Detection of chemiluminescence in lipid peroxidation of biological systems and its application to HPLC

A combined system of chemiluminescence detection and high performance liquid chromatography (CL–HPLC) was developed to determine primary peroxidation products in biological tissues, such as phosphatidylcholine hydroperoxide (PCOOH). The CL–HPLC assay consists of separation of lipid classes with HPLC and detection of hydroperoxide-specific chemiluminescence. Hydroperoxides react with heme compounds to produce oxidants as suggested by our early studies on tissue low-level chemiluminescence in which singlet molecular oxygen is generated as one of the excited species in several biological systems involving free radical events. In the CL–HPLC method, a cytochrome c–luminol mixture was used as a hydroperoxide-specific luminescent reagent, and the quantification of hydroperoxide was performed by detecting chemiluminescence due to the luminol oxidation caused by the oxidant produced during the lipid hydroperoxides with heme. The detection limit of PCOOH was 10 pmole hydroperoxide–O₂. PCOOH in normal human blood was found to be 10–500 pmol/ml plasma and significantly higher levels of PCOOH were observed in some hospitalized patients.     

Phospholipid hydroperoxide accumulation in liver of rats intoxicated with carbon tetrachloride and its inhibition by dietary alpha-tocopherol

The formation and accumulation of phospholipid hydroperoxides, especially of phosphatidylcholine hydroperoxide (PCOOH), a primary peroxidation product of phosphatidylcholine (PC), in livers of carbon tetrachloride-intoxicated rats was investigated. PCOOH in liver and blood plasma was measured by a chemiluminescence-high-performance liquid chromatography procedure originally developed by Miyazawa et al. (Anal. Lett. 20, 915, 1987; Free Radical Biol. Med. 7, 209, 1989). Male Sprague-Dawley rats (120 g body wt., 5 weeks of age) were used in the experiments. The amount of PCOOH in the liver of control rats (CCl4-untreated) was 160 +/- 20 pmol/100 mg protein (mean +/- SD) and the PCOOH/PC molar ratio was 1.1 +/- 0.1 X 10(-5). In CCl4 (0.1 ml/100 g body wt.)-dosed rats, the liver PCOOH was 289 +/- 65 pmol/100 mg protein (PCOOH/PC = 2.4 +/- 0.4 X 10(-5], 764 +/- 271 pmol/100 mg protein (PCOOH/PC = 5.2 +/- 1.7 X 10(-5], and 856 +/- 165 pmol/100 mg protien (PCOOH/PC = 6.0 +/- 0.8 X 10(-5] at 6 h, 24 h, and 1 week after the dose, respectively. Under such conditions, the liver phosphatidylethanolamine hydroperoxide (PEOOH) level was not altered and the concentration was less than 100 pmol/100 mg protein even after the dose. The increments of liver PCOOH were suppressed 56% by the oral supplementation of DL-alpha-tocopherol (5 mg/100 g body wt./day) for a week before CCl4 administration. A relatively larger amount of PEOOH was found after stimulation of PC hydroperoxidation in the liver of rats with a large amount of CCl4 (0.25 ml/100 g body wt.) rather than with the small amount of CCl4 (0.1 ml/100 g body wt.).(ABSTRACT TRUNCATED AT 250 WORDS)     

Fungal elicitor-induced retardation and its restoration of root growth in tobacco seedlings

In this study, we investigated responses of growing and intact tobacco (N. tabacum cv Xanthi) seedlings to a fungal elicitor, a xylanase from Trichoderma viride (TvX). In addition to the induction of defense gene expression, TvX treatment caused the retardation of growth of seedlings. In the TvX-treated seedlings, growth of primary roots was markedly reduced through repression of cell division and longitudinal cell elongation in a meristematic zone and an elongation zone, respectively. However, cell differentiation to form vascular bundles and root hairs continued. In the TvX-treated root cap, disappearance of starch granules in columella cells and aggregation of border cells were observed. Furthermore, the TvX-induced growth retardation was restored after removal of the elicitor, resulting in a plastic alteration of root architecture. Therefore, the fungal elicitor might act as an environmental cue that regulates root growth and development as well as the ordinary defense responses in plant seedlings. These findings suggest a novel aspect of plant growth regulation via a plant–microbe interaction in the rhizosphere.     

