Mass-spectrometric characterization of phospholipids and their hydroperoxide derivatives in vivo: effects of total body irradiation.

Combination of electrospray ionization mass spectrometry (ESI-MS), fluorescence high-performance liquid chromatography (HPLC), and 2D-high-performance thin-layer chromatography (2D-HPTLC) is a powerful approach to identify and quantitatively analyze oxidized phospholipids in vivo. We describe application of this methodology in assessments of phospholipid hydroperoxides using as an example their characterization and quantitative determinations in different tissues of mice exposed to total body irradiation (TBI, 10 and 15 Gy). Using ESI-MS, we identified individual molecular species - with particular emphasis on polyunsaturated molecules as preferred peroxidation substrates - in major classes of phospholipids: cardiolipin (CL), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidylinositol (PI) isolated from mouse brain, lung, muscles, small intestine, and bone marrow. We show that the pattern of phospholipid oxidation 24 h after TBI is nonrandom and does not follow the phospholipid abundance in tissues. The anionic phospholipids - CL, PS, and PI - are the preferred peroxidation substrates. We identified and structurally characterized individual hydroperoxides in these three classes of phospholipids. The protocols described may be utilized in studies of signaling functions of oxidized phospholipids in cell physiology and pathology.

Influence of bleaching by ultrasound on fatty acids and minor compounds of olive oil. Qualitative and quantitative analysis of volatile compounds (by SPME coupled to GC/MS).

The effects of bleaching using high power ultrasound (20 kHz) on the quality of olive oil were considered in this study, in order to verify the modifications that can occur in fatty acid composition and minor compounds. During the treatment of olive oil under ultrasonic waves, a rancid odour has been detected. Treated olive oils show no significant changes in their chemical composition but the presence of some volatile compounds, due to ultrasonic treatment. Some off-flavour compounds (hexanal, hept-2-enal and 2(E),4(E)-decadienal) resulting from the sonodegradation of olive oil have been identified. A wide variety of analytical techniques (GLC, HPLC and GC/MS) were used to follow the quality of bleached olive oils with ultrasonic waves by the determination of the amounts of certain minor compounds such as sterols and tocopherols. Steradienes, resulting from the dehydration of sterols, were detected with small quantities especially in severe conditions of sonication. Solid phase micro-extraction (SPME) coupled to gas chromatography was known to be a sensitive technique to follow changes in the oxidative state of vegetable oils by measuring the amount of volatile materials produced during the refining process.

Effect of intense pulsed-light exposure on lipid peroxides and thymine dimers in human skin in vivo.

BACKGROUND: Intense pulsed light (IPL) generates high-intensity short flashes of visible light and has been used for about 10 years to improve dermatological conditions such as telangiectasia, pigmented lesions, and skin aging. Although these systems deliver a moderate dose (10-30 J/cm(2)) of visible light, this dose is delivered during a short pulse (2-5 milliseconds), which implies a very high fluence rate (approximately 4000 W/cm(2)). For this reason, we speculated whether the Bunsen-Roscoe law of reciprocity could still be valid in these conditions. OBSERVATIONS: Nine healthy volunteers were exposed to IPL or UV-A or simulated solar UV radiation, and then thymine dimer and lipid peroxide concentrations were determined in skin biopsy specimens of the exposed sites. Only exposure to solar UV radiation (7-J/cm(2) UV-A + 80-mJ/cm(2) UV-B) produced measurable amounts of thymine dimers in DNA from skin biopsy specimens, whereas UV-A radiation (40 J/cm(2)) and IPL (9 J/cm(2)) induced 3-fold and 6-fold increases of cutaneous lipid peroxides, respectively. CONCLUSIONS: These preliminary results indicate that IPL, although filtered for wavelengths shorter than 500 nm, can generate oxidative stress, a typical hallmark of UV-A, but does not induce thymine dimers. This emphasizes the need for long-term studies involving IPL before using this technique in a recurrent manner.

Separation and quantitation of phospholipid hydroperoxide families using high-performance liquid chromatography with mercury cathode electrochemical detection.

High-performance liquid chromatography with mercury cathode electrochemical detection (HPLC-EC(Hg)) was used to separate and quantify various phospholipid hydroperoxide (PLOOH) families. Under the conditions used, baseline separation of four major biologically relevant PLOOH classes was achieved. Responsiveness was linear up to at least 1 nmol of PLOOH with a detection limit in the subpicomolar range (0.1-0.5 pmol). Applying this method to photodynamically stressed murine leukemia cells and mitochondria isolated from these cells, we identified and quantified PLOOHs derived from phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and cardiolipin. In terms of high sensitivity, specificity, and reliability, HPLC-EC(Hg) has a clear advantage over all other existing techniques for determining PLOOHs in complex biological systems.

