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Biomedical subjects

L Packer

Publications and source records attributed to L Packer.

At least 55 records · Page 3Linked to original sources
PubMed1996

Guanidino compounds generate reactive oxygen species.

Methylguanidine, guanidinoacetic acid and guanidinosuccinic acid are endogenous substances in body tissues. Extremely high levels of these substances are known to be related to the pathogenesis of epilepsy and renal failure such as uremia. In this study it was demonstrated that methylguanidine, guanidinoacetic acid and guanidinosuccinic acid, and arginine generate hydroxyl radicals in aqueous solution. These findings suggest that a high level of guanidino compounds accumulating near or within cells such as neurons (in an epileptogenic focus) or nephrons (in uremic patients) may cause free radical damage leading to these clinical disorders. Arginine may have a similar role in the pathogenesis of hyperarginemia.

Electron Spin Resonance Spectroscopy↗
PubMed1996

Proton gradients during salt adaptation of Synechococcus.

The ability of cells to energize their membranes during salt-adaptation as measured by delta pH was studied using the electron spin resonance (ESR) spin probe technique. Salt-adaptated cells have the following features: an increased stability of delta pH across the cytoplasmic membrane and a significantly lower rate of delta pH formation across the thylakoid membrane. Therefore, during salt-adaptation, the cytoplasmic membrane takes over the role of primary energizing membrane from the thylakoid membrane. In contrast to non-adapted cells, delta pH across the thylakoid membrane of salt-grown cells is higher under respiratory than under photosynthetic conditions.

Adaptation, Physiological↗
PubMed1996

Destruction of tocopherols, carotenoids, and retinol in human plasma by cigarette smoke.

The mechanisms by which exposure to cigarette smoke dramatically increase the incidence and severity of atherosclerosis and the incidence of lung cancer, chronic obstructive airways disease, and emphysema are incompletely understood. Epidemiologic evidence has suggested a modifying role for antioxidant micronutrients, including tocopherols and carotenoids, in these disease processes. It has been suggested that oxidants in cigarette smoke could be involved. We exposed freshly obtained human plasma to the gas phase of cigarette smoke to assess its effects on tocopherols, carotenoids, and retinol. Exposure to cigarette smoke led to the depletion of most of the lipophilic antioxidants in 20 mL human plasma. The order of disappearance was lycopene > alpha-tocopherol > trans-beta-carotene++ > (lutein + zeaxanthin) = cryptoxanthin > gamma-tocopherol = retinol. However, despite a substantial loss of alpha-tocopherol, there was very little peroxidative damage to lipids, and no detectable change in the content of polyunsaturated fatty acid-rich cholesterol esters. We conclude that a wide spectrum of lipophilic micronutrients undergo degradation when exposed to gas-phase cigarette smoke. The relevance of these in vitro findings to possible cigarette smoke-induced depletions of respiratory tract lipophilic antioxidants remains to be clarified.

Adult↗
PubMed1996

Antioxidant and redox regulation of gene transcription.

Reactive oxygen species (ROS) are implicated in the pathogenesis of a wide variety of human diseases. Recent evidence suggests that at moderately high concentrations, certain forms of ROS such as H202 may act as signal transduction messengers. To develop a better understanding of the exact mechanisms that underlie ROS-dependent disorders in biological systems, recent studies have investigated the regulation of gene expression by oxidants, antioxidants, and other determinants of the intracellular reduction-oxidation (redox) state. At least two well-defined transcription factors, nuclear factor (NF) kappa B and activator protein (AP) -1 have been identified to be regulated by the intracellular redox state. The regulation of gene expression by oxidants, antioxidants, and the redox state has emerged as a novel subdiscipline in molecular biology that has promising therapeutic implications. Binding sites of the redox-regulated transcription factors NF-kappa B and AP-1 are located in the promoter region of a large variety of genes that are directly involved in the pathogenesis of diseases, e.g., AIDS, cancer, atherosclerosis and diabetic complications. Biochemical and clinical studies have indicated that antioxidant therapy may be useful in the treatment of disease. Critical steps in the signal transduction cascade are sensitive to oxidants and antioxidants. Many basic events of cell regulation such as protein phosphorylation and binding of transcription factors to consensus sites on DNA are driven by physiological oxidant-antioxidant homeostasis, especially by the thiol-disulfide balance. Endogenous glutathione and thioredoxin systems, and the exogenous lipoate-dihydrolipoate couple may therefore be considered to be effective regulators of redox-sensitive gene expression. The efficacy of different antioxidants to favorably influence the molecular mechanisms implicated in human disease should be a critical determinant of its selection for clinical studies.