Singlet oxygen generation from phosphatidylcholine hydroperoxide in the presence of copper

This study pursued whether singlet oxygen ((1)O2) is generated from phosphatidylcholine hydroperoxide (PCOOH), the oxidized modification product of a major constituent of biomembranes and serum lipoproteins. The (1)O2 formation was detected, by utilizing the oxidation of 2,2,6,6-tetramethyl-4-piperidone (TMPD) by (1)O2 to yield 2,2,6,6-tetramethyl-4-piperidone-1-oxyl (TEMPONE), which generates electron spin resonance (ESR) signals. The TEMPONE signal was detected in human plasma with addition of PCOOH by ESR determination after introducing copper(II). The TEMPONE formation was proportional to the amounts of PCOOH added according to moles of active oxygen. The TEMPONE signal intensity was weakened significantly in the presence of beta-carotene and histidine in a concentration-dependent manner, but was not at all decreased by mannitol, Mn-superoxide dismutase and catalase. In addition, HPLC-chemiluminescence analysis demonstrated that incubation with the PCOOH/Cu(II) combination oxidized cholesterol, a relatively oxidation-resistant component, to the cholesterol hydroperoxide. These results reveal that (1)O2 is generated from PCOOH in contact with copper(II). In conclusion, this in-vitro study provides directly the (1)O2 formation in living organisms following the advancement of peroxidation of constitutive lipids.     

Early signaling events induced by elicitors of plant defenses

Plant pathogen attacks are perceived through pathogen-issued compounds or plant-derived molecules that elicit defense reactions. Despite the large variety of elicitors, general schemes for cellular elicitor signaling leading to plant resistance can be drawn. In this article, we review early signaling events that happen after elicitor perception, including reversible protein phosphorylations, changes in the activities of plasma membrane proteins, variations in free calcium concentrations in cytosol and nucleus, and production of nitric oxide and active oxygen species. These events occur within the first minutes to a few hours after elicitor perception. One specific elicitor transduction pathway can use a combination or a partial combination of such events which can differ in kinetics and intensity depending on the stimulus. The links between the signaling events allow amplification of the signal transduction and ensure specificity to get appropriate plant defense reactions. This review first describes the early events induced by cryptogein, an elicitor of tobacco defense reactions, in order to give a general scheme for signal transduction that will be use as a thread to review signaling events monitored in different elicitor or plant models.     

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.     

Cholic acid, a bile acid elicitor of hypersensitive cell death, pathogenesis-related protein synthesis, and phytoalexin accumulation in rice

When plants interact with certain pathogens, they protect themselves by generating various defense responses. These defense responses are induced by molecules called elicitors. Since long ago, composts fermented by animal feces have been used as a fertilizer in plant cultivation, and recently, have been known to provide suppression of plant disease. Therefore, we hypothesized that the compounds from animal feces may function as elicitors of plant defense responses. As a result of examination of our hypothesis, an elicitor of rice defense responses was isolated from human feces, and its structure was identified as cholic acid (CA), a primary bile acid in animals. Treatment of rice (Oryza sativa) leaves with CA induced the accumulation of antimicrobial compounds (phytoalexins), hypersensitive cell death, pathogenesis-related (PR) protein synthesis, and increased resistance to subsequent infection by virulent pathogens. CA induced these defense responses more rapidly than did fungal cerebroside, a sphingolipid elicitor isolated from the rice pathogenic fungus Magnaporthe grisea. Furthermore, fungal cerebroside induced both types of rice phytoalexins, phytocassanes and momilactones, whereas CA mainly induced phytocassanes, but not momilactones. In the structure-activity relationship analysis, the hydroxyl groups at C-7 and C-12, and the carboxyl group at C-24 of CA contributed to the elicitor activity. These results indicate that CA is specifically recognized by rice and is a different type of elicitor from fungal cerebroside. This report demonstrated that bile acid induced defense responses in plants.     

Nitric oxide is critical for inducing phosphatidic acid accumulation in xylanase-elicited tomato cells

Nitric Oxide (NO) is a second messenger related to development and (a)biotic stress responses in plants. We have studied the role of NO in signaling during plant defense responses upon xylanase elicitation. Treatment of tomato cell cultures with the fungal elicitor xylanase resulted in a rapid and dose-dependent NO accumulation. We have demonstrated that NO is required for the production of the lipid second messenger phosphatidic acid (PA) via the activation of the phospholipase C (PLC) and diacylglycerol kinase (DGK) pathway. Defense-related responses downstream of PA were studied. PA and, correspondingly, xylanase were shown to induce reactive oxygen species production. Scavenging of NO or inhibition of either the PLC or the DGK enzyme diminished xylanase-induced reactive oxygen species production. Xylanase-induced PLDbeta1 and PR1 mRNA levels decreased when NO or PA production were compromised. Finally, we have shown that NO and PA are involved in the induction of cell death by xylanase. Treatment with NO scavenger cPTIO, PLC inhibitor U73122, or DGK inhibitor R59022 diminished xylanase-induced cell death. On the basis of biochemical and pharmacological experimental results, we have shown that PLC/DGK-derived PA represents a novel downstream component of NO signaling cascade during plant defense.     