Oxidative and genotoxic damage after radio-iodine therapy of Graves' hyperthyroidism.

PURPOSE: To evaluate genetic damage and oxidative stress following a single therapeutic dose of 131I in Graves' disease patients monitored up to 180 days after treatment. MATERIALS AND METHODS: Genetic damage induction was estimated as the increase in micronuclei in peripheral lymphocytes of patients. As indicators of radiogenic oxidative stress, vitamin E and lipoperoxide levels were assessed in the plasma of patients, as well as the release of plasmic clastogenic factors measured by the induction of micronuclei in vitro in peripheral lymphocytes of a healthy donor. RESULTS: Vitamin E depletion lasted at least 3 days and the basal level was restored within 7 days. No statistically significant variations were observed in lipoperoxide plasma levels. A sharp increase of micronuclei in the peripheral lymphocytes of patients was correlated (p < 0.001) with the release of clastogenic factor in the plasma. The highest micronucleus value was negatively correlated (p < 0.03) with the lowest vitamin E level observed in each patient. CONCLUSIONS: Micronuclei induction was the direct consequence not only of the energy deposition of 131I on the genetic material, but also of oxidative stress, likely via the release of clastogenic factor.

Effects of ultraviolet B on epidermal morphology, shedding, lipid peroxide, and antioxidant enzymes in Cope's rat snake (Elaphe taeniura).

Cope's rat snakes (Elaphe taeniura) favor to expose under sunlight in order to increase their body temperature simultaneously increasing the risk of skin damage by ultraviolet B (UVB) irradiation. We have investigated the effects of UVB irradiation on their skin. Results show that the UVB transmission of the keratinous layer was only 5.1+/-0.36%. The peak of epidermal damage and malondialdehyde (MDA) content, a product of lipid peroxidation, simultaneously occurred 72-96, 48 or 24 h after exposure to 300, 500 and 800 mJ/cm2 of UVB radiation, respectively. Superoxide dismutase (SOD) activity was inhibited by UVB and the lowest activity occurred 24, 48, 12 and 12 h after exposure to 110, 300, 500 and 800 mJ/cm2 of UVB, respectively. SOD activity recovered later to some extent but mostly remained below control level. After exposure to different doses of UVB radiation, catalase (CAT) activity was inhibited immediately, and then gradually recovered and even increased to peak levels above control level. The highest CAT levels accompanied the most serious damage of skin morphology. Later on, CAT activity decreased and recovered again close to or below control level, which was accompanied by shedding off the damaged epidermal complex. This indicated that the epidermal damage induced by UVB is closely related to lipid peroxidation, where CAT acts as a primary antioxidant enzyme. Moreover, the keratinous layer protects the viable cell layer against UVB damage as well.

Radiation induced peroxidation of polyunsaturated fatty acids: recent results on formation of hydroperoxides.

Aqueous solutions of linoleic acid were irradiated in air with gamma-rays of 137Cs. High pressure liquid chromatography (HPLC) was been used to separate and measure the production of hydroperoxides. The results obtained after reverse phase chromatography, associated with a microperoxydase for hydroperoxide detection, indicate the presence of two different hydroperoxides. One type of hydroperoxide was the major product obtained when the initial linoleic concentrations were below the critical micellar concentration (2 mM), and the second type was produced when the concentrations were above 2 mM. A further separation carried out on the second hydroperoxide by direct phase HPLC showed that it contains three compounds, mainly HPODE 9 and 13.

[Radiation induced lipid peroxidation: factors which determine the oxidizability of lipids]. / De la peroxydation lipidique radioinduite : les facteurs déterminant l'oxydabilité des lipides.

Lipids are the essential components of cell membranes and lipoproteins. Their peroxidation plays an important role in numerous pathologies in which oxidative stress is involved. Lipid peroxidation occurs through a chain reaction that contributes to membrane damage in cells. It results in the conversion of fatty acids to polar hydroperoxides and leads to the breakdown or malfunction of the membrane. Lipids are amphiphilic molecules that aggregate in aqueous solutions into micelles and liposomes. The effect of this structural organization is significant in studies of radiation-induced peroxidation damage in highly ordered biological systems such as biological membranes. In this paper, a synthesis of the data concerning radioinduced lipid peroxidation is completed by an original review of the different parameters that determine lipid oxidizability. In addition, the influence of lipid aggregation and the effect of molecular packing are discussed.

