TBARS Production Research Articles

Pb 2+ promotes lipid oxidation and alterations in membrane physical properties

Experimental evidence suggests that cellular damage mediated by oxidants could be involved in the pathology associated with lead (Pb) toxicity. We investigated the effect of Pb 2+ on lipid oxidation in liposomes using different initiators. In the presence of Fe 2+, Pb 2+ (12.5–200 μM) stimulated lipid oxidation in phosphatidylcholine:phosphatidylserine-containing liposomes, measured as 2-thiobarbituric acid-reactive substances (TBARS) and conjugated dienes. This stimulatory effect depended on the presence of membrane negative charges and on bilayer integrity. Pb 2+ did not stimulate TBARS formation in the presence of 25 mM 2,2′-azo-bis (2,4 dimethylvaleronitrile (AMVN) and 2,2′ azobis (2-amidinopropane) (AAPH). Pb 2+ significantly stimulated TBARS production and NADH oxidation in the presence of photoactivated rose Bengal. The use of specific inhibitors indicated that several reactive oxygen species were involved in the pro-oxidant action of Pb 2+. Pb 2+ (12.5–200 μM) caused membrane lateral phase separation and this effect was positively correlated with its capacity to stimulate Fe 2+ and rose Bengal-initiated TBARS production. Pb 2+ could bind to the membrane and act to stimulate lipid oxidation by causing changes in membrane physical properties. Through this mechanism Pb 2+ would favor the propagation of lipid oxidation. By causing lateral phase separation and/or by increasing lipid oxidation rates, Pb 2+ could be cytotoxic by altering membrane-related processes.

Vitamin B6 deficiency affects antioxidant defences in rat liver and heart.

We have evaluated the effects of a diet containing normal amounts of lipids and a marginal content of vitamin B6 on lipid peroxidation. Pyridoxal phosphate concentrations of plasma and liver indicated that an initial deficiency state was reached. Vitamin B6 deficiency led to peroxidative stress: TBARS production was higher in the liver (+18.6%) and even more in the heart (+61%) of deficient rats as compared with controls. Furthermore, significant stimulation of glutathione-dependent enzymes occurred in both heart and liver of deficient rats: glutathione peroxidase activity increased in heart (+144%) and liver (+505%); glutathione reductase increased in heart (+54.9%) and liver (+15.5%). No difference in the total glutathione content of the organs of the two groups was observed. The reduced glutathione/oxidized glutathione ratio was significantly lower in deficient rats. Although the activity of glutathione-dependent enzymes was significantly greater in deficient rats than in controls, this stimulation was only partially able to counteract the peroxidative damage due to vitamin B6 deficiency.

Selective effects of different antioxidants on oxidation of lipoproteins from rats.

Lipoprotein oxidation may contribute to development of atherosclerosis, and supplementation with antioxidants may reduce risk for atherosclerotic events. Genistein, a major isoflavone from soy protein, and catechins from green tea have important antioxidant properties. This study compared the effects of various diets containing antioxidant-rich foods or supplements on serum lipids and lipoprotein oxidation of male Sprague-Dawley rats. The control diet used was devoid of vitamin E. Test diets included these ingredients: green tea powder, 20 g/kg; beta-carotene, 250 mg/kg; a low isoflavone soy protein isolate; a genistein-rich soy protein isolate; and vitamin E, 4000 mg/kg. Ten-week-old rats were acclimatized for 1 week on a special custom diet without vitamin E. Following randomization and allocation to different diet groups, rats were fed the test diets for 3 weeks. Blood was drawn by cardiac puncture, and the plasma was separated by centrifugation. The VLDL-LDL fraction was isolated by ultracentrifugation. Oxidation kinetics of the VLDL-LDL fraction were determined by measuring the lag phase and formation of conjugated dienes, lipid peroxides, and TBARS. The vitamin E diet (P < 0.001) and high-genistein diet profoundly decreased all parameters of lipoprotein oxidation. The following alterations were noted with the high-genistein diet compared to the control diet: the lag phase was 49% longer (P=0.002); conjugated diene formation was decreased by 28% (P=0.01); lipid peroxide formation was decreased 31% (P=0.0059); and TBARS production was 35% lower (P=0.019). The low-isoflavone diet increased the lag phase by 43% (P=0.0019) but did not significantly alter other measures of oxidation. Green tea increased only the lag phase by 33% (P=0.012) compared to the control diet. Beta-Carotene had no significant effect on the oxidation of lipoproteins. The effect of genistein-rich soy protein isolates on lipoprotein oxidation in vitro suggests that either soy isoflavones or other antioxidants derived from soy protein, like vitamin E, may be transported in these lipoproteins. The minimal effects of the isoflavone-poor soy protein isolate suggests that either the small amount of isoflavones present have a potent effect or other components of soy protein are exerting these effects. Further studies are required to examine these results.