Antioxidant activity of palm oil carotenes in peroxyl radical-mediated peroxidation of phosphatidyl choline liposomes

The antioxidant efficacy of alpha-carotene and comparison with beta-carotene in multilamellar liposomes prepared from egg yolk phosphatidyl choline (EYPC) exposed to the lipid soluble 2,2'-azobis (2,4-dimethyl valeronitrile) (AMVN) was investigated. Lipid peroxidation was measured as thiobarbituric acid reacting substances (TBARS) at 532 nm or as hydroperoxide formation at 234 nm after separation of phosphatidyl choline hydroperoxide (PCOOH) by high-pressure liquid chromatography (HPLC). Lutein and zeaxanthin, the hydroxyl derivatives of alpha- and beta-carotenes, and the chain breaking antioxidant alpha-tocopherol were also included in the study. AMVN being a lipid soluble, non polar azo initiator penetrates into the hydrophobic interior of the phospholipid bilayer, forming peroxyl radicals which peroxidate the phospholipid leading to PCOOH accumulation. All the carotenoids tested at 1 mol% relative to EYPC significantly suppressed the formation of PCOOH compared to control samples. In this system, alpha-carotene retarded PCOOH formation better than beta-carotene. Similarly, lutein was a better antioxidant than is zeaxanthin. But lutein and zeaxanthin were more effective antioxidants than alpha- and beta-carotenes, respectively. After 1 h of incubation of the carotenoid with AMVN, alpha-, beta-carotene, lutein and zeaxanthin limited PCOOH formation by 77%, 68%, 85% and 82%, respectively, while alpha-tocopherol elicited 90% reduction. AMVN incubated with EYPC for 2 h induced the formation of TBARS compared to control (P < 0.001). alpha-Carotene significantly suppressed the TBARS formation by 78% whilst beta-carotene, lutein, zeaxanthin and alpha-tocopherol elicited 60%, 91% and 80% reductions, respectively. Increasing the concentration of the carotenoid > 1 mol% to EYPC did not significantly increase protection of the membrane against free radical attack. Our findings suggest that alpha-carotene is a better antioxidant than is beta-carotene in phosphatidyl choline vesicles. It may, therefore, be useful in limiting free radical mediated peroxidative damage against membrane phospholipids in vivo.     

Role of myeloperoxidase in the neutrophil-induced oxidation of low density lipoprotein as studied by myeloperoxidase-knockout mouse

Low density lipoprotein was oxidized by neutrophils derived from either C57BL/6 mice or myeloperoxidase (MPO)-knockout mice. The generation of superoxide from neutrophils of MPO-knockout mice was about 70% of that from wild-type mice. The extent of the oxidation of human low density lipoprotein (LDL) by phorbol myristate acetate (PMA)-activated neutrophils of wild-type and MPO-knockout mice was assessed by measuring consumption of a-tocopherol and formation of phosphatidylcholine hydroperoxide (PCOOH) and cholesteryl ester hydroperoxide (CEOOH). Little consumption of a-tocopherol was observed in both oxidations. It was found, however, that lipid hydroperoxides were accumulated with time in both oxidations and that the rates of formation of PCOOH and CEOOH in the oxidation by MPO-knockout neutrophils were about 66 and 44% of those by wild-type neutrophils, respectively. The lipid peroxidation was completely inhibited by adding superoxide dismutase (SOD) in both cases. The addition of L-tyrosine and SOD enhanced lipid peroxidation of LDL induced by wild-type neutrophils but not by MPO-knockout ones. These results suggest that, regardless of their MPO activity, neutrophils induce lipid peroxidation of LDL by a superoxide-dependent pathway, and that MPO-catalyzed lipid peroxidation is enhanced by the presence of an appropriate amount of free tyrosine and further enhanced by SOD.     