Studies of methyl 2-nitroimidazole-1-acetohydroxamate (KIN-804) 2: effect on certain antioxidant enzyme systems in mice bearing Ehrlich ascites carcinoma.

In order to decrease toxicity and/or increase radiosensitizing activity, a new 2-nitroimidazole derivative, methyl 2-nitroimidazole-1-acetohydroxamate (KIN-804), was synthesized to solve the problem of tumor hypoxia. Evaluation of the efficiency of KIN-804 was carried out through studying the antioxidant enzyme system: The superoxide dismutase (SOD), catalase and lipid peroxide levels provide a rough index of the balance between free radical generation and scavenging. Female albino mice were inoculated with Ehrlich ascites carcinoma (EAC) in the thigh. The administration of KIN-804 (i.p. 80 mg/kg body weight) was carried out 20 min before localized irradiation of 10 Gy. In general, the data revealed that KIN-804 administration, followed or not by gamma irradiation, exerted significant inhibition of SOD and catalase activities accompanied by a significant increase in lipid peroxide level in tumor-bearing mice.

A theoretical formalism for aggregation of peroxidized lipids and plasma membrane stability during photolysis.

The objective of this investigation was to examine, from a theoretical perspective, the mechanism underlying the lysis of plasma membranes by photoinduced, chemically mediated damage such as is found in photolysis. Toward this end, a model is presented which relates the membrane lifetime to the thermodynamic parameters of the membrane components based upon the kinetic theory of aggregate formation. The formalism includes a standard birth/death process for the formation of damaged membrane components (i.e., peroxidized lipids) as well as a terminating condensation process for the formation of aggregates of peroxidized plasma membrane lipids. Our theory predicts that 1) the membrane lifetime is inversely correlated with predicted rate of membrane damage; 2) an upper limit on the duration of membrane damage exists, above which the mean and variance of the membrane lifetime is independent of further membrane damage; and 3) both the mean and variance of the time of membrane lifetime distribution are correlated with the number of sites that may be damaged to form a single membrane defect. The model provides a framework to optimize the lysis of cell membranes by photodynamic therapy.

[The trends and intensity of lipid peroxidation processes initiated by the action of "Chernobyl" factors]. / Izuchenie napravlennosti i intensivnosti protsessov perekisnogo okisleniia lipidov, initsiirovannykh deistviem "Chernobyl'skikh" faktorov.

The 45-day's action of nutritional radionuclides (137Cs, 90Sr, 40K) by inside organism irradiation at doses: 0.16 sGy, 0.55 cGy, 5.5 cGy was investigated. The blood, muscles, heart, kidneys, lung, liver, brain, spleen and testis were studied. The content of dienes, trienes, oxodienes, tetraenes conjugates and malondialdehyde were researched. The level of dienes increased in blood and kidneys; MDA--in spleen than more, that more of dose irradiation. Content of oxodienes, trienes and tetraenes (the biological activity eicosanoids) decreased at kidneys, lung, brain and spleen. At total, these parameters by organism's adaptational systems controlled at physiological conditions.

DNA cleavage induced by oxyl radicals generated in the photosensitized decomposition of fatty ester hydroperoxides derived from oleic and linoleic acid.

The xanthone-sensitized photodecomposition of the fatty ester hydroperoxides 1 and 2 in the presence of pBR 322 DNA was investigated as a chemical model system to assess whether this process may cause DNA damage through oxyl radicals. Unequivocally, oxyl radicals are formed in the xanthone-sensitized photodecomposition of the hydroperoxides 1 and 2, as confirmed by EPR studies. Indeed, both hydroperoxides 1 and 2 induce DNA single-strand breaks upon uv-A irradiation in the presence of the exogenous sensitizer xanthone. Under similar reaction conditions, the corresponding alcohol 3 of the hydroperoxide 1 was ineffective. Mannitol as radical scavenger inhibited significantly the formation of DNA single-strand breaks in the xanthone-sensitized decomposition of the hydroperoxides 1 and 2. Irradiation of xanthone alone or the hydroperoxides 1 and 2 without sensitizer did not cause any detectable DNA single-strand breaks. These results confirm that photosensitization of the fatty ester hydroperoxides 1 and 2 induces DNA modifications by oxyl radicals. We suspect that the combination of endogenous photosensitizers, solar uv radiation, and lipid hydroperoxides may damage cellular DNA through oxyl radicals.

[The action of sublethal doses of ionizing radiation on the endogenous radioresistance background]. / Issledovanie deistviia subletal'nykh doz ioniziruiushchego izlucheniia na éndogennyi fon radiorezistentnosti.