Free-radical scavenging actions of natural antioxidants

A dose-dependent inhibitory effect of wheat, alfalfa and ginkgo biloba (EGb) extracts on TBARS production was measured. The half-inhibition concentration (IC50) of the tested antioxidants were 2.7±0.2, 1.3±0.1, and 0.20±0.02 mg/ml for wheat, alfalfa and EGb extracts, respectively. Lipid radicals combined with the spin trap POBN resulted in adducts that gave a characteristic EPR spectrum. The IC50 of the tested antioxidants on lipid radical content, were 12.4±0.2, 7.7±0.3, and 1.20±0.06 mg/ml for wheat, alfalfa and EGb extracts, respectively. Rat liver microsomes in the presence of DMPO, NADPH and iron-citrate generate an EPR spectra with characteristics of the DMPO-OH spin adduct. The basic system, without the addition of any scavenger showed an area of 3.5 AU/mg protein. The areas in the presence of 1.5 mg/ml EGb, 4 mg/ml wheat or alfalfa, were of 1.7±0.2, 3.4±0.3, and 3.6±0.2 AU/mg protein, respectively. O 2 − generation rate by the microsomes exposed to EGb extract was decreased by 40%, as compared to the rate measured in microsomes incubated in the absence of the extract. However, the supplementation of rat liver microsomes with either wheat or alfalfa extracts did not affect microsomal generation of O 2 −. Iron reduction rate was not affected by the addition of any of the tested extracts. The data presented here showed that EGb extracts were able to limit lipid peroxidation and scavenge lipid radicals in rat liver microsomes more efficiently than alfalfa and wheat bran extracts. Moreover, wheat and alfalfa extracts were not able to inhibit O 2 − and ·OH generation by biological membranes, suggesting that their potentiality to be successfully used in human health in the treatment of diseases involving free radical and oxidative damage are not as promising as that for the use of EGb extracts.

Effect of nicotine and cotinine on the susceptibility to in vitro oxidation of LDL in healthy non smokers and smokers

Cigarette smoke of which the major component is nicotine plays an important role in the development of cardiovascular diseases. To study the effect of in vitro incubation of LDL with nicotine and its metabolite, cotinine on a copper-induced peroxidation, we monitored the formation of conjugated dienes, hydroperoxides and thiobarbituric acid-reactive substances production. The LDL studied were taken from six non-smokers (aged 41.5 years) and six smokers who consumed at least ten cigarettes per day (40.7 years). LDL oxidation with CuSO 4 showed that cigarette smoking promotes LDL susceptibility to peroxidative modification. During the peroxidation of LDL with nicotine (0 to 5 mmol/l) and CuSO 4 (5 μmol/l), the formation of hydroperoxides decreased when nicotine concentrations increased and the production of TBARS increased in a concomitant manner. The results showed that the presence of nicotine destabilized the production of hydroperoxides in LDL and increased the formation of secondary oxidation products. On the other hand, cotinine had no effect on LDL oxidative susceptibility in smokers and non-smokers.

Membrane composition can influence the rate of Al3+-mediated lipid oxidation: effect of galactolipids.

