Chemiluminescent simultaneous determination of phosphatidylcholine hydroperoxide and phosphatidylethanolamine hydroperoxide in the liver and brain of the rat.

The quantification of phospholipid hydroperoxides in biological tissues is important in order to know the degree of peroxidative damage of membrane lipids. For this purpose, optimal conditions for the chemiluminescent simultaneous assay of phosphatidylcholine hydroperoxide (PCOOH) and phosphatidylethanolamine hydroperoxide (PEOOH) in rat liver and brain were determined. A chemiluminescence detection-high performance liquid chromatography (CL-HPLC) method that incorporates cytochrome c and luminol as a post-column hydroperoxide-specific luminescent reagent was used (Miyazawa et al. 1987. Anal. Lett. 20: 915-925; Miyazawa. 1989. Free Radical Biol. Med. 7: 209-217). An n-propylamine-bound silica column with hexane-2-propanol-methanol-water 5:7:2:1 (v/v/v/v) (flow rate 1.0 ml/min) as eluant was used to determine both PCOOH and PEOOH, which were separated from each other and from other lipids and lipid-soluble antioxidants. High reproducibility and sensitivity as low as 10 pmol hydroperoxide-O2 were observed with a mixture of 10 micrograms/ml cytochrome c and 2 micrograms/ml luminol in 50 mM borate buffer (pH 10.0, flow rate 1.1 ml/min) as luminescent reagent and a post-column mixing joint temperature of 40 degrees C. Using the established analytical conditions, it was confirmed that both PCOOH (1324 +/- 122 pmol/g liver, 114 +/- 18 pmol/g brain, mean +/- SD) and PEOOH (728 +/- 89 pmol/g liver, 349 +/- 60 pmol/g brain, mean +/- SD) are present in the liver and brain of Sprague-Dawley rats bred on a slightly modified AIN-76A semisynthetic diet for 3 months. The phospholipid hydroperoxide content in the rat liver was shown to be affected by dietary oils, but not significantly affected in the brain.     

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)     

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.     

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.     

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.     

A critical evaluation of the effect of sorbitol on the ferric-xylenol orange hydroperoxide assay

Measurement of hydroperoxide concentration by the ferric-xylenol orange assay has the advantages of simplicity, convenience, and inertness to oxygen (Gay, C., et al. (1999) Anal. Biochem. 273, 149-155). However, its sensitivity is limited by the molar absorption coefficients, which are less than 50,000 M(-1) cm(-1) for most hydroperoxides. An earlier report showed that this could be significantly enhanced by the inclusion of 100 mM sorbitol in the assay solution, resulting in an increase of the apparent absorption coefficient for H(2)O(2) from 4.46 x 10(4) to 2.24 x 10(5) M(-1) cm(-1) (Wolff, S. P. (1994) Methods Enzymol. 233, 182-189). It was claimed that the technique was also valid for other hydroperoxides, such as butyl and cumyl. In an attempt to extend this modification to a wider range of hydroperoxides, we have confirmed the enhancement of the assay of H2O2 by sorbitol. However, the sensitivity of the measurements of butyl, cumyl, amino acid, protein, and human blood serum hydroperoxides was only approximately doubled by the inclusion of sorbitol. A mechanism explaining the difference in the assay of H2O2 and the organic hydroperoxides is proposed.     

Detection and time-course analysis of phospholipid hydroperoxide in soybean seedlings after treatment with fungal elicitor, by chemiluminescence-HPLC assay.

Direct evidence for the existence of phosphatidylcholine hydroperoxide (PCOOH) and the determination of the changes in PCOOH after treatment of soybean seedlings with fungal elicitor, were examined using chemiluminescence-HPLC of hydroperoxide specific assay. PCOOH was transiently accumulated within 6 h after elicitor treatment but such an event was not observed in control plants, thus suggesting that accumulation of PCOOH was a response to fungal elicitor in the host plant. The result suggested that singlet oxygen had participated in the lipid hydroperoxidation, since non-conjugated fatty acid hydroperoxide isomers due to the singlet oxygen oxidation were detected in the phospholipid fraction. The reaction of lipid hydroperoxidation, preceding the induction of a defense compound, may be involved in the triggering of a plant defense mechanism against fungal invasion.     