Sublethal doses of X radiation (0.5 Gy and 1 Gy) caused the alterations in levels of main components of endogenous radioresistance background in rat tissues. There were demonstrated the decrease of serotonin content in stomach mucosa and spleen, adrenalin, noradrenalin and corticosteroid contents in adrenal glands, nonprotein thiols content in spleen, and the increase of lipid peroxide level in serum on the 3-14 days after irradiation. The recovery of the investigated parameters was occurred to the 21 day after exposure.

[Changes in the intensity of lipid peroxidation in the blood plasma of sheep under repeat gamma irradiation after chronic gamma exposure]. / Izmenenie intensivnosti perekisnogo okisleniia lipidov v plazme krovi ovets pri povtornom gamma-obluchenii posle khronicheskogo gamma-vozdeistviia.

The indices of lipid peroxidation and the contents of alpha-tocopherol and retinol in the blood plasma of sheep exposed to repeated gamma irradiations were studied. It was shown that a repeated acute gamma irradiation of sheep with sublethal and lethal doses after 9 months of chronic low-dose gamma irradiation adversely affected the activity of the lipid free-radical oxidation. A radiologic pathology in the repeatedly irradiated animals developed as a decrease in the intensity of spontaneous chemiluminescence. Also, a tendency to an increase in the concentration of malonic dialdehyde was observed along with weak changes in the contents of triene and diene conjugates and the levels of alpha-tocopherol and retinol.

L-ascorbic acid inhibits UVA-induced lipid peroxidation and secretion of IL-1alpha and IL-6 in cultured human keratinocytes in vitro.

We investigated the antioxidative effect of L-ascorbic acid on lipid peroxidation and on secretion and mRNA expression of IL-1alpha and IL-6 after UVA irradiation (20 J/cm2) in cultured human keratinocytes. Lipid peroxidation was measured by (i) high performance liquid chromatography with UV detection of malondialdehyde (MDA) at 256 nm and (ii) spectrometric measurement of thiobarbituric acid-reactive substances (TBARS). To evaluate UV-induced cytotoxicity, we assessed cell membrane damage by measuring lactate dehydrogenase (LDH) release. UVA-induced lipid peroxidation in cultured human keratinocytes was inhibited by ascorbic acid in a concentration-dependent manner: MDA protein equivalent was reduced by 47% (10(-6)), compared to keratinocytes not exposed to L-ascorbic acid (p < 0.05), and the TBARS showed a concentration-dependent decrease of 49% (10(-6) M) in L-ascorbic acid-supplemented cultures compared to controls (p < 0.05). LDH release was decreased by 45% in L-ascorbic acid-supplemented keratinocyte cultures, indicating protection against cell death (p < 0.05). L-Ascorbic acid was able to downregulate IL-1alpha mRNA expression in both UVA-irradiated and nonirradiated cells; however, IL-6 mRNA expression remained unaffected. The secretion of these cytokines was reduced nearly to normal in the presence of L-ascorbic acid. These findings indicate a major cell-protective effect of L-ascorbic acid on UVA-induced lipid peroxidation and the secretion of pro-inflammatory cytokines by UVA-irradiated human keratinocytes.

Changes in lipid peroxide levels and the activity of reactive oxygen scavenging systems in thyroid tissue after exposure to radioactive iodine in rats.

Exposure to ionizing radiation causes radiolysis of water in tissues leading to generation of reactive oxygen species (ROS), which are known to affect the antioxidant defense systems and induce lipid peroxidation (LP). Use of radioactive iodine (131I) for diagnosis and therapy of thyroid disorders may also generate ROS in the thyroid. Early (24 and 48 hours) and late (18 days) effects of subablation doses of 131I (370/555/1110 kBq) on the antioxidant defense and LP in the thyroid tissues have been studied. LP was elevated in all 131I treated groups by 10% to 41%. Although there was no change in catalase (CAT), the superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities showed evidence of change from 48 hours onwards--SOD decreased by 32% to 56% and GPx increased by 15% to 43%. Nonprotein thiols (reduced glutathione, GSH) showed an elevation of 16% at 24 hours, but later declined by 15% by day 18 after 370 KBq of 131I. Thus, the increase in LP observed may be due to beta irradiation induced ROS by 131I. The parallel decrease in SOD could be due to inactivation by ROS. The increase in GPx may be a consequence of induction due to elevated LP and/or ROS, which may be inadequate to lower the LP. In spite of elevated LP, the thyroid function appears to be normal.