In the first part of the present study we investigated the effects of pre-natal and early postnatal exposure of mice to high levels of dietary Al3+ on myelin lipid composition and lipid oxidation. We found: (1) a significantly higher (104%; P<0.01) content of brain myelin galactolipids in the high-Al3+ group than in controls, and, (2) a significant correlation (r2=0.70; P<0.01) between the concentration of myelin galactolipids and TBARS (2-thiobarbituric acid-reactive substances) content, a parameter of lipid oxidation. Based on these results, we evaluated in an in vitro model (liposomes) whether galactolipids could affect the capacity of Al3+ to stimulate Fe2+-initiated lipid oxidation, and whether this effect could be due to the promotion of changes in membrane physical properties (membrane phase separation and rigidification). The presence of galactolipids (10-40 mol%) in the liposomes caused a concentration-dependent increase in the stimulatory effect of Al3+ on Fe2+-induced TBARS production, and on the ability of Al3+ to induce phase separation and membrane rigidification. The capacity of Al3+ (10-100 microM) to induce lateral phase separation in liposomes composed of phosphatidylcholine/phosphatidylserine/galactolipid (36:24:40, molar ratio) was correlated significantly (r2=0.99; P<0. 001) with the stimulatory action of Al3+ on Fe2+-induced TBARS production. We propose that the high content of galactolipids found in myelin from Al3+-intoxicated mice could favour Al3+-induced changes in membrane physical properties, with the subsequent acceleration of lipid oxidation rates.

Protective Effects of the Lazaroid U-74389G Against Hyperoxia in Rat Type II Pneumocytes

The aims of this study were to investigate the effect of hyperoxia on O2−·, H2O2and·NO generation and iNOS mRNA levels in rat type II pneumocytes in vitro and the possible protective effect of the lazaroid U-74389G. Rat type II pneumocytes were exposed, 36 h after isolation, to air, 60% or 85% O2for 48 h. At the beginning of the experiment and 24 h later, the cells were exposed for 30 min to either 30 μmU-74389G or only the vehicle for the lazaroid (control). Exposure to 60% and 85% O2decreased nitrite production 2.9-fold and 3.9-fold, and increased O2−·and H2O2generation 4.6-fold and 6.7-fold, respectively. In the 85% O2-exposed cells, hyperoxia increased lipid peroxidation (thiobarbituric acid reactive substances, TBARS production) 2-fold and iNOS mRNA production 5.4-fold. U-74389G prevented the decrease in nitrite and the rise in O2−·and H2O2production, the increase in TBARS and the rise in iNOS mRNA after hyperoxia. We conclude that exposure of type II pneumocytes in vitro to subtoxic oxygen levels leads to a disturbance in the·NO-O2−·balance despite increased iNOS mRNA levels. The lazaroid U-74389G appears to be a useful compound in the protection of hyperoxic lung injury by restoration of this·NO-O2−·balance and prevention of TBARS formation.

An ethanolic-aqueous extract of Curcuma longa decreases the susceptibility of liver microsomes and mitochondria to lipid peroxidation in atherosclerotic rabbits.

Atherosclerosis is characterized by oxidative damage which affects lipoproteins, the walls of blood vessels and subcellular membranes. This study evaluates the antioxidant capacity of a Curcuma longa extract on the lipid peroxidation of liver mitochondria and microsome membranes in atherosclerotic rabbits. Male rabbits fed a 3% (w/w) lard and 1.3% (w/w) cholesterol diet were randomly assigned to three groups. Two groups were treated with different dosages of a turmeric extract (A and B) and the third group (control) with a curcumin-free solution. Basal and in vitro 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH)-induced hydroperoxide and TBARS productions in liver mitochondria and microsomes were analyzed. Group A had the lowest concentration of mitochondrial hydroperoxides. In microsomes, the basal hydroperoxide levels were similar in all groups but, after the induction of oxidation, group C registered the highest value; TBARS production followed the same trend in mitochondria. These findings suggest that active compounds in curcuma extract may be protective in preventing lipoperoxidation of subcellular membranes in a dosage-dependent manner.

