Antioxidants and total peroxyl radical-trapping ability of olive and seed oils.

The presence of the oxidized and reduced forms of ubiquinones Q(9) and Q(10) was determined in commercial extra virgin olive and seed oils, where the amounts of alpha- and gamma-tocopherols and beta-carotene were also quantitated. Very high concentrations of ubiquinones were found in soybean and corn oils. Furthermore, the total antioxidant capability of each oil was evaluated by measuring total radical-trapping antioxidant parameters (TRAP) in tert-butyl alcohol and using egg lecithin as the oxidizable substrate. These values decreased in the order sunflower > corn > peanut > olive; the highest TRAP, which was found in sunflower oil, was related to the very high amount of alpha-tocopherol. Olive oil, because of the low content of alpha-tocopherol, exhibited a TRAP value approximately one-third that of sunflower oil. TRAP values of corn and soybean oils, in which low amounts of alpha-tocopherol but very high contents of gamma-tocopherol and reduced ubiquinones were present, were intermediate. gamma-Tocopherol exhibited a poor ability of trapping peroxyl radicals in tert-butyl alcohol. This behavior was probably due to the effects of the solvent on the rate of hydrogen abstraction from this phenol.

Formation of micelles and liposomes from carnitine amphiphiles.

Esterification of the carboxy and/or the hydroxy groups of (R)-carnitine (3-hydroxy-4-trimethylammonium butanoate) produces interesting classes of (cationic or zwitterionic) surfactants whose CMC values are in general predictable from their molecular structure. In fact similar relationships between CMC and the number of carbon atoms, Cn, have been found for three classes of such surfactants. However the sensitivity of CMC to Cn for the diesters is considerably lower than that calculated from literature values for the monoesters (either in their cationic or zwitterionic forms). The CMC values for the diesters have been determined by tensiometric, conductimetric and spectrophotometric methods, both in pure water and in 0.154 M NaCl solutions, at 25 degrees C. In particular the tensiometric results provide evidence that double-chain diesters undergo self assembly into structures more complex than simple micelles if the two chains are of comparable length. EPR and electron microscopy experiments show that the aggregates spontaneously formed by these surfactants are a mixture of multilamellar vescicles.

Size matters: the relationship between MMP-9 expression and aortic diameter.

BACKGROUND: Despite a wealth of data detailing increased metalloproteinase (MMP)-9 expression and activity in abdominal aortic aneurysms (AAAs), no studies examine the relationship between aortic size and MMP-9 expression. Because elastolysis occurs early in AAA formation, we hypothesized that MMP-9 expression would vary with aortic diameter. The purpose of this study was to measure MMP-9 mRNA levels in AAAs of various diameters and define the relationship between AAA size and MMP-9 expression. METHODS AND RESULTS: MMP-9 mRNA levels were measured by competitive polymerase chain reaction (PCR) using gene-specific external standards with cDNA from AAAs (n= 19) and normal aortas (n=4). Levels were normalized to GAPDH mRNA, determined separately via competitive PCR, to control for efficiency of reverse transcription. AAA size was measured on CT scans obtained within 6 weeks of surgery. MMP-9/GAPDH mRNA transcript levels in AAAs were expressed as mean+/-SEM and analyzed by ANOVA with a Tukey adjustment. There was a fourfold elevation in MMP-9/GAPDH mRNA transcript levels in 5.0- to 6.9-cm AAAs (98.06+/-15.19) compared with small (3.0- to 4.9-cm) AAAs (20.87+/-5.15, P<.03), large (>7-cm) AAAs (27.16+/-14.56, P<.01), or normal aortas (3.57+/-1.13, P<.003). The results did not change when they were normalized to patient height, nor were there significant differences in risk factors, age, or sex in each AAA group. CONCLUSIONS: MMP-9 mRNA expression is significantly higher in moderate-diameter (5- to 6.9-cm) AAAs than either small (<4.0-cm) or large (>7.0-cm) AAAs. Increased MMP-9 expression may account for the propensity of AAAs >5 cm to continue to expand, in contrast to smaller aneurysms. Lower levels in AAAs >7 cm suggest that increases in other enzymes or in diameter-dependent mechanical stress on the aortic wall are responsible for their characteristic rapid expansion and high rupture rates.