[The estimate of the free-radical lipid oxidative indices of the blood plasma in irradiated sheep]. / Otsenka pokazatelei svobodnoradikal'nogo okisleniia lipidov v plazme krovi obluchennykh ovets.

External alpha-irradiation of sheep with doses of 51.6 and 154.8 mC/kg initiated free-radical oxidation of lipids in blood plasma, which was characterized by increase of spontaneous chemiluminescence intensity, malonaldehyde, conjugated diene and triene as well as of alpha-tocopherol concentration, while retinol level simultaneously decreased. On the contrary, chronic irradiation of sheep with doses of 2.58 and 12.9 mC/kg resulted in decrease of malonaldehyde content, conjugated diene and triene level as well as natural antioxidant alpha-tocopherol level in blood plasma.

UV-initiated autoxidation of methyl linoleate in micelles studied by optical absorption.

The UV-induced dissociation of lipid hydroperoxides, naturally present in methyl linoleate and methyl linoelaidate, was monitored by absorption spectroscopy. The decay of hydroperoxides in homogeneous solution, for a fluence rate of 0.82 mW.cm-2 at 240 nm, followed an exponential course with an average rate constant of k = (2.0 +/- 0.1) . 10(-3) s-1. Addition of alpha-tocopherol led to an increase in the magnitude of k until it reached a limiting value of (3.4 +/- 0.2) . 10(-3)s-1. When methyl linoleate (containing adventitous traces of hydroperoxides) was incorporated in SDS micelles, sustained UV irradiation caused initially an increase in the amounts of lipid hydroperoxides, due to linoleate autoxidation, followed by an exponential decrease due to photodissociation. From the initial rate of hydroperoxide formation (due to linoleate autoxidation) and the rate constant of the subsequent hydroperoxide decay, an oxidizibility of 0.61 . 10(-2) (Ms)-1/2 of methyl linoleate in micelles was deduced. It is suggested that UV irradiation and optical absorption afford an easy and reliable means for studying autoxidation of polyunsaturated fatty acids.

Recovery of antioxidants and reduction in lipid hydroperoxides in murine epidermis and dermis after acute ultraviolet radiation exposure.

In previous studies we have found that a single acute dose of ultraviolet radiation to murine skin causes a large degree of destruction of enzymic and non-enzymic antioxidants immediately after irradiation. In the present study, we wished to elucidate the recovery of antioxidants after a single dose of ultraviolet (UV) radiation. We measured antioxidants and lipid hydroperoxides (as a marker of membrane damage) in murine epidermis and the dermis at 0, 3, 12, 24, 72 and 120 h after exposure to UV radiation (25 J/cm2, UVA+UVB). Lipid hydroperoxides showed the highest values immediately after UV exposure and returned to control values within 24 h in both epidermis and dermis. The activities of catalase, glutathione peroxidase and glutathione reductase showed the lowest activities immediately after UV exposure; superoxide dismutase activities reached a minimum at 3 h postexposure. The pattern of recovery was different for each enzyme and for epidermis and dermis. The activities of superoxide dismutase and catalase decreased remarkably and recovered slowly. Superoxide dismutase in the dermis recovered full activity by 120 h and in the epidermis by 12 h. Catalase activity in both epidermis and dermis had returned to only 50% of control activity at 120 h, although the epidermis showed a temporary increase (to 93%) at 24 h. Glutathione peroxidase and glutathione reductase were slightly decreased immediately after irradiation, recovered to 100% at 3 h and then increased to 200-250% in both the epidermis and the dermis at various times; values had returned to 100% in epidermis by 120 h but remained elevated in dermis.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of low-dose X-ray irradiation on biomembrane in brain cortex of aged rats.

We previously found that low-dose X-ray irradiation or radon (weak alpha-ray) inhalation increases SOD activities and reduces lipid peroxide levels in various organs of 7-week-old rats or rabbits. In this study, we examined how the changes of SOD activity, lipid peroxide level, and membrane fluidity of the cerebral cortex in aged male Wistar rats (65 and 91 weeks old) were affected by low-dose X-ray irradiation (100 cGy or under) compared with those in 7-week-old rats, to elucidate the mechanism of aging inhibition. The following results were obtained: Although radiation sensitivity was observed to decreases with age, low-dose irradiation changed the Mn-SOD activity, lipid peroxide level, and membrane protein fluidity parameter of the cerebral cortex in the age rats to be closer to those in the youth. These findings suggest that the increased SOD activity induced by low-dose irradiation enhances biomembrane functions, and that the decrease of lipid peroxide level enhances the membrane protein fluidity.