S.U. Weber, J.J. Thiele , J. Choi, and L. Packer
251 LSA, Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, CA 94720-3200, USA
As the outermost layer of the skin the stratum corneum (SC) plays an important role in the protection against oxidative stressors, such as UV-radiation, ozone and chemicals. Previously we have shown that Vitamin E is the major lipophilic SC antioxidant. We hypothesized that facial skin, which is chronically exposed to the environment, possesses increased antioxidant levels.
To test this we applied a tapeipping technique to compare the upper SC tocopherol concentrations of exposed (cheek) versus non- exposed (upper arm) skin sites. The a-tocopherol concentration in the cheek was 3.5 times higher than in the upper arm (p<0.05). While in the upper arm we found a decrease of both a- and g-tocopherol towards the outside layers, the reverse gradient was found in the face, with the highest tocopherol concentration in the skin surface lipids and top SC layers. Interestingly, the same pattern was observed for cholesterol and squalene, a known lipid marker for sebaceous glands. Furthermore, the sebum output was measured using SebutapesTM and found to be highest in the cheek > forehead > arm (p < 0.001). Importantly a- and g-tocopherol as well as cholesterol and squalene were excreted continuously in high amounts during a period of 135 min. (cae 53 ± 34.4 pmol/mg sebum, cge 6.74 ± 5.6). Their concentration correlated closely to the sebum output (n=63, p < 0.05).
In summary, we demonstrate that the sebaceous gland secretion is a major pathway for the delivery of a- and g-tocopherol to the facial surface lipids and the SC. This mechanism may serve to protect the facial skin from environmental oxidative stress.
W. N. Wicomb
Department of Organ Preservation, California Pacific Medical Center R310, 2200 Webster St., San Francisco, CA 94115
Polyethylene glycol (PEG) has frequently been used in organ preservation solutions to protect tissues from ischemic injury. In heart preservation studies we have evaluated numerous antioxidants, but none of these compounds had the efficacy of PEG in improving heart function. The mechanism of action of PEG remains unknown, but may involve spontaneous peroxide formation developing adjacent to Œether linkages¹ along the length of the molecule. In earlier studies we investigated various characteristics of PEG, including the influence of Œshelf aged flake¹ on stored heart function and the effect of molecular size and anoxia (flake or solution) on the stability of PEG. In this study we evaluate the redox state of PEG and used it to predict heart function following storage. The redox state was determined spectrophotometrically by monitoring the reaction between PEG and the Fenton reagents. The Fenton reaction was measured at wavelength 362 nanometers and the reaction is shown in equation (a) below. A spectrophotometric scan of PEG also suggests the presence of a Œreduced¹ component that has a maximum absorbance occurring at approximately 362 nanometers. The change in absorbance was correlated with in vitro heart function.
In vitro heart function is shown in the table below comparing PEG with low absorbance data (delta low) Vs PEG with high absorbance data (delta high) following 24 hour ice storage. Control unstored hearts were tested immediately, without an intervening period of preservation. Statistical analysis included the Mann-Whitney test for non-parametric data.
| 24 hr storage parameters | Delta Low | Delta High | Unstored Controls |
| Cardiac Output (ml/min) | 163 (15.9) | 256 (12.34) | 307 (7.0) |
| Absorbance (units) | 72 (3.4) | 113 (2.8) | |
| n | 22 | 22 | 8 |
PEG containing solutions that demonstrated decreased activity (delta low) (72 absorbance units) in the Fenton-like reaction resulted in poor heart function, whereas PEG demonstrating increased activity (p < 0.0001) in the Fenton reaction (113 absorbance units) showed improved heart function (see table) (p <0.0001). This study shows that the oxidation state of PEG correlates with heart function.
Christine C. Winterbourn, Hendrikje Buss, Timothy P. Chan, Matthew Clarke*, and Graham Hill*
Department of Pathology, Christchurch School of Medicine, Christchurch, and *Department of Surgery, University of Auckland School of Medicine and Health Sciences, Auckland, New Zealand
Trauma patients and those who develop sepsis after major surgery or injury have high mortality that is frequently associated with adult respiratory distress syndrome (ARDS) and multiple organ failure. Generation of free radicals and associated oxidative injury are proposed to be important contributors to these events. To seek direct evidence for oxidative injury, plasma and bronchial lavage from 22 patients with sepsis and 8 patients with trauma who were admitted to the intensive care unit at Auckland Hospital were collected over 10 days. Analyses were performed for protein carbonyls by a newly developed ELISA, thiobar bituric acid reactive substances (TBARS) and myeloperoxidase as respective markers of protein oxidation, lipid peroxidation and neutrophil activation.