真菌诱导子对悬浮培养南方红豆杉细胞态势及紫杉醇合成的影响

在南方红豆杉细胞悬浮培养过程中,研究了在指数生长期的末期加入真菌诱导子(尖孢镰刀菌的胞壁组分粗提物)对细胞态势及紫杉醇合成的影响。结果表明,真菌诱导子能在短期内激发细胞的防御性应答反应而产生氧迸发,细胞的氧化还原态势发生了明显的变化,培养基发生碱化现象,表征酶含量的蛋白质含量明显提高,SOD、POD、CAT的活力出现波动性变化,并具有一定的时序性。同时,南方红豆杉悬浮培养体系中产次生代谢途径中重要的酶PAL的活力得到提高,紫杉醇的合成被加强,产量得到了显著提高,达到了对照组的5倍左右。     

Presence of phosphatidylcholine hydroperoxide in human plasma

A chemiluminescence-high performance liquid chromatography (CL-HPLC) system was newly developed and used for the hydroperoxide-specific determination of phosphatidylcholine hydroperoxide (PCOOH) in human plasma. The method involves separation of phosphatidylcholine derivatives from plasma lipids by normal phase HPLC and subsequent detection of hydroperoxide-dependent chemiluminescence (CL) of PCOOH. CL was produced through luminol oxidation during the reaction of the hydroperoxide and cytochrome c-heme. The high specificity for the hydroperoxide allows the sensitive assaying of a large PCOOH range over a concentration range of 50-2,000 pmol of hydroperoxide-O2. Using this method, the occurrence of PCOOH in normal human plasma was strongly suggested and was confirmed quantitatively.     

Enhancement of stress tolerance in transgenic tobacco plants overexpressing Chlamydomonas glutathione peroxidase in chloroplasts or cytosol

To evaluate the physiological potential of the defense system against hydroperoxidation of membrane-lipid components caused by environmental stresses in higher plants, we generated transgenic tobacco plants expressing a glutathione peroxidase (GPX)-like protein in the cytosol (TcGPX) or chloroplasts (TpGPX). The activities toward alpha-linolenic acid hydroperoxide in TcGPX and TpGPX plants were 47.5-75.3 and 32.7-42.1 nM min(-1) mg(-1) protein, respectively, while no activity was detected in wild-type plants. The transgenic plants showed increased tolerance to oxidative stress caused by application of methylviologen (MV: 50 microM) under moderate light intensity (200 micro E m(-2) sec(-1)), chilling stress under high light intensity (4 degrees C, 1000 microE m(-2) sec(-1)), or salt stress (250 mM NaCl). Under these stresses, the lipid hydroperoxidation (the production of malondialdehyde (MDA)) of the leaves of TcGPX and TpGPX plants was clearly suppressed compared with that of wild-type plants. Furthermore, the capacity of the photosynthetic and antioxidative systems in the transgenic plants remained higher than those of wild-type plants under chilling or salt stress. These results clearly indicate that a high level of GPX-like protein in tobacco plants functions to remove unsaturated fatty acid hydroperoxides generated in cellular membranes under stress conditions, leading to the maintenance of membrane integrity and increased tolerance to oxidative stress caused by various stress conditions.     

Antioxidant activity of palm oil carotenes in peroxyl radical-mediatedperoxidation of phosphatidyl choline liposomes

Abstract

The antioxidant efficacy of α-carotene and comparison with β-carotene in multilamellar liposomes prepared from egg yolk phosphatidyl choline (EYPC) exposed to the lipid soluble 2,2′-azobis (2,4-dimethyl valeronitrile) (AMVN) was investigated. Lipid peroxidation was measured as thiobarbituric acid reacting substances (TBARS)at 532 nm or as hydroperoxide formation at 234 nm after separation of phosphatidyl choline hydroperoxide (PCOOH) by high-pressure liquid chromatography (HPLC). Lutein and zeaxanthin, the hydroxyl derivatives of α- and β-carotenes, and the chain breaking antioxidant α-tocopherol were also included in the study.AMVN being a lipid soluble, non polar azo initiator penetrates into the hydrophobic interior of the phospholipid bilayer, forming peroxyl radicals which peroxidate the phospholipid leading to PCOOH accumulation. All the carotenoids tested at 1 mol% relative to EYPC significantly suppressed the formation of PCOOH compared to control samples.In this system, α-carotene retarded PCOOH formation better than β-carotene. Similarly, lutein was a better antioxidant than is zeaxanthin. But lutein and zeaxanthin were more effective antioxidants than α- and β-carotenes, respectively. After 1 h of incubation of the carotenoid with AMVN, α-, β-carotene, lutein and zeaxanthin limited PCOOH formation by 77%, 68%, 85%and 82%, respectively, while α-tocopherol elicited 90%reduction.AMVN incubated with EYPC for 2 h induced the formation of TBARS compared to control (P <0.001). α-Carotene significantly suppressed the TBARS formation by 78% whilst β-carotene, lutein, zeaxanthin and α-tocopherol elicited 60%, 91%and 80% reductions, respectively. Increasing the concentration of the carotenoid >1 mol% to EYPC did not significantly increase protection of the membrane against free radical attack.Our findings suggest that α-carotene is a better antioxidant than is β-carotene in phosphatidyl choline vesicles. It may, therefore, be useful in limiting free radical mediated peroxidative damage against membrane phospholipids _{in vivo}.     