Use and detection of nonradioactive iodine-labeled antibodies for immunoassay

Traditionally radioactively labeled iodine has been used as a reporter group for the detection of antibodies in immunoassay. We have recently developed a microassay system for the detection of very low concentrations of iodide which eliminates the need for the use of radiolabeled iodine (O'Kennedy, R. et al. 1989, Anal. Biochem. 179, 138-144). The successful application of this assay for the detection of mouse IgG is described. The performance of this system compared with enzyme immunoassay was evaluated.     

Ion-trap tandem mass spectrometric analysis of Amadori-glycated phosphatidylethanolamine in human plasma with or without diabetes

Peroxidized phospholipid-mediated cytotoxicity is involved in the pathophysiology of diseases [i.e., an abnormal increase of phosphatidylcholine hydroperoxide (PCOOH) in plasma of type 2 diabetic patients]. The PCOOH accumulation may relate to Amadori-glycated phosphatidylethanolamine (Amadori-PE; deoxy-D-fructosyl phosphatidylethanolamine), because Amadori-PE causes oxidative stress. However, the occurrence of lipid glycation products, including Amadori-PE, in vivo is still unclear. Consequently, we developed an analysis method of Amadori-PE using a quadrupole/linear ion-trap mass spectrometer, the Applied Biosystems QTRAP. In positive ion mode, collision-induced dissociation of Amadori-PE produced a well-characterized diglyceride ion ([M+H-303]+) permitting neutral loss scanning and multiple reaction monitoring (MRM). When lipid extract from diabetic plasma was infused directly into the QTRAP, Amadori-PE molecular species could be screened out by neutral loss scanning. Interfacing liquid chromatography with QTRAP mass spectrometry enabled the separation and determination of predominant plasma Amadori-PE species with sensitivity of approximately 0.1 pmol/injection in MRM. The plasma Amadori-PE level was 0.08 mol% of total PE in healthy subjects and 0.15-0.29 mol% in diabetic patients. Furthermore, plasma Amadori-PE levels were positively correlated with PCOOH (a maker for oxidative stress). These results show the involvement between lipid glycation and lipid peroxidation in diabetes pathogenesis.     

Singlet oxygen production by bleomycin. A comparison with heme-containing compounds

Fe(III)-bleomycin catalyzes the decomposition of 13-hydroperoxylinoleic acid and of 15-hydroperoxyarachidonic acid to produce small quantities of singlet oxygen. No singlet oxygen is produced when hydrogen peroxide, ethyl hydroperoxide, cumene hydroperoxide, and t-butyl hydroperoxide are used as substrates. The heme-containing catalysts, methemoglobin and hematin, have identical hydroperoxide substrate requirements for singlet oxygen production. The hydroperoxide requirements for singlet oxygen production correlate with those reported by Dix et al. (Dix, T.A., Fontana, R., Panthani, A., and Marnett, L.J. (1985) J. Biol. Chem. 260, 5358-5365) for the production of peroxyl radicals in the hematin-catalyzed decomposition of hydroperoxides. The bimolecular reaction of peroxyl radicals is a plausible reaction mechanism for the singlet oxygen production in the systems studied.     

Effect of simulated microgravity on human lymphocytes

During space flight the function of the immune system changes significantly. Several papers reported that postflight the number and the proportion of circulating leukocytes in astronauts are modified (Leach, 1992), the in vitro mitogen induced T cell activation is depressed (Cogoli et al., 1985; Konstantinova et al. 1993) and there are detectable differences in cytokine production of leukocytes as well (Talas et al. 1983; Batkai et al. 1988; Chapes et al. 1992). One of the possible modifying forces is the microgravity condition itself. Our aim was to analyse mechanisms responsible for changing leukocyte functions in low gravity environment. For terrestrial simulation of microgravity we used a Rotary Cell Culture System (RCCS) developed by NASA. We investigated the effect of simulated microgravity on separated human peripheral blood mononuclear cells (PBMCs). We detected the populations of different cells by antibodies conjugated to fluorofors using a Flow Cytometer. Since space flight reduces the number of peripheral blood lymphocytes (Stowe et al., 1999) we supposed that apoptotic (programmed cell death) processes might be involved. This hypothesis was supported by the result of our earlier experiment demonstrating that simulated microgravity increased the level of secreted Tumor Necrosis Factor-alpha (TNFalpha, a known apoptotic signal molecule) significantly (Batkai et al. 1999).     