Antioxidant activity of reduced menadione in solvent solution and in model membranes.

The antioxidant activity of reduced menadione was investigated and compared with that of alpha-tocopherol both in solvent solution and in large unilamellar vesicles by using azocompounds as free radical generators. The results show that: i) reduced menadione behaves as a chain-breaking antioxidant; ii) its inhibition rate constant is similar to that of alpha-tocopherol in homogeneous solution, whereas it is 4 times larger in egg yolk lecithin vesicles; iii) the stoichiometric factor is found lower than 1 in both systems, since a substantial portion of menadiol is consumed by autoxidation and does not contribute to radical trapping; iv) when both alpha-tocopherol and menadiol are present in vesicles, reduced menadione can spare alpha-tocopherol. Data presented here suggest that the reduced form of vitamin K may protect, when present, cellular membranes from free radical damage.

Elevations of tissue-type plasminogen activator and differential expression of urokinase-type plasminogen activator in diseased aorta.

PURPOSE: Elevations of plasmin have been implicated in the pathogenesis of abdominal aortic aneurysms (AAA) because of its ability to digest extracellular matrix proteins. Plasminogen activators regulate the conversion of plasminogen to plasmin. Tissue-type plasminogen activator (tPA) is more important in modulation of fibrinolysis, and urokinase-type plasminogen activator (uPA) is predominant in tissue remodeling. The purpose of this study was to determine the levels of plasminogen activators in diseased aorta because they may be responsible for the increased plasmin levels previously described in AAA. METHODS: Levels of tPA and uPA in AAA, occlusive, and normal (organ donor) aorta were studied in tissue explant supernatants. Supernatant tPA and uPA levels were measured with an enzyme-linked immunosorbent assay. Northern analysis was used to quantitate uPA messenger RNA (mRNA) levels in aortic tissue. RESULTS: Levels of tPA in the supernatants were similar in occlusive (20 +/- 4 ng/ml) and AAA (23 +/- 8) aorta, but threefold higher than in normal aorta (7 +/- 5; p < 0.005 for normal vs occlusive and p < 0.001 for normal vs AAA). In contrast, uPA supernatant levels were differentially expressed, with the highest level existing in AAA (9.7 +/- 2.7 ng/ml), followed by occlusive (4.9 +/- 3.5), and the lowest levels in normal aorta (1.2 +/- 0.7; p < 0.05 for normal vs occlusive, p < 0.001 for normal vs AAA, and p < 0.005 for occlusive vs AAA). Inhibition of protein or RNA synthesis by addition of cyclohexamide or actinomycin D, respectively, revealed no significant difference between treated and control supernatants, suggesting that the increases were caused by protein release rather than active synthesis. Levels of uPA mRNA followed the same trend as the supernatant uPA levels (AAA 1.07 +/- 0.54, occlusive 0.54 +/- 0.08, and normal aorta 0.01 +/- 0.01). CONCLUSIONS: Levels of tPA were similar in aneurysmal and occlusive aorta, but exhibited a threefold increase over normal aorta, suggesting that the elevations of tPA are associated with the arteriosclerosis present in both aneurysmal and occlusive disease. Differences in uPA levels were significant between all three groups, with the highest levels in AAA and the lowest levels in normal specimens. Northern analysis of uPA mRNA followed the same trend, suggesting that the increase in uPA may be regulated at the level of transcription. As uPA plays an important role in tissue remodeling, our findings may also reflect the relative tissue repair activities in these three types of specimens and may explain the previously reported increased levels of plasmin seen in AAA.

Plasminogen activator levels are influenced by location and varicosity in greater saphenous vein.