Determination of phospholipid hydroperoxides in human blood plasma by a chemiluminescence-HPLC assay

A chemiluminescence-high performance liquid chromatography (CL-HPLC) system was developed (Miyazawa, T. et al., Anal. Lett., 20, 915-925, 1987) and applied for the hydroperoxide-specific determination of phosphatidylcholine hydroperoxide (PCOOH) in biological tissues such as human blood plasma (Miyazawa, T. et al., Anal Lett 21:1033-1044, 1988; J. Biochem. 103:744-746; 1988). This system involves separation of phosphatidylcholines from plasma total lipids with normal phase silica gel HPLC and post-column detection of hydroperoxide-dependent chemiluminescence of PCOOH. The chemiluminescence is produced by luminol oxidation during a reaction of hydroperoxide and cytochrome c-heme. The high specificity for hydroperoxide base enables a sensitive assay for a large range of PCOOH, with the detection limit of 10 picomole of hydroperoxide-O2. By use of this assay system, the presence of PCOOH in human blood plasma is confirmed quantitatively. The PCOOH concentration of healthy plasma is in the range below 10 nM to 500 nM, and much higher concentrations (500-9000 nM) of PCOOH are observed in the plasma of unhealthy donors.     

Induction of defense responses in cultured parsley cells by plant cell wall fragments

Cell suspension cultures of parsley (Petroselinum crispum) accumulated coumarin phytoalexins and exhibited increased β-1,3-glucanase activity when treated with either a purified α-1,4-d-endopolygalacturonic acid lyase from Erwinia carotovora or oligogalacturonides solubilized from parsley cell walls by endopolygalacturonic acid lyase. Coumarin accumulation induced by the plant cell wall elicitor was preceded by increases in the activities of phenylalanine ammonia lyase (PAL), 4-coumarate:CoA ligase (4CL) and S-adenosyl-l-methionine:xanthotoxol O-methyltransferase (XMT). The time courses for the changes in these three enzyme activities were similar to those observed in cell cultures treated with a fungal glucan elicitor. The plant cell wall elicitor was found to act synergistically with the fungal glucan elicitor in the induction of coumarin phytoalexins. As much as a 10-fold stimulation in coumarin accumulation above the calculated additive response was observed in cell cultures treated with combinations of plant and fungal elicitors. The synergistic effect was also observed for the induction of PAL, 4CL, and XMT activities. These results demonstrate that plant cell wall elicitors induce at least two distinct biochemical responses in parsley cells and further support the role of oligogalacturonides as important regulators of plant defense.     

Direct Measurement of Lipid Hydroperoxides in Iron-Dependent Spinal Neuronal Injury

Abstract: The relationship between iron-dependent fetal mouse spinal cord neuron injury and the generation of endogenous lipid hydroperoxides (LOOHs) has been investigated. Cultured spinal cord neurons were incubated with ferrous iron (3–200 µM). Cell viability was measured in terms of the uptake of α-[methyl-³H]aminoisobutyric acid ([³H]AIB). Both endogenously and iron-generated LOOH, i.e., free fatty acid hydroperoxide (FFAOOH), phosphatidylethanolamine hydroperoxide (PEOOH), and phosphatidylcholine hydroperoxide (PCOOH), were measured directly by an HPLC-chemiluminescence (HPLC-CL) assay. The FFAOOH, PEOOH, and PCOOH levels in neurons incubated with 200 µM Fe²⁺ for 40 min were, respectively, 22-, 158-, and sevenfold higher than those in non-iron-exposed cultures, demonstrating that phosphatidylethanolamine (PE) was most sensitive to peroxidation. The dose-response and time course of Fe²⁺-induced generation of these LOOHs were also established. In both experiments, the LOOH levels were correlated directly with loss of neuronal viability, suggesting strongly a direct relationship between lipid peroxidation and cell injury. On examination of the time course of the LOOH generation, an immediate increase in PEOOH and PCOOH levels with only 30 s of Fe²⁺ incubation was observed. In contrast, a lag phase in the increase in FFAOOH level (2 min after Fe²⁺ addition) suggested a delay in the activation of phospholipase A₂ (PLA₂) required for the hydrolysis and generation of FFAOOH. This culture system provides an excellent model for screening antioxidant neuroprotective compounds with regard to their ability to protect against iron-dependent peroxidative injury and the relationship of the neuroprotection to inhibition of lipid peroxidation and/or PLA₂.