Neopterin and 7,8-Dihydroneopterin Interfere With Low Density Lipoprotein Oxidation Mediated by Peroxynitrite and/or Copper

Low density lipoprotein (LDL) oxidation within the artery wall likely represents a key event in the formation of atherosclerotic lesions. Oxidatively modified LDL particles exert chemotactic properties on macrophages, and the uncontrolled uptake of modified LDL by macrophages leads to the formation of lipid-loaded foam cells, a hallmark of early stage atherosclerosis. Human macrophages stimulated by interferon- &#110 generate reactive oxygen species (ROS), neopterin, and 7,8-dihydroneopterin. Higher concentrations of neopterin were found in atherosclerosis, and earlier studies have provided evidence that these neopterin derivatives are able to interfere with reactive species. We therefore investigated whether they also modulate LDL oxidation mediated by Cu(II) and/or peroxynitrite (ONOO &#109 ). By means of UV-absorption recording the formation of conjugated dienes in the course of lipid oxidation as well as by measuring the relative electrophoretic mobility of oxidized LDL, we found that neopterin is capable of enhancing ONOO &#109 - as well as Cu(II)-mediated LDL oxidation, whereas 7,8-dihydroneopterin mainly protects LDL from oxidation. However, in case of Cu(II)-mediated LDL oxidation, an initial prooxidative effect of 7,8-dihydroneopterin could be observed. We hypothesize that 7,8-dihydroneopterin may chemically reduce Cu(II) to Cu(I) thereby increasing its oxidative capacity. After total reduction of Cu(II), excess 7,8-dihydroneopterin may block the oxidative potential of Cu(I) and thus decrease the oxidation of LDL. These findings confirm the general behavior of pteridines in redox processes and suggest an in vivo contribution to the process of LDL oxidation.     

Neopterin and 7,8-dihydroneopterin interfere with low density lipoprotein oxidation mediated by peroxynitrite and/or copper

Low density lipoprotein (LDL) oxidation within the artery wall likely represents a key event in the formation of atherosclerotic lesions. Oxidatively modified LDL particles exert chemotactic properties on macrophages, and the uncontrolled uptake of modified LDL by macrophages leads to the formation of lipid-loaded foam cells, a hallmark of early stage atherosclerosis. Human macrophages stimulated by interferon- γgenerate reactive oxygen species (ROS), neopterin, and 7,8-dihydroneopterin. Higher concentrations of neopterin were found in atherosclerosis, and earlier studies have provided evidence that these neopterin derivatives are able to interfere with reactive species. We therefore investigated whether they also modulate LDL oxidation mediated by Cu(II) and/or peroxynitrite (ONOO -). By means of UV-absorption recording the formation of conjugated dienes in the course of lipid oxidation as well as by measuring the relative electrophoretic mobility of oxidized LDL, we found that neopterin is capable of enhancing ONOO -- as well as Cu(II)-mediated LDL oxidation, whereas 7,8-dihydroneopterin mainly protects LDL from oxidation. However, in case of Cu(II)-mediated LDL oxidation, an initial prooxidative effect of 7,8-dihydroneopterin could be observed. We hypothesize that 7,8-dihydroneopterin may chemically reduce Cu(II) to Cu(I) thereby increasing its oxidative capacity. After total reduction of Cu(II), excess 7,8-dihydroneopterin may block the oxidative potential of Cu(I) and thus decrease the oxidation of LDL. These findings confirm the general behavior of pteridines in redox processes and suggest an in vivo contribution to the process of LDL oxidation.     

Macrophage mediated protein hydroperoxide formation and lipid oxidation in low density lipoprotein are inhibited by the inflammation marker 7,8-dihydroneopterin