Modulation of UVA-induced lipid peroxidation and suppression of UVB-induced ornithine decarboxylase response by all-trans-retinoic acid in human skin fibroblasts in vitro.

Although clinical evidence of improvement of human skin photodamage by all-trans-retinoic acid (atRA) has accumulated, evidence for its preventative effects against photodamage is limited. Here we studied human skin fibroblasts in vitro. For determination of atRA uptake and metabolism, cells were treated with 3 or 10 microM atRA and retinoids analyzed by HPLC. After 16h a peak of cellular retinoid levels was reached, mainly atRA and 13-cis-RA. At this time point cells were irradiated with UVA (1-20 J/cm2) or UVB (5-500 mJ/cm2). TBARS in medium supernatant were used as an indicator of lipid peroxidation; ornithine decarboxylase (ODC) activity served as a marker of the mutagenic and carcinogenic effects of UV light. 1 h post irradiation (p.i.) with 20 J/cm2 UVA, TBARS production was enhanced by a 3 microM atRA treatment (121+/-5% of vehicle treated cells) and decreased by a 10 microM atRA treatment (75+/-2% of vehicle treated cells), and not significantly altered in UVB irradiated cells. 24 h p.i. with 50 mJ/cm2 UVB, ODC activity peaked in vehicle treated cells at a 7.4+/-0.2-fold increase compared to sham irradiated control cells, and was reduced to a 4.9+/-0.2-fold increase by 3 microM atRA. Treatment with 10 microM atRA further decreased ODC activity (3.7+/-1.0-fold increase) and this delayed activity peak occurred at 36 h p.i. ODC activity was not significantly enhanced by UVA irradiation. These results suggest that in normal human skins fibroblast atRA and/or its metabolites influence the UVA-induced lipid peroxidation by at least two distinct antagonistic mechanisms, while the ODC response to UVB-induced DNA damage possibly involves a ROS-independent, retinoid-sensitive regulatory pathway.

Autoxidation of Rat Brain Homogenate: Evidence for Spontaneous Lipid Peroxidation. Comparison with the Characteristics of Fe2+- and Ascorbic Acid-Stimulated Lipid Peroxidation

Aerobically-incubated brain homogenates are known to undergo autoxidation characterized by spontaneous TBARS production, presumably as a result of lipid peroxidation. However, TBARS measurement alone, because of its lack of specificity, is not sufficient to demonstrate the occurrence of lipid peroxidation in complex biological systems. This study, undertaken to determine whether or not spontaneous oxidation of rat brain homogenate is due to lipid peroxidation, measured different specific markers of this process (fatty acids, lipid aldehydes and the formation of fluorescence products) and studied changes in α-tocopherol. Incubation of rat brain homogenates at 37°C under air led to spontaneous TBARS formation, which was accompanied by lipid aldehydes and lipid fluorescence products as well as polyunsaturated fatty acid (PUFA) degradation. Alpha-tocopherol was also consumed. On the whole, these results demonstrate that autoxidation of brain homogenate is a spontaneous lipid peroxidation process. When homogenates were exposed to Fe2+ and ascorbic acid-induced oxidative stress, lipid peroxidation was enhanced. However, spontaneous and stimulated peroxidation showed similar patterns not characteristic of classical lipid peroxidation, i.e. without the lag and accelerating phases typical of a propagating chain reaction. PUFA degradation was limited despite stimulation of peroxidation.

Lipid peroxidation induced by ultrasonication in Ehrlich ascitic tumor cells

The mechanism of sonodynamic action in tumor cells is poorly investigated. It is known that ultrasound generates free radicals in phosphatidylcholine liposomes used as a membrane model. The participation of lipid peroxidation products in the mechanisms of physiological suppression of cell multiplication has been investigated for some tumor cells. In the present work ultrasound-induced lipid peroxidation in Ehrlich ascitic tumor cells was studied. Ultrasonication increased the level of lipid peroxidation quantified by the TBARS method in homogenates from Ehrlich ascitic tumor cells. Changes in the fatty acid composition of lipids from Ehrlich ascitic tumor cells irradiated by sonication were observed. TBARS production obtained by ultrasound was compared to TBARS production obtained by widely used chemical inductors. The free-radical processes evoked by ultrasound are of interest in antitumor therapy.

