peroxidizability is related to lipoxidative protein damage.

Carboxymethylated phosphatidylethanolamine in mitochondrial membranes of mammals--evidence for intracellular lipid glycoxidation.

The non-enzymatic modification of aminophospholipids with lipoperoxidation-derived aldehydes and glycoxidation-derived products have been reported previously. However, it remains unknown whether intracellular membranes are damaged by these glycoxidation-derived products. To investigate this issue, we tested whether aminophospholipids from mitochondrial membranes are damaged by glycoxidative stress the mitochondrion being identified as the major site of reactive-species production in the cell. We have used a selected-ion-monitoring/gas-chromatography/mass-spectrometry assay for carboxymethylethanolamine (CM-Etn) detection, and provide evidence for the presence of CM-Etn in mitochondrial phospholipids. Further, as a physiological approach to evaluate the influence of mitochondrial oxidative stress in CM-Etn formation, we also present the comparative levels of CM-Etn in mitochondrial membranes of ten mammalian species ranging in maximum life-span from 3.5 years to 100, since the rate of mitochondrial reactive-oxygen-species production is inversely correlated to the maximum life-span. Our results show that CM-Etn levels correlate in a logarithmic fashion with the maximum-life-span [[CM-Etn] = 0.51 + 0.50 x', where x' = log(maximum-life-span); r = 0.81, P < 0.004]. The data demonstrate the intracellular occurrence of glycoxidative processes affecting membrane lipids. Moreover, these data show that longer-lived mammals contain higher levels of CM-Etn in mitochondrial membrane aminophospholipids. This trend could result from differences in rates of CM-Etn accumulation and/or phospholipid turnover.

Increased concentrations of serum pentosidine in rheumatoid arthritis.

Advanced glycosylation end products (AGEs) are thought to play an important role in the development of diabetic complications. Oxidative reactions are essential for the formation of some AGEs, termed glycoxidation products. Increased concentrations of pentosidine, one of such products, are found in tissue and serum in diabetes mellitus and in end-stage renal disease, suggesting that hyperglycemia and impaired renal function are important factors in AGE accumulation. We hypothesized that increased concentrations of pentosidine would also be found in pathological conditions associated with increased oxidative stress. We measured pentosidine in sera of patients with rheumatoid arthritis (RA), systemic lupus erythematosus, and diabetes. Increased serum pentosidine was found in RA (108.4 +/- 146.5 nmol/L, P < 0.002) and in diabetes (69.6 +/- 42.4 nmol/L, P < 0.001) as compared with healthy subjects (48.3 +/- 12.0 nmol/L). These results prove that AGEs may accumulate in the absence of hyperglycemia or impaired kidney function.

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.

Lipoxidation products as biomarkers of oxidative damage to proteins during lipid peroxidation reactions.

Oxidative stress is implicated in the pathogenesis of numerous disease processes including diabetes mellitus, atherosclerosis, ischaemia reperfusion injury and rheumatoid arthritis. Chemical modification of amino acids in protein during lipid peroxidation results in the formation of lipoxidation products which may serve as indicators of oxidative stress in vivo. The focus of the studies described here was initially to identify chemical modifications of protein derived exclusively from lipids in order to assess the role of lipid peroxidative damage in the pathogenesis of disease. Malondialdehye (MDA) and 4-hydroxynonenal (HNE) are well characterized oxidation products of polyunsaturated fatty acids on low-density lipoprotein (LDL) and adducts of these compounds have been detected by immunological means in atherosclerotic plaque. Thus, we first developed gas chromatography-mass spectrometry assays for the Schiff base adduct of MDA to lysine, the lysine-MDA-lysine diimine cross-link and the Michael addition product of HNE to lysine. Using these assays, we showed that the concentrations of all three compounds increased significantly in LDL during metal-catalysed oxidation in vitro. The concentration of the advanced glycation end-product N epsilon-(carboxymethyl)lysine (CML) also increased during LDL oxidation, while that of its putative carbohydrate precursor the Amadori compound N epsilon-(1-deoxyfructose-1-yl)lysine did not change, demonstrating that CML is a marker of both glycoxidation and lipoxidation reactions. These results suggest that MDA and HNE adducts to lysine residues should serve as biomarkers of lipid modification resulting from lipid peroxidation reactions, while CML may serve as a biomarker of general oxidative stress resulting from both carbohydrate and lipid oxidation reactions.