The formation of oxidised low density lipoprotein (LDL) within the atherosclerotic plaque appears to be a factor in the development of advanced atherosclerotic plaques. LDL oxidation is dependent on the balance of oxidants and antioxidants within the intima. In addition to producing various oxidants, human macrophages release 7,8-dihydroneopterin which in vivo is oxidised to the inflammation marker neopterin. Using macrophage-like THP-1 cells and human monocyte-derived macrophages, we demonstrate that 7,8-dihydroneopterin is a potent inhibitor of cell-mediated LDL oxidation. 7,8-Dihydroneopterin scavenges the chain propagating lipid peroxyl radical, inhibiting both lipid and protein hydroperoxide formation. A significant amount of the hydroperoxide formed during cell-mediated LDL oxidation was protein hydroperoxide. 7,8-Dihydroneopterin oxidation to 7,8-dihydroxanthopterin was only observed in the presence of both cells and LDL, showing that 7,8-dihydroneopterin had no effect on initiating oxidant generation by the cells. 7,8-Dihydroneopterin did not regenerate alpha-tocopherol but competed with it for the lipid peroxyl radical. Although stimulation of both cell types with gamma-interferon failed to produce sufficient 7,8-dihydroneopterin to inhibit LDL oxidation in tissue culture, analysis of advanced atherosclerotic plaque removed from patients showed that total neopterin levels could reach low micromolar concentrations. This suggests that 7,8-dihydroneopterin synthesis by macrophages could play a significant role in the development of atherosclerotic plaques.     

Oxidation of 7,8-dihydroneopterin by hypochlorous acid yields neopterin

In vitro, interferon-gamma stimulates primate monocytes/macrophages to produce the pteridines neopterin and 7,8-dihydroneopterin. These pteridines are capable of modulating the oxidative potential of reactive species. Neopterin is pro-oxidative whereas 7, 8-dihydroneopterin is an effective antioxidant. In the presence of oxygen, 7,8-dihydroneopterin is rapidly oxidized and after loosing the side chain 7,8-dihydroxanthopterin is formed. It is considered that under physiological conditions, 7,8-dihydroneopterin cannot be a source for neopterin production. In this study it is demonstrated that hypochlorous acid is capable to oxidize 7,8-dihydroneopterin yielding neopterin. Neopterin is less affected by hypochlorous acid, and in a mixture of both pteridines similar to the in vivo situation, only 7,8-dihydroneopterin is oxidized, thereby increasing the ratio towards neopterin. The findings may beat relevance for the in vivo situation since hypochlorous acid shifts the neopterin/7, 8-dihydroneopterin ratio towards the side of neopterin, hence probably increasing the oxidative potential in a micro-environment.     

Pteridine-dependent oxygen activation in neutrophils

We investigated the influence of neopterin and 7,8-dihydroneopterin on the myeloperoxidase activity and secretory degranulation in neutrophils and interaction of pteridines with its major substrate (hydrogen peroxide) and intermediate product of halogenation cycle (hypochlorous acid). It was shown that, in neutrophils, the redox-pair, neopterin and 7,8-dihydroneopterin, control oxygen activation, which is regulated by myeloperoxidase. Pteridines influence the secretion of myeloperoxidase depending on concentration and decrease the level of hydrogen peroxide and hypochlorous acid, which are the substrate and intermediate product of the enzyme, respectively. It was found that, in micromolar concentrations, 7,8-dihydroneopterin is a noncompetitive inhibitor of myeloperoxidase. We suppose that myeloperoxidase facilitates 7,8-dihydroneopterin oxidation by hypochlorous acid and results in an increase in neopterin concentration. These changes modify the concentration of intracellular and extracellular reactive oxygen species.     

Radioimmunoassay for neopterin in body fluids and tissues

Specific antibodies against D-erythroneopterin have been prepared in rabbits using a conjugate of D-erythroneopterin to bovine serum albumin (D-erythroneopterinylcaproyl-bovine serum albumin). The antiserum distinguished D-erythroneopterin from other pteridines, i.e., three stereoisomers of neopterin, L-erythrobiopterin, folic acid, xanthopterin, and four other synthetic pteridines. Using this specific antiserum, a radioimmunoassay for D-erythroneopterin has been developed to measure the neopterin concentrations in urine and tissues. The conjugate of D-erythroneopterin with tyramine (NP-Tyra) was synthesized and labeled with 125I as the labeled ligand NP-[125I]tyra for the radioimmunoassay. The minimal detectable amount of neopterin was about 0.1 pmol. The concentration of total neopterin (neopterin, 7,8-dihydroneopterin, quinonoid dihydroneopterin, and tetrahydroneopterin) in the biological samples was obtained by iodine oxidation under acidic conditions prior to the radioimmunoassay, and that of neopterin plus 7,8-dihydroneopterin by oxidation under alkaline conditions. Total neopterin values in human urine obtained by this new radioimmunoassay showed a good agreement with those obtained by high-performance liquid chromatography with fluorescence detection. With rat tissue samples which contained very low concentrations of neopterin as compared to biopterin, biopterin was simultaneously determined by our previously reported radioimmunoassay, and neopterin values were corrected for the cross-reactivity (0.1%). The neopterin concentrations obtained by this method agreed with the values obtained by the radioimmunoassays for neopterin and biopterin after their separation by high-performance liquid chromatography. This very small amount of neopterin, as compared with biopterin, in rat tissues could not be determined by high-performance liquid chromatography-fluorometry alone due to the masking of the neopterin peak by a large biopterin peak.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protein Hydroperoxides are a Major Product of Low Density Lipoprotein Oxidation During Copper,Peroxyl Radical and Macrophage-mediated Oxidation