Effects of Sangre de Drago from Croton lechleri Muell.-Arg. on the production of active oxygen radicals

The total reactive antioxidant potential (TRAP) of ‘Sangre de Drago’ from Croton lechleri (Euphorbiaceae) was determined by monitoring the intensity of luminol enhanced chemiluminescence enhanced by peroxyl radicals derived from thermolysis of 2,2'-azo-bis(2-amidinopropane). The TRAP index was calculated as 935.4 ± 141 μM, measured as equivalents of Trolox concentration. On the other hand, the additive incorporation of lower concentrations yielded an instantaneous increase in chemiluminescence, suggesting a prooxidant activity at these levels. DNA sugar damage induced by Fe(II) salts was also used to determine the capacity of the latex to suppress hydroxyl radical-mediated degradation of DNA. As in the case of luminol enhanced chemiluminescence, Sangre de Drago was highly effective in reducing oxidation of DNA at higher concentrations, but showed an increase in the production of TBARS at lower doses, as compared to the control. Finally, antioxidant activity was tested using hydroperoxide-initiated chemiluminescence in rat liver homogenates, and the latex showed an increase in light emission, suggesting the presence of prooxidant compounds.

Evaluation of thein vitro antioxidant activity in extracts ofUncaria tomentosa (Willd.) DC.

Different extracts of U. tomentosa were tested in vitro for their antioxidant activity utilizing tert -butyl-hydroperoxide-initiated chemiluminescence in rat liver homogenates. Methanol fractions of both stem-bark and roots were capable of exerting antioxidant activity by this technique. The presence of different concentrations of bark and root methanol extracts also prevented TBARS production and free radical-mediated DNA-sugar damage. © 1997 John Wiley & Sons, Ltd.

Effect of aliphatic aldehydes on the lipid peroxidation and chemiluminescence of biological systems under oxidative stress.

The effect of several aliphatic aldehydes on lipid peroxidation was evaluated by measuring the oxygen uptake rate, thiobarbituric acid-reactive products formation and the emitted visible chemiluminescence intensity. Measurements were carried out in brain homogenates and erythrocyte plasma membrane and liver microsomal fractions. In all systems studied, aldehydes (25 mmol/L) (e.g. acetaldehyde, 2,2-dimethylpropanal), increased the intensity of the luminescence associated with the oxidation process. In contrast, aldehyde incorporation decreased TBARS production and the rate of oxygen uptake. The increased luminescence intensity is explained in terms of secondary reactions of aldehyde derived free radicals. These results clearly indicate that extreme care must be exercised in the interpretation of chemiluminescence data in the presence of aldehydes.

Radiation-induced red cell damage: role of reactive oxygen species.

Cellular blood components are irradiated to prevent graft-versus-host disease in transfusion recipients at risk for this syndrome. Because gamma radiation can result in the production of reactive oxygen species, the role of reactive oxygen species was investigated in radiation-induced red cell damage. Whole blood from normal donors was exposed to various doses of t-butyl hydroperoxide (0-1 mM) and/or to gamma-radiation (0-50 Gy). Oxidative damage was assessed by the extent of lipid peroxidation (measured by thiobarbituric acid-reactive substances [TBARS]) and hemoglobin oxidation. Fresh blood was divided into three parts-one initially irradiated and stored, another stored with portions irradiated weekly, and a third stored without irradiation. TBARS and hemoglobin oxidation were measured weekly. As expected, t-butyl hydroperoxide induced TBARS formation and hemoglobin oxidation in a dose-dependent fashion. The gamma-radiation not only increased hemoglobin oxidation and TBARS formation, but also enhanced the t-butyl hydroperoxide effect on red cells. Red cell storage increased TBARS generation and hemoglobin oxidation in a time-dependent fashion. When radiation was administered either initially or after weekly storage, TBARS production and hemoglobin oxidation were increased over that measured in unirradiated paired controls. Gamma radiation at clinically used doses increases lipid peroxidation and hemoglobin oxidation in human red cells. The effect of gamma-radiation is accentuated by blood storage and induces damage independent of time of storage.