PURPOSE: The plasminogen system, which includes tissue type plasminogen activator (tPA), urokinase type plasminogen activator (uPA), and their main inhibitor, plasminogen activator inhibitor type 1 (PAI-1), plays a major role in both fibrinolysis and tissue remodeling. This study compares the levels of tPA, uPA, and PAI-1 at the groin and ankle in normal and varicose greater saphenous vein (GSV). METHODS: GSV was collected from patients undergoing varicose vein (VV) removal and from normal vein (NV) from arterial bypass procedures. Portions of the GSV at the groin and the ankle were minced and placed in serum-free media for 48 hours. Assays of the supernatants were obtained for tPA, uPA, and PAI-1 protein by enzyme-linked immunosorbent assay. Cyclohexamide and actinomycin D were also added to the media of the VV tissue explant supernatants to inhibit protein and RNA synthesis, respectively. RESULTS: Levels of tPA were significantly higher at the groin (11 +/- 2) than the ankle (5 +/- 1) in the VV (p < 0.005), and this trend was also seen in the NV (groin 10 +/- 2 and ankle 7 +/- 3). Levels of uPA were significantly higher in the groin VV (14 +/- 4.3) than in NV (3.0 +/- 0.8, p < 0.05). This difference, although not statistically significant, applied to the ankle as well (VV 14.5 +/- 6.3 and NV 5.3 +/- 2.7). No significant difference was seen between NV and VV for PAI-1 (NV, groin 155 +/- 73 and ankle 113 +/- 53, VV, groin 161 +/- 20 and ankle 142 +/- 38) or tPA. Inhibitor studies revealed no significant difference among control, cyclohexamide, and actinomycin D supernatants for tPA, suggesting release of protein rather than active synthesis. In contrast, inhibitor supernatants were significantly lower for uPA and PAI-1 than control supernatants (p < 0.05), suggesting that uPA and PAI-1 were actively synthesized. CONCLUSIONS: In the tissue explant supernatant model uPA and PAI-1 are actively synthesized, but tPA is not. Levels of PAI-1 were comparable in all four groups. Levels of uPA in the varicose GSV were higher than in NV, suggesting a role for uPA in the pathologic makeup of VV. Levels of tPA were higher at the groin versus the ankle position, potentially explaining the previously described increased fibrinolytic activity seen at the groin.

Paramagnetic resonance in detecting carcinogenic risk from cytochrome P450 overexpression.

BACKGROUND: Despite the increasing interest in the role of oxygen radicals on human degenerative disorders including cancer, oxidative stress status is not yet measurable in vivo, largely precluding clinical application. Limited semi-quantitative assays of damage to broad classes of biomolecules such as lipids, proteins, and DNA are currently available. The detection of radicals in humans by a whole-body electron paramagnetic resonance (EPR) technique has not yet been developed, although this possibility has long fascinated free radical investigators. METHODS: While the EPR spin trapping procedure can be used to detect carbon centered or hydroxyl radical in human tissues, the most common spin traps are much less useful for capturing the superoxide anion (O2). To overcome these limitations, we propose a whole-body-harvest approach that utilizes a highly lipophilic spin scavenger that when injected in the animal is capable of trapping the O2 generated in vivo throughout the body with formation of a stable nitroxide measurable by EPR in the urine. A process known to generate the O2 is the induction of certain cytochrome P450 (CYP) isozymes by drugs or environmental pollutants. RESULTS: We report: 1) a correlation between the induction of each CYP gene family and the O2 yield; 2) support to an observation reported previously that the tumor promoting ability of CYP inducers is mainly mediated by the O2; and 3) the description of a method for nitroxide mediated O2 detection in vivo. CONCLUSION: These findings could open the way for using electron spin resonance in diagnostic practice.

Elevated levels of plasminogen-activator inhibitor type 1 in atherosclerotic aorta.