Damage to apoB100 on low density lipoprotein (LDL) has usually been described in terms of lipid aldehyde derivatisation or fragmentation. Using a modified FOX assay, protein hydroperoxides were found to form at relatively high concentrations on apoB100 during copper, 2,2′-azobis(amidinopropane) dihydrochloride (AAPH) generated peroxyl radical and cell-mediated LDL oxidation. Protein hydroperoxide formation was tightly coupled to lipid oxidation during both copper and AAPH-mediated oxidation. The protein hydroperoxide formation was inhibited by lipid soluble α-tocopherol and the water soluble antioxidant, 7,8-dihydroneopterin. Kinetic analysis of the inhibition strongly suggests protein hydroperoxides are formed by a lipid-derived radical generated in the lipid phase of the LDL particle during both copper and AAPH mediated oxidation. Macrophage-like THP-1 cells were found to generate significant protein hydroperoxides during cell-mediated LDL oxidation, suggesting protein hydroperoxides may form in vivo within atherosclerotic plaques. In contrast to protein hydroperoxide formation, the oxidation of tyrosine to protein bound 3,4-dihydroxyphenylalanine (PB-DOPA) or dityrosine was found to be a relatively minor reaction. Dityrosine formation was only observed on LDL in the presence of both copper and hydrogen peroxide. The PB-DOPA formation appeared to be independent of lipid peroxidation during copper oxidation but tightly associated during AAPH-mediated LDL oxidation.     

7,8 Dihydroneopterin Inhibits Low Density Lipoprotein Oxidation in Vitro. Evidence That This Macrophage Secreted Pteridine is an Anti-Oxidant

Neopterin and its reduced form, 7,8 dihydroneopterin afe pteridines released from macrophages and monocytes when stimulated with interferon gamma in vivo. The function of this response is unknown though there is an enormous amount of information available on the use of these compounds as clinical markers of monocyte/macrophage activation. We have found that in vitro 7,8-dihydroneopterin dramatically increases, in a dose dependent manner, the lag time of low density lipoprotein oxidation mediated by Cu⁺⁺ ions or the peroxyl radical generator 2,2'-azobis (2-amidino propane) dihydrochloride (AAPH). 7,8-Dihydroneopterin also inhibits AAPH mediated oxidation of linoleate. The kinetic of the inhibition suggests that 7,8-dihydroneopterin is a potent chain breaking antioxidant which functions by scavenging lipid peroxyl radicals. No anti-oxidant activity was observed in any of the oxidation systems studied with the related compounds neopterin and pterin.     

Protein hydroperoxides are a major product of low density lipoprotein oxidation during copper, peroxyl radical and macrophage-mediated oxidation