Targeting of proteins into the peroxisomal matrix

Summary During the last few years much has been learned regarding signals that target proteins into peroxisomes. The emphasis in the near future will undoubtedly shift towards the elucidation of the mechanism of import. The use of mammalian and yeast cells deficient in peroxisome assembly and/or import (Zoeller & Raetz, 1986; Erdmann et al., 1989; Cregg et al., 1990; Morand et al., 1990; Tsukamoto, Yokota & Fujiki, 1990) should provide a handle on the genes (Erdmann et al., 1991; Tsukamoto et al., 1991) involved in these processes. This will have to be coupled with further development of in vitro systems which will permit the dissection of the steps in the translocation of proteins into peroxisomes. Though some progress has been made in the development of such assays (Imanaka et al., 1987; Small et al., 1987, 1988; Miyazawa et al., 1989), the fragility of peroxisomes and the absence of biochemical hallmarks of import (such as protein modifications or proteolytic processing) have hindered progress. Since peroxisomes exist in the form of a reticulum in mammalian cells (Gorgas, 1984), all peroxisome purification schemes (from mammalian cells at least) must undoubtedly rupture the peroxisomes, which then reseal to form vesicular structures. Additionally, the reliance on the latency of catalase alone as a major criterion for the integrity of peroxisomes ignores the fact that many other matrix proteins leak out of peroxisomes at vastly different rates during purification of the organelles (Thompson & Krisans, 1990). In view of these problems, the development of peroxisomal transport assays with semi-intact cells would also constitute an important advance. It is very likely that in the next few years we will witness some major advances in our understanding of the mechanism by which proteins enter this organelle.I would like to thank all the members of my lab and my collaborators, past and present, whose hard work provided the material for this review. This work has been supported by grants from the March of Dimes Foundation (#1081) and the NIH (DK41737).     

Demonstration of glycosylation variants of human fibrinogen, using the new technique of glycoprotein lectin immunosorbent assay (GLIA)

A new highly sensitive method, incorporation the ELISA technique (enzyme-linked immunosorbent assay), is described for the quantitation of the glycan residues of glycoproteins. With the aid of this "glycoprotein-lectin immunosorbent assay (GLIA)", it is possible to determine the nature of the glycan residues of a single protein in a glycoprotein mixture, without prior purification. The GLIA can be used for the accurate determination of the inhibitor constant for the interaction of any monosaccharide with any lectin. Using the described technique, glycosylation of human fibrinogen from plasma and amniotic fluid were compared. In fibrinogen from amniotic fluid a "fetal" glycosylation type could be demonstrated. In addition, evidence is presented for the first time that plasma fibrinogen possesses (GlcNAc beta 1----4Man beta) residues (bisecting GlcNAc) and O-glycosidically bound carbohydrate units. Preliminary results were published as abstract (E. K?ttgen et al. (1988) Fresenius Z. Anal. Chem. 330, 448).     

Photochemiluminescent detection of antiradical activity. VII. Comparison with a modified method of thermo-initiated free radical generation with chemiluminescent detection.

The method of photosensitized chemiluminescence (PCL) allows the quantification of water- and lipid-soluble antioxidants and activity of superoxide dismutase (SOD) in the same measuring system. However, it needs a special device, which we have described in a previous paper in this series. Another method suitable for the assay of water- and lipid-soluble antioxidants is the thermo-initiated decay of azo-compounds combined with the measurement of O2 consumption (Niki, 1985; Wayner et al., 1985). Its long duration and the complicated measuring procedure is not acceptable for routine medical applications. We show that a modification using CL detection of free radicals with luminol, has results comparable with PCL for the determination of non-enzymic water- and lipid-soluble antioxidants, SOD activity and oxidative modification of proteins. In contrast to PCL, it is possible to use any luminometer with a heatable measuring cell and to investigate coloured samples. While the new method has an overall higher sensitivity and is scalable to microtitre plates, PCL measurements can be made at different pH. The advantages and analytical information content of certain components of the integral antioxidative capacity of blood plasma are discussed in comparison with other methods.     

Detection and characterization of lipid hydroperoxides at picomole levels by high-performance liquid chromatography

A new method for the detection of various lipid hydroperoxides and hydrogen peroxide at the picomole level has been developed by combining an HPLC system with an ultrasensitive analytical system based on the detection of chemiluminescence emitted by isoluminol in the presence of hydroperoxide and microperoxidase. This HPLC separation removes interfering antioxidants so that the method can be applied to biological samples such as blood plasma lipids. Several HPLC conditions are described which allow simple identification of different lipid hydroperoxides.     