PURPOSE: Plasminogen activator inhibitor type I (PAI-1) inhibits the plasminogen activators that convert plasminogen to plasmin. In addition to initiating fibrinolysis, plasmin activates tissue matrix metalloproteinases, which cause degradation of the extracellular matrix (ECM) in the arterial wall. Elevated levels of PAI-1 ultimately decrease plasmin formation and may lead to an accumulation of ECM and arteriosclerosis. METHODS: PAI-1 was studied by four methods in atherosclerotic (aneurysmal and occlusive) and normal (organ donor) aorta: (1) PAI-1 secretion by tissue explant supernatants, including time course and inhibition studies; (2) tissue PAI-1 by protein extraction; (3) PAI-1 mRNA was quantitated by Northern analysis using glyceraldehyde-3-phosphate dehydrogenase to normalize for RNA loading; and (4) in situ hybridization was used to localize the cells that produced PAI-1 mRNA. RESULTS: Supernatant PAI-1 levels at 48 hours were 776 +/- 352, ng/ml in 11 atherosclerotic aortas and 248 +/- 98 ng/ml in 8 normal aortas (p < 0.005). Tissue PAI-1 levels per 100 mg of tissue were 99 +/- 58 ng in 11 atherosclerotic aortas and 38 +/- 20 ng in 5 normal aortas (p < 0.05). PAI-1 mRNA levels by Northern analysis were 0.91 +/- 0.49 in seven atherosclerotic aortas and 0.44 +/- 0.27 in five normal aortas. Supernatant time-course experiments revealed that PAI-1 increased over time. Inhibitor studies revealed that PAI-1 decreased to approximately one third of control values when cycloheximide or actinomycin D were added to the media, indicating that active synthesis of PAI-1 had occurred. In-situ hybridization localized PAI-1 mRNA predominately to endothelial cells and a few scattered vascular smooth muscle and inflammatory cells. Subgroup analysis revealed no statistically significant differences between aneurysmal and occlusive PAI-1 levels in any of the experiments. CONCLUSION: PAI-1 secretion, as measured by tissue explant supernatants, and total tissue PAI-1 in the protein extracts were significantly increased in atherosclerotic aorta. This elevation was also observed in the mRNA, which suggests that the increase is controlled at the level of transcription. PAI-1 mRNA was localized to endothelial, vascular smooth muscle, and inflammatory cells. We conclude that elevated levels of PAI-1 exist in diseased aorta. These elevated levels may lead to an accumulation of ECM, thereby contributing to the arteriosclerosis found in aortic occlusive and aneurysmal disease.

Injury of rat thymocytes caused by exogenous peroxyl radicals in vitro.

The aim of this study was to investigate oxidative cell injury in rat thymocytes under conditions of radical generation exterior to the cell utilizing the thermolabile azocompound 2,2'-azobis(2-amidinopropane) dihydrochloride to generate peroxyl radicals at a constant and reproducible rate. This initiator, being water-soluble and endowed with a positive charge, is suitable for studies on oxidative damage of biomembranes induced in the external water environment. The relationship between cell viability, lipid and thiol oxidation and chain-breaking antioxidant depletion was studied. During the first hour of treatment cell viability decreased slightly, protein sulfhydryl groups were consumed slowly and no significant production of conjugated dienes occurred. After 90 min of incubation, when thymocyte permeability started to increase, the concentration of alpha-tocopherol decreased gradually, significant changes of polyunsaturated fatty acids occurred and a rapid phase of thio oxidation commenced. It can be concluded that, under conditions of an exogenous oxidant challenge, initially the cell membrane provides a physical barrier to the entrance of radicals to the thymocyte. When peroxyl radicals gain access to the membrane and the molecular barrier begins to disorganize, the oxidizable cellular components become susceptible to massive attack.

In situ localization and quantification of mRNA for 92-kD type IV collagenase and its inhibitor in aneurysmal, occlusive, and normal aorta.