Animals↗
PubMed1996

2D NMR of the metabolic antioxidant dihydrolipoic acid and its derivatives.

Dihydroplipoate and lipoate are physiological thiols which in addition to their coenzyme functions exhibit antioxidant activity. For NMR investigations of their protective mechanism in biological and model systems it is very important to know the full assignment of proton and carbon spectra of these molecules in water (D2O). An unambiguous assignment of proton and carbon NMR spectra has been made for dihydrolipoate and its short chain derivatives bisnor- and tetranor-lipoic acid in D2O and CDCl3 solutions using 2D NMR methods. Oxidation of dihydrolipoic acid produces substantial electron density deshielding of the carbons nearest to the SH groups with the largest shift found at the inner SH group (17.79 ppm in D2O, 16.93 in CDCl3) and almost no changes in the tail portion of the molecule. However, bisnor-dihydrolipoic acid and especially tetranor-dihydrolipoic acid have more carbon deshielding near the outer SH group of the molecule which correlates with their known diminished ion chelating activity. Moreover, the proton triplet at position 2 of lipoic acid has strong pH dependence (pK = 4.58) due to the close proximity to the carboxylic group and this feature may be used for monitoring pH.

Antioxidants↗
PubMed1996

Simultaneous determination of tissue tocopherols, tocotrienols, ubiquinols, and ubiquinones.

A tissue-specific distribution of the various vitamin E forms, tocotrienols and tocopherols, has been found, suggesting that these forms have unique roles in cellular functions. A sensitive procedure is described for the simultaneous determination of individual tocopherols, tocotrienols, ubiquinols, and ubiquinones using gradient high pressure liquid chromatography (HPLC) and electrochemical detection for vitamin E homologues and ubiquinols, and in-line UV detection for ubiquinones. Using this method, the lipophilic antioxidant complement of a variety of hairless mouse tissues was analyzed. Of the vitamin E forms, brain contained virtually only alpha-tocopherol (5.4 +/- 0.1 nmol/g; 99.8%) and no detectable tocotrienols were found. By contrast, skin contained nearly 15% tocotrienols and 1% gamma-tocopherol. In other tissues, the alpha-tocopherol content was higher (20 nmol/g), while each of the other forms represented about 1% of the total (gamma-tocopherol 0.2 to 0.4 nmol/g, alpha-tocotrienol 0.1, gamma-tocotrienol 0.2). Ubiquinol-9 concentrations were highest in kidney (81 nmol/g) and in liver (42 nmol/g), while the highest ubiquinone-9 concentrations were found in kidney (301 +/- 123 nmol/g) and heart (244 +/- 22 nmol/g). Liver contained nearly identical concentrations of each of the redox couple (ubiquinol-9 (41 +/- 16 nmol/g) and ubiquinone-9 (46 +/- 18 nmol/g). The unique distribution of these various antioxidants in the tissues measured suggests their distribution may be dependent upon selective mechanisms for maintaining antioxidant defenses in each tissue.

Animals↗
PubMed1996

Amplified determination of lipoyl groups by lipoamide dehydrogenase in the presence of oxidized glutathione.

Lipoyl group determination by lipoamide dehydrogenase (NADH: lipoamide oxidoreductase; EC 1.8.1.4) was examined using lipoyl lysine as a substrate. The reaction was monitored by the coupled oxidation of NADH at 340 nm absorbance. Dehydrogenase-mediated NADH oxidation was too slow to be used for the quantification of lipoyl groups in the concentration range 1 to 10 microM. However, when glutathione disulfide (GSSG) was added to the reaction mixture to regenerate the oxidized substrate for the enzyme, NADH oxidation was markedly enhanced. This GSSG-dependent enhancement of NADH oxidation was strongly dependent upon the lipoyl substrate, but was only slightly dependent on the amounts of GSSG without the substrate. In the presence of excess GSSG, NADH oxidation was linearly correlated to the concentration of lipoyl lysine up to 10 microM; this assay is suitable for determining micromolar concentrations of the lipoyl moiety.

Dihydrolipoamide Dehydrogenase↗
PubMed1996

Efficacy of Bio-Catalyzer alpha.rho no.11 (Bio-Normalizer) supplementation against peroxyl radical-induced oxidative damage in rat organ homogenates.