In vitro influence of ascorbate on lipid peroxidation in rat testis and heart microsomes.

Lipid peroxidation (LPO) in rat testis and heart microsomes was compared using the ADP/Fe2+ as initiator with and without ascorbate at different concentrations. The extent of LPO was estimated by the levels of TBARS and PUFA. Without ascorbate, LPO was higher in heart than in testis despite elevated levels of catalase in heart. With increased ascorbate concentrations, a biphasic effect of LPO was observed. For a concentration < or = 0.2 mM, ascorbate acted as pro-oxidant and increased TBARS correlated with decreased PUFA were observed both in testis and heart. Above 0.2 mM, ascorbate acts as antioxidant but differences in the rate of LPO were observed. In heart decreased TBARS correlated with increased PUFA whereas in testis TBARS only decreased, PUFA were not significantly modified. These results suggest different mechanisms in LPO initiation in the two organs. Increasing concentrations of H2O2 produced directly elevated TBARS levels in testis while a lag phase was observed in heart before the increase, suggesting that H2O2 was the essential ROS produced by ascorbate-ADP/Fe2+. The effects of scavengers such as catalase and ethanol showed an inhibitory effect on TBARS production only in testis, suggesting the role of H2O2/OH. as an initiator of LPO. In heart, catalase produced a slight increase in TBARS levels whereas no modification was observed with ethanol, suggesting a possible direct activation by ADP/Fe2+ through a metal-oxo intermediate.

Garlic compounds protect vascular endothelial cells from oxidant injury

Oxygen radical injury and lipid peroxidation have been suggested as major causes of cancer, atherosclerosis and the aging process. We examined in vitro the effect of garlic on H2O2-induced oxidant injury in bovine pulmonary artery endothelial cells (PAEC). After overnight preincubation with Aged Garlic Extract (AGE, from Wakunaga Pharmaceutical Co., Ltd., Japan) or S-allyl cysteine (SAC), PAEC monolayers were exposed to H2O2 for 3 h. Cell viability (MTT assay), lactate dehydrogenase (LDH) release, and lipid peroxidation (TBA-RS) were measured to assess oxidant injury. AGE (1-4 mg/ml) pretreatment significantly reduced the loss of cell viability induced by 50-100 microM of H2O2. AGE and SAC exhibited dose dependent inhibition of both LDH release and TBA-RS production induced by 50 microM of H2O2. The results show that AGE and SAC can protect vascular endothelial cells from oxidant injury. Numerous garlic compounds could be involved in the antioxidant properties of garlic, while there could be some prooxidant compounds derived from garlic. It is important to keep an array of antioxidant compounds to develop good herbal preparation, like AGE.

α-Tocopherol Enrichment of High-Density Lipoproteins: Stabilization of Hydroperoxides Produced During Copper Oxidation

In the aim to study the effect of an in vitro enrichment of high-density lipoprotein (HDL) with α-tocopherol in alcoholic solution on a copper-induced peroxidation, we monitored several markers of lipid peroxidation ( α-tocopherol consumption, formation of conjugated dienes and of fatty acid hydroperoxides, production of thiobarbituric acid-reactive substances) and the integrity of apolipoprotein A-I. High-density lipoproteins (1.063 < d < 1.21) with a mean of 0.58 α-tocopherol molecules per HDL particle were enriched whith α-tocopherol in alcoholic solution to obtain an average of 3.7 and 21 α-tocopherol molecules per HDL particle. HDL oxidation with 5 μM CuSO 4 at 37°C resulted in the total disappearance of endogenous α-tocopherol after 2 h, but after 24 h about 19% of α-tocopherol remained in the most enriched HDL. In agreement with the tocopherol-mediated peroxidation, the formation of conjugated dienes and of fatty acid hydroperoxides was very fast and increased with α-tocopherol concentration, whereas TBARS production decreased. These results showed that α-tocopherol enrichment stabilized the production of hydroperoxides in HDL and decreased the formation of secondary oxidation products. These latter products are known for deleterious effects towards apolipoproteins. This could explain why we observed that the apolipoprotein A-I of the most enriched HDL was only slightly altered after incubation with CuSO 4. Copyright © 1996 Elsevier Science Inc.