Ninety-two-kilodalton type IV collagenase (MMP-9) is present in aortic aneurysms and may be important to the pathogenesis of this disease. Alteration in expression of MMP-9 or its inhibitor, the tissue inhibitor of metalloproteinase type 1 (TIMP-1), could increase degradation of extracellular matrix and lead to aneurysm formation. The purpose of this study was (1) to measure tissue levels of MMP-9 and TIMP-1 mRNA in aneurysmal (AAA), atherosclerotic occlusive (AOD), and normal (NL) human infrarenal aorta; (2) to test for their expression by cultured AAA and NL vascular smooth muscle cells (VSMCs); and (3) to locate in situ the cells responsible for mRNA production within AAA, AOD, and NL aortic wall. Total RNA extracted from AAA (n = 8), AOD (n = 8), and NL (n = 7) tissue was subjected to Northern analysis. Signals for MMP-9 and TIMP-1 were normalized to alpha-tubulin. Mean values +/- SEM were compared by ANOVA. NL and AAA VSMCs were cultured, passaged, and grown to confluence before RNA extraction and Northern analysis. In situ hybridization with digoxigenin-labeled RNA probes localized cells responsible for MMP-9 and TIMP-1 mRNA expression within sections of AAA (n = 5), AOD (n = 2), and NL (n = 2) aorta. MMP-9 mRNA levels were significantly greater in AAA (0.855 +/- 0.180) than NL (0.046 +/- 0.23) (P < .02), but differences between AOD (0.406 +/- 0.196) and AAA or AOD and NL were not significant.(ABSTRACT TRUNCATED AT 250 WORDS)

Interleukin-1 beta induces differential gene expression in aortic smooth muscle cells.

PURPOSE: Abdominal aortic aneurysms are characterized by an accelerated turnover of extracellular matrix proteins and by an inflammatory infiltrate that releases the cytokines interleukin-1 beta and tumor necrosis factor-alpha. We examined the gene expression of human aneurysmal aortic smooth muscle cells and normal aortic smooth muscle cells after treatment with interleukin-1 beta and tumor necrosis factor-alpha by measuring the changes in smooth muscle cell collagen, elastin, collagenase, and tissue inhibitor of metalloproteinase messenger ribonucleic acid levels in response to these cytokines. METHODS: Biopsy of aneurysmal aorta (n = 6) and donor normal aorta (n = 3) was obtained at operation. Medial smooth muscle cells were cultured, passaged (P2 to P4), and incubated with 0, 10, 100, or 1000 pg/ml interleukin-1 beta, tumor necrosis factor-alpha, or platelet-derived growth factor for 24 hours. Total ribonucleic acid was harvested. Percentage changes in messenger ribonucleic acid from control levels for type I and type III procollagen, elastin, collagenase, 72 kDa type IV collagenase, tissue inhibitor of metalloproteinase-1, and tissue inhibitor of metalloproteinase-2 were measured by Northern hybridization. Analyses were performed with analysis of variance (p < 0.05). All comparisons between aneurysmal aortic smooth muscle cells and normal aortic smooth muscle cells represent comparisons between one aneurysmal aorta and one normal aorta. RESULTS: Added interleukin-1 beta resulted in significant, dose-dependent increases in the collagenase messenger ribonucleic acid level at all concentrations tested in both aneurysmal aorta and normal aorta. The increase in the collagenase messenger ribonucleic acid level ranged from a minimum increase of 123% for 10 pg/ml interleukin-1 beta in aneurysmal aortic smooth muscle cells to a maximum of 450% for 1000 pg/ml interleukin-1 beta in normal aortic smooth muscle cells. Interleukin-1 beta caused a significant decrease in the steady-state messenger ribonucleic acid levels for type 1 procollagen in both aneurysmal and normal aorta. The greatest reduction in type 1 procollagen messenger ribonucleic acid levels occurred at 100 pg/ml interleukin-1 beta in both aneurysmal aortic smooth muscle cells (-39%) and normal aortic smooth muscle cells (-48%). The only observed qualitative difference between aneurysmal aortic smooth muscle cells and normal aortic smooth muscle cells was the change in tissue inhibitor of metalloproteinase-1 messenger ribonucleic acid levels in response to added interleukin-1 beta. In aneurysmal aortic smooth muscle cells interleukin-1 beta at 1000 pg/ml significantly increased messenger ribonucleic acid levels by 82%, whereas levels of tissue inhibitor of metalloproteinase-1 messenger ribonucleic acid in normal aortic smooth muscle cells did not change in response to added interleukin-1 beta. Interleukin-1 beta did not alter messenger ribonucleic acid levels for type III procollagen, elastin, type IV collagenase, or tissue inhibitor of metalloproteinase-2 in aneurysmal aorta or normal aorta. When tumor necrosis factor-alpha or platelet-derived growth factor were added, this did not significantly change aneurysmal aortic smooth muscle cells messenger ribonucleic acid levels for collagenase, type IV collagenase, tissue inhibitor of metalloproteinase-1, tissue inhibitor of metalloproteinase-2, and type I and type III procollagen. CONCLUSIONS: These findings suggest that interleukin-1 beta, through its effect on smooth muscle cell collagenase and collagen gene expression, mediates the increased matrix turnover observed in aneurysms. Macrophages may induce changes in aortic smooth muscle cell gene expression in a paracrine manner that could lead to aneurysm formation.