To better delineate the antioxidant potential of Bio-Catalyzer alpha.rho No.11 (Bio-Normalizer), a natural food supplement recently proposed as an antioxidant agent, we investigated the efficacy of Bio-Normalizer supplementation to protect rat organ homogenates against oxidative damage induced in vitro by peroxyl radicals generated in the hydrophobic or in the hydrophilic phase. Bio-Normalizer supplementation efficiently protected rat kidney homogenates against the accumulation of thiobarbituric reactive substances (TBARS), the formation of protein carbonyl derivatives and the depletion of alpha-tocopherol induced by peroxyl radicals generated from the hydrophobic azo-initiator 2,2'-azobis (2,4-dimethylvaleronitrile) (AMVN). It also protected the heart but not the liver or the brain homogenates. Bio-Normalizer supplementation did not have effect in any organ homogenates when peroxyl radicals were generated from the hydrophilic azo-initiator 2,2'-azobis (2-amidinopropane) dihydrochloride) (AAPH). In vitro direct addition of aqueous solutions of Bio-Normalizer to the organ homogenates was ineffective against AMVN or AAPH-induced oxidative damage. Our findings expand previous reports on the antioxidant activity of Bio-Normalizer. They confirm that supplemented Bio-Normalizer protects against peroxyl radical-induced oxidative damage and suggest that its antioxidant action depends on in vivo bioactivation, it is organ specific and it is limited to damage induced by peroxyl radicals generated in the hydrophobic phase.

Animals↗
PubMed1996

Decomposition of alpha-lipoic acid derivatives by photoirradiation-formation of dihydrolipoic acid from alpha-lipoic acid-.

Due to its strained five-membered ring, alpha-lipoic acid (LA) has an absorption band around 330 nm, which is used to quantify its concentration. In order to obtain information for the homolytic rupture of the S-S bond and the formation of dihydrolipoic acid (DHLA), the photochemical reaction of lipoic acid was examined in the presence or absence of ascorbic acid upon exposure to UVA light. The absorption band of alpha-lipoic acid at around 330 nm disappeared by photoirradiation, which corresponds to the rupture of S-S bond of the 1,2-dithiolane ring in lipoic acid. HPLC-Electrochemical Detection (ECD) analysis of lipoic acid showed significant formation of dihydrolipoic acid and other thiols. The formation of thiols from the photoreaction of lipoic acid was also confirmed by the Ellman method. The formation of thiols from lipoic acid was completely time-dependent and the formation of the thiols increased upto 55%. Similar results were obtained in the photochemical reactions of short-chain analogues, bisnor- and tetranor-lipoic acid. On the other hand, beta-lipoic acid was quite stable, no photodecomposition of beta-lipoic acid was observed in the UV region. The formation of thiols including dihydrolipoic acid from lipoic acid can be explained by considering the rupture of S-S bond, which results in the formation of the dithiyl radicals of alpha-lipoic acid. It is proposed that intra- and intermolecular hydrogen abstraction of the dithyl radical produces thiols including dihydrolipoic acid as final products.

Antioxidants↗
PubMed1995

The phorbol 12-myristate 13-acetate (PMA)-induced oxidative burst in rat peritoneal neutrophils is increased by a 0.1 mT (60 Hz) magnetic field.

Magnetic fields (MF) may affect biological systems by increasing free radical concentrations. To test this, we have investigated whether low frequency (60 Hz) low intensity (0.1 mT) MF can modulate the phorbol 12-myristate 13- acetate (PMA) induced respiratory burst in primed rat peritoneal neutrophils, followed in real time using the dye 2',7'-dichlorofluorescin (DCFH), which reacts with free radical-derived oxidants such as H2O2 (which is formed from the dismutation of superoxide) to become 2',7'-dichlorofluorecein (DCF), a highly fluorescent compound. In the presence of the MF, a 12.4% increase in the fluorescence signal was observed in PMA-stimulated neutrophils (n = 5, P < 0.02, 18 pairs of measurements). We believe this represents the first experimental observation of MF influencing events involving free radical species generated during signal transduction in living cells.

Animals↗
PubMed1995

Role of ascorbate in protection by nitecapone against cardiac ischemia-reperfusion injury.