Damage to apoB100 on low density lipoprotein (LDL) has usually been described in terms of lipid aldehyde derivatisation or fragmentation. Using a modified FOX assay, protein hydroperoxides were found to form at relatively high concentrations on apoB100 during copper, 2,2'-azobis(amidinopropane) dihydrochloride (AAPH) generated peroxyl radical and cell-mediated LDL oxidation. Protein hydroperoxide formation was tightly coupled to lipid oxidation during both copper and AAPH-mediated oxidation. The protein hydroperoxide formation was inhibited by lipid soluble alpha-tocopherol and the water soluble antioxidant, 7,8-dihydroneopterin. Kinetic analysis of the inhibition strongly suggests protein hydroperoxides are formed by a lipid-derived radical generated in the lipid phase of the LDL particle during both copper and AAPH mediated oxidation. Macrophage-like THP-1 cells were found to generate significant protein hydroperoxides during cell-mediated LDL oxidation, suggesting protein hydroperoxides may form in vivo within atherosclerotic plaques. In contrast to protein hydroperoxide formation, the oxidation of tyrosine to protein bound 3,4-dihydroxyphenylalanine (PB-DOPA) or dityrosine was found to be a relatively minor reaction. Dityrosine formation was only observed on LDL in the presence of both copper and hydrogen peroxide. The PB-DOPA formation appeared to be independent of lipid peroxidation during copper oxidation but tightly associated during AAPH-mediated LDL oxidation.     

Neopterin derivatives modulate toxicity of reactive species on Escherichia coli

Neopterin and 7,8-dihydroneopterin are released by human monocytes/macrophages upon stimulation with interferon-γ. In parallel, a panel of highly reactive species is produced by macrophages as part of their cytotoxic armature, which is directed against microbial and viral challenge and against malignant growth. Recently, neopterin and 7,8-dihydroneopterin were shown to modulate the action of reactive species in vitro. In this study we investigated the impact of neopterin and 7,8-dihydroneopterin on the toxicity of reactive species, namely chloramine-T, H₂O₂, hypochlorite, nitrite, and formaldehyde, respectively. We studied the growth inhibition of Escherichia Coli (E. coli) by these toxic agents and its modulation by neopterin and 7,8-dihydroneopterin. Bacterial growth was monitored by optical density of suspension cultures at 600 nm. Compared to control experiments, neopterin enhanced toxicity of all reactive species tested except formaldehyde, while 7,8-dihydroneopterin reduced activity of hypochlorite and chloramine-T. No significant impact of the pteridines could be established for H₂O₂-mediated and formaldehyde-mediated growth inhibition. The data support the concept that neopterin and 7,8-dihydroneopterin produced during immune response in humans could be important to modulate the action of reactive species released in parallel.     

人血浆低密度脂蛋白亚组分氧化反应敏感性的比较

本文对３种ＬＤＬ亚组分在体外对Ｃｕ＾２＋催化氧化反应敏感性进行了比较。结果表明,随氧化时间延长,各ＬＤＬ亚组分的电泳迁移率均增加。测定脂质过氧化物的含量以及用结合二烯法测定氧化反应的潜伏期,发现较高密度的ＬＤＬ亚组分更易氧化。荧光免疫测定结果显示,较高密度ＬＤＬ中载脂蛋白Ｂ上新生的４－羟壬烯醛抗原决定簇的表达高于较低密度的ＬＤＬ,从而证明较高密度的ＬＤＬ亚组分对氧化反应的敏感性高于较低密度的亚组分     

Inhibition of THP-1 cell-mediated low-density lipoprotein oxidation by the macrophage-synthesised pterin, 7,8-dihydroneopterin

Human macrophages release the pterin, 7,8-dihydroneopterin when exposed to the immune stimulant gamma-interferon (IFN-gamma). Previous in vitro studies have shown 7,8-dihydroneopterin is a potent antioxidant, which inhibits copper- and peroxyl-radical mediated low-density lipoprotein (LDL) oxidation. Using THP-1 cells, a human derived monocyte-like cell line, we have found that low micromolar concentrations of 7,8-dihydroneopterin inhibit cell mediated oxidation of LDL, as measured by electrophoretic mobility, alpha-tocopherol loss, and lipid oxidation. Stimulation of the THP-1 cells with IFN-gamma caused a significant reduction in the cells' ability to oxidise LDL. The extracellular pterin concentration increased from 0 to 16 nM with IFN-gamma stimulation, while the intracellular concentration increased from 0.21 to 1.69 nmol/mg cell protein.     