Characterization of large unilamellar vesicles as models for studies of lipid peroxidation initiated by azocompounds.

The aim of this work was to characterize large unilamellar vesicles (LUVETs) prepared by a hand-driven extrusion device in order to use them for studies of lipid peroxidation and antioxidant activity. Vesicle structure and size were examined by electron microscopy. Lipid and antioxidant content was determined before and after the extrusion procedure. Then LUVETs were subjected to autoxidation initiated by both the lipid-soluble 2,2'-azobis(2,4-dimethylvaleronitrile) and the water-soluble 2,2'-azobis(2-amidinopropane hydrochloride) azocompounds. The results demonstrated that: i) LUVETs prepared with lipid concentrations ranging between 25 and 150 mM were essentially unilamellar and reasonably homogeneous, with an average diameter of 90 nm; ii) the phospholipid, cholesterol and antioxidant amounts retained by filters were about 10-15%; iii) LUVETs were suitable for autoxidation studies initiated by the water-soluble azocompound both in the absence and presence of antioxidants. The lipid-soluble azocompound could be used only at low concentrations and its vesicle content had to be determined since part of the initiator was not incorporated into the lipid bilayer. These data suggest that LUVETs seem to be recommended for studies of lipid peroxidation and antioxidant activity.

Peroxidation potential of rat thymus during development and involution.

The thymus of rats of ages between 1 and 7 months was homogenised and subjected to oxidative stress induced by iron salts. Lipid peroxidation, protein thiols and glutathione status were evaluated. The thymus of rats of 1 month of age exhibited lower susceptibility to the radical attack with respect to the thymus of rats between 3 and 7 months of age. This susceptibility was correlated with the content of polyunsaturated fatty acids and of lipophylic chain-breaking antioxidants.

Autoxidation and antioxidant activity of ubiquinol homologues in large unilamellar vesicles.

The antioxidant activity of ubiquinol homologues with different side-chain length such as ubiquinol-3 and ubiquinol-7 was compared with that of alpha-tocopherol when peroxidation was induced by the water-soluble initiator 2,2'-azobis-(2-amidinopropane hydrochloride). In large unilamellar vesicles containing equal amounts of alpha-tocopherol, ubiquinol-3 and ubiquinol-7 the rates of inhibition were very similar but the stoichiometric factor of quinols was approximately 1. To explain this low value, which is one-half of that found when the autoxidation was performed in apolar solvents (Chem. Phys. Lipids (1992) 61, 121-130), the oxidation of alpha-tocopherol and ubiquinol-3 initiated by the azocompound was studied both in methanol and in dimiristoyl-lecithin vesicles. The results obtained show that the ubiquinol homologues undergo a radical chain reaction taking place at the polar interface and suggest that the average preferred location of both quinol headgroups is near to the outer surface of the bilayer.