The antioxidant properties of nitecapone, a catechol derivative and an inhibitor of catechol-O-methyltransferase, were reported recently. In the present study, the influence of nitecapone on isolated rat heart ischemia-reperfusion injury was investigated to elucidate its cardioprotective role. Nitecapone, administered in the perfusion buffer from the beginning of the pre-ischemic phase, significantly improved recovery of cardiac mechanical function, suppressed enzyme leakage in the coronary effluent, and minimized loss of ascorbate, compared with the control group. In rats fed a diet containing 4% ascorbate, myocardial ascorbate content in ascorbate-fed rats after ischemia-reperfusion was higher than that in control rats fed a normal diet without ischemia. However, supplemented rats did not show any beneficial effects on cardiac mechanical recovery or enzyme leakage, suggesting that maintenance of tissue ascorbate level is not the cause, but the result of the protective effects of nitecapone against cardiac ischemia-reperfusion injury. The iron-chelating effect of nitecapone was also tested. It was confirmed, using electron spin resonance, that 50 microM nitecapone chelates the same concentration of iron released from the heart into the coronary effluent. Hence, the iron-chelating ability of nitecapone may be responsible, at least in part, for its cardioprotective effects in ischemia-reperfusion injury.

Animals↗
PubMed1995

Ginkgo biloba extract (EGb 761) protects human low density lipoproteins against oxidative modification mediated by copper.

The antioxidant effects of Ginkgo biloba extract (EGb 761) on copper-mediated human low density lipoprotein (LDL) oxidative modification were evaluated by several techniques. Human LDL (0.5 mg/ml) in phosphate buffered saline, pH 7.4, was incubated with 10 microM cupric sulfate at 37 degrees C under air for 8 hours and 24 hours in the presence of varying concentrations of EGb 761. Increases in LDL apoB carbonylation, lipid peroxidation, apoB electrophoretic mobility and LDL fluorescence were all inhibited when the incubation mixture contained EGb 761 at concentrations less than 100 micrograms/ml. This inhibition was EGb 761-concentration-dependent. Thus, EGb 761 has powerful antioxidant effects on copper-mediated LDL oxidative modification.

Apolipoproteins B↗
PubMed1995

Reduction and transport of lipoic acid by human erythrocytes.

Reduction of exogenous lipoic acid to dihydrolipoate is known to occur in several mammalian cells and tissues. Dihydrolipoate is a potent radical scavenger, and may provide significant antioxidant protection. Because lipoic acid appears in the bloodstream after oral administration, we have examined the reduction of exogenous lipoate by human erythrocytes. Normal human erythrocytes reduced lipoate to dihydrolipoate only in the presence of glucose; deoxyglucose did not substitute for glucose, indicating that the reduction of lipoate requires glucose metabolism. Furthermore, the reduction was shown to be NADPH dependent. Erythrocytes isolated from a human subject with a genetic deficiency of glucose-6-phosphate dehydrogenase (and, therefore, deficient in the formation of NADPH) did not reduce lipoate. Dehydroepiandrosterone, a specific inhibitor of glucose-6-phosphate dehydrogenase, inhibited lipoate reduction. Our findings imply that some of the reduction of exogenous lipoic acid is catalysed by glutathione reductase, a flavoprotein dehydrogenase; mitomycin C, an inhibitor of FAD-dependent reductases, inhibited lipoate reduction by erythrocytes, and glutathione reductase purified from human erythrocytes was observed to reduce lipoic acid in a cell-free system. We further explored these findings with erythrocyte ghosts and liposomes. Our results indicate that a transport system exists for alpha-lipoic acid and dihydrolipoate; resealed erythrocyte ghosts, containing trapped lipoamide dehydrogenase and pyridine nucleotides, reduced externally added lipoate. By contrast, liposomes prepared with enzyme and pyridine nucleotides did not catalyze reduction of lipoate. This work indicates that uptake of exogenous lipoate and reduction to dihydrolipoate by normal human erythrocytes may contribute to oxidant protection in the human bloodstream.

Biological Transport↗
PubMed1995

The spin-trap N-tert-alpha-phenyl-butylnitrone prolongs the life span of the senescence accelerated mouse.

Free radicals and oxidative damage have been proposed as underlying factors in aging, in chronic and degenerative diseases of aging and in acute clinical conditions. To test involvement of free-radicals in such processes, spin trapping agents which quench more reactive radicals to produce long-lived stable radical adducts have been used as an experimental strategy. Spin traps protect against oxidatively induced injury in numerous in vitro and in vivo model systems involving different organs. A model system for mammalian aging is afforded by the senescence accelerated mouse (SAM-P8), which exhibits many features characteristic of mammalian aging but with a much shortened lifespan. Daily intraperitoneal injection of the spin trap N-tert-alpha-phenyl-butylnitrone (PBN) was administered to male or female mice after they reached maturity at 3 months of age. PBN treated animals as compared with control sham injected animals revealed a remarkable extension of the mean life span in both male and female populations. Overall, a 50% mean survival rate was found of 42 weeks for control as compared to 56 weeks for the PBN administered groups. These results show that the spin trap PBN can prolong lifespan and support the free radical theory of aging.