Reduced pteridine derivatives induce apoptosis in PC12 cells

In cerebrospinal fluid of patients with cerebral infections, elevated concentrations of the pteridine compounds neopterin and 7,8-dihydroneopterin were detected. Here, the potential of pteridines to induce apoptosis of the rat pheochromocytoma cells (PC12) was investigated. In contrast to aromatic pteridines like neopterin, the reduced forms 7,8-dihydroneopterin, 5,6,7,8-tetrahydrobiopterin and 7,8-dihydrobiopterin led to a significant increase of apoptotic cells. After terminal differentiation, cells were less sensitive to incubation with pteridines. A noticeable augmentation of apoptosis was observed upon incubation with 7,8-dihydroneopterin and 7,8-dihydrofolic acid. Antioxidants partly protected PC12 cells from pteridine-induced apoptosis, suggesting the involvement of reactive oxygen intermediates. Exposure of cells to 7,8-dihydroneopterin led to activation of the mitogen-activated protein (MAP) kinase and to a lesser degree also of JUN/SAP kinase. Results implicate that high concentrations of reduced pteridines, might contribute to the pathogenesis involved in neurodegeneration.     

Tea catechins inhibit cholesterol oxidation accompanying oxidation of low density lipoprotein in vitro

Endogenous oxidized cholesterols are potent atherogenic agents. Therefore, the antioxidative effects of green tea catechins (GTC) against cholesterol oxidation were examined in an in vitro lipoprotein oxidation system. The antioxidative potency of GTC against copper catalyzed LDL oxidation was in the decreasing order (-)-epigalocatechin gallate (EGCG)=(-)-epicatechin gallate (ECG)>(-)-epicatechin (EC)=(+)-catechin (C)>(-)-epigallocatechin (EGC). Reflecting these activities, both EGCG (74%) and ECG (70%) inhibited the formation of oxidized cholesterol, as well as the decrease of linoleic and arachidonic acids, in copper catalyzed LDL oxidation. The formation of oxidized cholesterol in 2,2'-azobis(2-amidinopropane) hydrochloride (AAPH)-mediated oxidation of rat plasma was also inhibited when the rats were given diets containing 0.5% ECG or EGCG. In addition, EGCG and ECG highly inhibited oxygen consumption and formation of conjugated dienes in AAPH-mediated linoleic acid peroxidative reaction. These two species of catechin also markedly lowered the generation of hydroxyl radical and superoxide anion. Thus, GTC, especially ECG and EGCG, seem to inhibit cholesterol oxidation in LDL by combination of interference with PUFA oxidation, the reduction and scavenging of copper ion, hydroxyl radical generated from peroxidation of PUFA and superoxide anion.     

Oxidized low-density lipoprotein is chemotactic for arterial smooth muscle cells in culture

The effects of human native and Cu2(+)-oxidized low-density lipoprotein (LDL) were tested on the migration of cultured bovine aortic smooth muscle cells (SMCs) in blind-well chambers. LDL oxidation was controlled by measuring the formation of conjugated dienes and lipid hydroperoxides, and by agarose gel electrophoresis. Oxidized LDL stimulated SMC migration, and the effect was dose-dependent up to 200 microgram/ml. The stimulation was chemotactic in nature. Native LDL was without significant activity. The results suggest that oxidized LDL may contribute to the migration of medial SMCs into the intima during atherogenesis.     

Glucose Accelerates Copper- and Ceruloplasmin-induced Oxidation of Low-density Lipoprotein and Whole Serum

Glucose at pathophysiological concentrations was able to accelerate copper-induced oxidation of isolated low-density lipoprotein (LDL) and whole serum. The efficiency of glucose was favored under the following circumstances: (a) when LDL oxidation was induced by low copper concentration, (b) when LDL was partly oxidized, i.e. enriched with lipid peroxides. The glucose derivative methyl- &#102 - d -glucoside was ineffective on Cu 2+ -induced LDL oxidation, pointing out the essential role of the reactivity of the aldehydic carbon for the pro-oxidative effect. When LDL oxidation was induced by a peroxyl radical generator, as a model of transition metal independent oxidation, glucose was ineffective. Glucose was found to stimulate oxidation of LDL induced by ceruloplasmin, the major copper-containing protein of human plasma. Thus, glucose accelerated oxidation of LDL induced by both free and protein bound copper. Considering the requirement for catalytically active copper and for the aldehydic carbon, the pro-oxidative effect of glucose is likely to depend on the increased availability of Cu + ; this is more efficient in decomposing lipid peroxide than Cu 2+ , accounting for acceleration of LDL oxidation. The possible biological relevance of our work is supported by the finding that glucose was able to accelerate oxidation of whole serum, which was assessed by monitoring low-level chemiluminescence associated with lipid peroxidation.     