Homologous potyvirus and flavivirus proteins belonging to a superfamily of helicase-like proteins

Comparison of the nucleoside triphosphate-binding motif(NTBM)-containing proteins of two groups of apparently distantly related positive-strand RNA viruses (potyvirus and flavivirus), revealed significant sequence similarity. In addition, these two groups of viral proteins show amino acid motifs in common with those conserved in a group of five NTBM-containing proteins from prokaryotic and eukaryotic cells, some of which have been experimentally related to helicase activity. Here we propose that the proteins mentioned above constitute a superfamily of helicase-like proteins, distinct from the one previously described [Gorbalenya et al., FEBS Lett. 235 (1988) 16-24; Hodgman, Nature 333 (1988) 22-23; 578], which includes the NTBM-containing proteins from another group of positive-strand RNA viruses, the 'Sindbis-like' viruses.     

Amide bond cleavage monitored continuously through detection of a dansylcadaverine leaving group.

The transglutaminase-catalyzed incorporation of the fluorescent amine, dansylcadaverine, into casein derivatives, such as N,N-dimethylcasein, is accompanied by a large increase in intensity of emission (Lorand et al., Anal. Biochem. 44, 221-231, 1971). We have sought to make use of this sensitive detection device for the continuous, on-line monitoring of an amide-splitting reaction in which dansylcadaverine served as the leaving group. The transglutaminase-coupled test system comprised gamma-glutamyldansylcadaverine as the first substrate and gamma-glutamylamine cyclotransferase as the enzyme responsible for releasing dansylcadaverine from the gamma-amide. At close to saturating levels of transglutaminase, the measured rate of increase of fluorescence, i.e. the steady-state rate of dansylcadaverine incorporation into N,N-dimethylcasein, showed a near-linear relationship with the concentration of gamma-glutamylamine cyclotransferase present in the assay mixture. The general approach developed may be applicable to the assay of other amide cleaving enzymes.     

Sensitivity of mouse lung fibroblasts heterozygous for GPx4 to oxidative stress

Phospholipid hydroperoxide glutathione peroxidase (GPx4) is a member of the family of selenium-dependent enzymes that catalyze the reduction of cell membrane-bound phospholipid hydroperoxides in situ and thus protects against membrane damage. Overexpression of GPx4 protects cultured cells from phosphatidylcholine hydroperoxide (PCOOH)-induced loss of mitochondrial membrane potential and blocks cell death induced by treatment with various apoptotic agents. We have generated mice that are heterozygous for a GPx4 null allele (GPx4 +/-); the homozygous null genotype is embryonic lethal. We report that cultured lung fibroblasts (LFs) isolated from adult GPx4 +/- mice had approximately 50% of the GPx4 activity of LFs from GPx4 +/+ mice and were significantly more susceptible to H2O2, cadmium, and cumene hydroperoxide-induced cytotoxicity, as measured by neutral red assay. Both GPx4 +/+ and GPx4 +/- LFs were susceptible to PCOOH-induced cytotoxicity at a high PCOOH concentration. We also found that GPx4 +/- LFs have lower mitochondrial membrane potential, greater cardiolipin oxidation, and lower amounts of reduced thiols relative to GPx4 +/+ LFs, but are more resistant than GPx4 +/+ LFs to further decrements in these endpoints following PCOOH treatment. These results suggest that adult lung fibroblasts deficient in GPx4 may have upregulated compensatory mechanisms to deal with the highly oxidized environment in which they developed.     

Neutrophils promote the release of alpha-6-fucosyltransferase from blood platelets through the action of cathepsin G and elastase

Human blood platelets release alpha-6-fucosyltransferase during coagulation of blood or after stimulation with thrombin or other agonists that cause platelet activation (Antoniewicz et al., FEBS Lett. 244 (1989) 388-390). However, in the absence of neutrophils the thrombin-stimulated platelets release only a small fraction of alpha-6-fucosyltransferase activity (Ko?cielak et al., Acta Biochim. Polon. 42 (1995) 35-40). We show that the effect of neutrophils is reproduced by cathepsin G or (less efficiently) by elastase, the two enzymes that are released by neutrophils during coagulation of blood. We have also localized alpha-6-fucosyltransferase to membrane and alpha-granule fractions of platelets that had been disrupted by nitrogen cavitation. It is concluded that thrombin-activated neutrophils release cathepsin G and elastase that promote degranulation of platelets and hence the secretion of alpha-6-fucosyltransferase.     

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.