Bilirubin sensitized photooxidation of human plasma low density lipoprotein

Previous investigations have shown that the bile pigment bilirubin can act as peroxyl radicals scavenger and transition metals trap, but also as a prooxidant, to erythrocyte ghost membranes through 102driven photooxidation. In the present study we examined the changes occurring in the lipoprotein particle following bilirubin-sensitized photooxidation of isolated plasma LDL. The oxidative stress resulted in increased TBA reactivity, diene formation, free cholesterol oxidation, apo B fragmentation and enhanced uptake of the modified particle by the mouse macrophage scavenger receptors as well as the decrease binding to the native B, E-receptor on fibroblasts. The marked increase in TBARS production in D20-enriched medium and the inhibition of lipid peroxidation of azide is consistent with singlet oxygen involvement in the oxidation process. The apo B-bound Cu2+ appears to become redox active during photooxidation since the presence of EDTA in the reaction mixture greatly reduced protein fragmentation. It was also found that BHT inhibited almost completely the lipid peroxidation, as determined by the TBA reaction but could not totally abolish the formation of 5 a-hydroxycholesterol, which is the main product formed by the direct attack of 102 on cholesterol. The results of this work strongly suggest that, through photooxidation by light-activated bilirubin, the lipoprotein particle may be modified in the blood stream as well, besides being modified in the well known oxidation site within the arterial wall. Our findings provide the rationale for extending these studies to clinical investigations, which aim at developing strategies for minimizing damage to arterial tissue following phototherapy of hyperbilirubinemic newborns or cancer patients after systemic administration of photosensitizers.

Effect of aluminum ion on Fe(2+)-induced lipid peroxidation in phospholipid liposomes under acidic conditions.

The effects of Al3+ on Fe(2+)-induced lipid peroxidation in phospholipid liposomes consisting of phosphatidylcholine (PC) and phosphatidylserine (PS) were examined under acidic conditions. The stimulatory effect of Al3+ on Fe(2+)-induced lipid peroxidation in the liposomes showed a biphasic response against pH variation, and the maximum stimulation was observed around pH 6.0. In addition, it was found that the stimulatory effect of Al3+ on the lipid peroxidation was dependent on the proportion of PS in the liposomes. On the other hand, the lipid peroxidation in PC liposomes was not stimulated by the addition of Al3+. From these findings, it is suggested that the Al3+ effect on Fe(2+)-induced lipid peroxidation under acidic conditions is largely dependent on the phospholipid composition. Trivalent cations such as Tb3+ and Ga3+ also stimulated Fe(2+)-induced lipid peroxidation in PC/PS liposomes under acidic conditions, but divalent cations (Zn2+ and Mn2+) showed no stimulatory effect. The extents of Fe2+ disappearance and Fe3+ formation during the reaction were enhanced by the addition of Al3+ or Ga2+, but Tb3+ had no effect on Fe2+ disappearance. The results with 1,6-diphenyl-1,3,5-hexatriene (DPH) showed that the fluorescence anisotropy of DPH-labeled PC/PS liposomes under acidic conditions was increased by the addition of Al3+. Furthermore, there is a relation between the extents of the fluorescence anisotropy of the complex and TBARS production. In contrast, the fluorescence anisotropy of DPH molecules embedded in PC liposomes was not changed by the addition of Al3+. Based on these results, a possible mechanism of the stimulatory effect of Al3+ on Fe(2+)-induced lipid peroxidation under acidic conditions is discussed.