Metal Ion Release from Mechanically-Disrupted Human Arterial Wall. Implications for the Development of Atherosclerosis

Oxidation of low density lipoproteins (LDL) in blood vessel walls plays a significant role in the development of atherosclerosis. LDL oxidation in vitro is greatly accelerated by the presence of “catalytic” iron or copper ions, which have already been shown to be present within advanced atherosclerotic lesions. We demonstrate here that mechanical damage to human arterial wall samples (both normal and early or intermediate atherosclerotic lesions) causes release of “catalytic” iron and copper ions, to an extent increasing with the damage. It may be that traumatic (e.g. during angioplasty) or other injury to the vessel wall contributes to the generation of metal ions that can facilitate LDL oxidation and other free radical reactions, so promoting atherosclerosis.     

Mechanism of the antioxidant to pro-oxidant switch in the behavior of dehydroascorbate during LDL oxidation by copper(II) ions

Oxidised low density lipoprotein (LDL) may be involved in the pathogenesis of atherosclerosis. We have therefore investigated the mechanisms underlying the antioxidant/pro-oxidant behavior of dehydroascorbate, the oxidation product of ascorbic acid, toward LDL incubated with Cu(2+) ions. By monitoring lipid peroxidation through the formation of conjugated dienes and lipid hydroperoxides, we show that the pro-oxidant activity of dehydroascorbate is critically dependent on the presence of lipid hydroperoxides, which accumulate during the early stages of oxidation. Using electron paramagnetic resonance spectroscopy, we show that dehydroascorbate amplifies the generation of alkoxyl radicals during the interaction of copper ions with the model alkyl hydroperoxide, tert-butylhydroperoxide. Under continuous-flow conditions, a prominent doublet signal was detected, which we attribute to both the erythroascorbate and ascorbate free radicals. On this basis, we propose that the pro-oxidant activity of dehydroascorbate toward LDL is due to its known spontaneous interconversion to erythroascorbate and ascorbate, which reduce Cu(2+) to Cu(+) and thereby promote the decomposition of lipid hydroperoxides. Various mechanisms, including copper chelation and Cu(+) oxidation, are suggested to underlie the antioxidant behavior of dehydroascorbate in LDL that is essentially free of lipid hydroperoxides.     

Dissociation of neopterin and 7,8-dihydroneopterin from plasma components before HPLC analysis

Measurement of plasma neopterin by HPLC with fluorescence detection is used clinically as a marker of immune cell activation in the management of a number of disease pathologies. HPLC analysis of neopterin requires the acidic removal of plasma proteins but we have found that 7,8-dihydroneopterin is oxidised to neopterin with varying yield. Using acetonitrile as the precipitant, we have measured substantially higher quantities of both total neopterin (7,8-dihydroneopterin and neopterin) and neopterin from plasma of healthy and septicemia patient's. Total neopterin concentrations were on average 50% and 200% greater in healthy and septicemia subjects, respectively, when measured after acetonitrile precipitation compared to trichloroacetic acid. Our data suggests that some pterin co-precipitates with proteins during acid treatment.     

Acetylcholine esterase protects LDL against oxidation

Acetylcholine esterase (AChE) and paraoxonase 1 (PON1) are both serum ester hydrolases, which are associated with the prevalence of myocardial infarction. Both genes are located in close proximity on chromosome 7q21-22. As PON1 was suggested to protect against cardiovascular diseases secondary to its ability to break down oxidized lipids and to inhibit LDL oxidation, we examined AChE capacity to protect LDL against oxidation. Preincubation of LDL with AChE retarded the onset of copper ion-induced LDL oxidation in a concentration-dependent manner. AChE significantly reduced the formation of lipid peroxides and TBARS during the course of LDL oxidation, by up to 45%. This effect was associated with AChE-mediated hydrolysis of lipid peroxides, which accounts for the inhibition in the onset of LDL oxidation, the oxidative propagation phase, and aldehyde formation. We conclude that AChE, similar to PON1, can hydrolyze lipid peroxides and thus may prevent the accumulation of oxidized LDL and attenuate atherosclerosis development.