Aging↗
PubMed1995

Peroxyl radical scavenging activity of Ginkgo biloba extract EGb 761.

Antioxidant mechanisms have been proposed to underlie the beneficial pharmacological effects of EGb 761, an extract from Ginkgo biloba leaves used for treating peripheral vascular diseases and cerebrovascular insufficiency in the elderly. In vitro evidence has been reported that EGb 761 scavenges various reactive oxygen species, i.e. nitric oxide, and the superoxide, hydroxyl, and oxoferryl radicals. However, the ability of EGb 761 to scavenge peroxyl radicals (reactive species mainly involved in the propagation step of lipid peroxidation) has not been investigated. To characterize further the antioxidant action of EGb 761, we measured the protective effects of EGb 761 during: (1) the oxidation of B-phycoerythrin by peroxyl radicals generated in aqueous solution by 2,2'-azobis (2-amidinopropane) hydrochloride (AAPH); and (2) the reaction of luminol or cis-parinaric acid with peroxyl radicals generated from 2,2'-azobis (2,4-dimethylvaleronitrile) (AMVN) in liposomes or in human low density lipoprotein (LDL), respectively. To evaluate the peroxyl radical scavenging activity of EGb 761 in a more physiologically relevant model of damage to lipid-containing systems, we also analyzed the effect of the extract on the oxidation of human LDL exposed to the azo-initiators in terms of: (1) accumulation of cholesterol linoleate ester hydroperoxides, (2) depletion of alpha-tocopherol and beta-carotene, and (3) changes in intrinsic tryptophan fluorescence. EGb 761 afforded protection against oxidative damage in all the systems we analyzed; thus, it is an efficient scavenger of peroxyl radicals. This result extends the oxygen radical scavenging properties of the extract and supports the hypothesis of an antioxidant therapeutic action of EGb 761.

Amidines↗
PubMed1995

Transient overexpression of catalase does not inhibit TNF- or PMA-induced NF-kappa B activation.

H2O2 has been proposed as a second messenger involved in cell signaling for NF-kappa B activation. In the present study, this hypothesis was tested by transiently overexpressing catalase, a specific scavenger of H2O2, in COS-1 cells. A mammalian expression vector was constructed by incorporating catalase gene from pCAT10 clone into the unique EcoRI site of the pSG5 vector which contains the SV-40 promoter. Transient transfection of the catalase expression vector by the DEAE-dextran method led to a four-fold increase in catalase activity and catalase content as detected by immunoblot analysis. This level of increase was detected in both nuclear/mitochondrial- and cytosolic/microsomal fractions. Overexpression of catalase, however, did not block TNF- or PMA-induced NF-kappa B activation. These results weaken the hypothesis that H2O2 is a second messenger for TNF- and PMA-signaling for NF-kappa B activation.

Animals↗
PubMed1995

Elucidation of antioxidant activity of alpha-lipoic acid toward hydroxyl radical.

The photosensitive organic hydroperoxide, NP-III, which produces hydroxyl radicals on illumination by UVA light, was used to examine the antioxidant activity of alpha-lipoic acid and its derivatives toward hydroxyl radical. Apolipoprotein (apo-B) of human low density lipoprotein (LDL) and bovine serum alubumin (BSA) were irradiated with UVA in the presence of NP-III and alpha-lipoic acid. The oxidation of BSA and the apo-B protein of LDL by NP-III was completely suppressed by alpha-lipoic acid. ESR studies using dimethylpyrroline oxide (DMPO) as a spin trapping reagent also revealed that in the presence of alpha-lipoic acid, the DMPO-OH adduct produced from the irradiation of NP-III and DMPO completely disappeared. DMPO-OH quenching experiments were performed in the presence or absence of desferoxamine but no change in the signal intensity was found. Hence, the quenching activity of alpha-lipoic acid is not due to its chelating activity toward transition metals (ferrous ions). The results lead us to conclude that alpha-lipoic acid is an efficient hydroxyl radical quencher owing to the disulfide bond in the dithiolane ring.

Antioxidants↗