Protein carbonyls in plasma and bronchial lavage were highly elevated in both patient groups at the time of entry into the study (in plasma on average more than 10 times normal). They declined progressively from about day 2 to day 8, but plasma concentrations stay-ed above the normal range. TBARS concentrations in lavage samples also showed a decline over time, but in plasma they remained within the normal range. Myeloperoxidase activity in lung aspirates from both groups were initially high and decreased over time. Plasma con-centrations were above normal over the time period studied. Using Spearman¹s rank sum test, there was a strong correlation between lung and plasma carbonyls collected on the same day (0.66, sepsis; 0.59, trauma, p = 0.001), and for carbonyls and TBARS against MPO activity in lung lavage.
These findings provide strong evidence for oxidative reactions occurring in the early stages of trauma and sepsis, and for protein carbonyl measurements providing a sensitive measure of the process. The association with MPO activity suggests that neutrophils may contribute to oxidative injury in the lung, but the high carbonyl content of plasma shows that oxidation is not restricted to this one organ.
Lipid and protein oxidation during oxidative stress in isolated mitochondria
I. Wiswedel, T. Reinheckel, D. Hirsch, H. Noack, and W. Augustin
Institute of Clinical Chemistry, Department of Pathological Biochemistry, Otto-von-Guericke-University, Magdeburg, Germany
The kinetics of lipid peroxidation and oxidative protein modification with iron/ascorbate was investigated in isolated rat brain and liver mitochondria. The formation of malondialdehyde as a result of lipid peroxidation is a well known marker of oxidative stress. Further products of lipid peroxidation in minor concentrations were represented by the monofunctional aldehydes 4-hydroxynonenal (4-HNE), 4-hexanal, n-pentanal, n-nonenal, n-heptanal, 2-octenal and 4-hydro xydecenal. Hexanal and 4-HNE belonged to the predominant monoaldehydes which were formed. The kinetics of aldehyde generation showed an initiation phase which precedes an exponential increase of aldehydes. In order to look for earlier formed very specific marker of lipid peroxidation we developed a quantitative GC-MS- method for the sensitive analysis of monohydroxy fatty acids derived from arachidonic acid in mitochondria. In freshly isolated rat brain mitochondria we found a typical pattern with 2-, 3-, 5- and 15-HETEs as major and 8+9- 11+12- and 20-HETE as minor products in very small amounts (about 0.2 % of the total arachidonic acid content). After induction of an oxidative stress several hydroxy fatty acids showed steep characteristic increases, which seemed to precede the formation of TBARS.
Oxidative protein modification was measured as formation of DNPH reactive protein bound carbonyls (spectrophotometrically and by Western blot analysis). Carbonyl formation occured during the initiation phase of lipid peroxidation in parallel to the functional decline (respiratory rate, membrane potential, loss of antioxidants) and represents an early event during iron induced oxidative stress.
Alice Wong1, Franco Taroni2, Cinzia Gellera2, and Gino Cortopassi1
1Department of Molecular Biosciences, University of California, Davis, CA 96516 and 2Department of Biochemical Genetics, Instituto Neurologico ³Carlo Besta², Milan, Italy
Freidreich¹s ataxia (FA) is an autosomal recessive neurodegener ative disease characterized by progressive gait and limb ataxia, dysarthria, and cardiomypathy. Expansions of the of the triplet repeat GAA in the first intron of the frataxin gene cause FA, presumably by reducing expression of frataxin mRNA. Frataxin has recently been localized to the mitochondria, and deletion of the frataxin homolog in yeast cells results in increased mitochondrial free iron (Babcock, et al., 1997; Foury and Cazzalini, 1997). Because free iron could potentially participate in Fenton chemistry by reacting with hydrogen peroxide and producing hydroxyl radical, neurodegeneration in FA could result from excessive oxidative stress, and thus we evaluated the sensitivity of human FA cells to oxidant stress. FA cells were significantly more sensitive to hydrogen peroxide than control cells. Iron alone induced death in FA cells, and addition of deferoxamine rescued FA cells but not control cells from hydrogen peroxide-induced death. Removal of Ca2+ from the media or treatment of cells with BAPTA, an intracellular calcium chelator, rescued the cells from oxidant-induced death. Treatment of cells with apoptotic inhibitors also rescued cells from death, suggesting a possible role of the biochemical apoptosis machinery in the pathophysiology of FA.
Munehisa Yabuki*İ, Rumi Ishisaka*, Tatsuji Yasudaİ, Tamotsu Yoshioka*, and Kozo Utsumi*
*Institute of Medical Science, Kurashiki Medical Center, Kurashiki 710, Japan and İDepartment of Cell Chemistry, Institute of Molecular and Cell Biology, Okayama University Medical School, Okayama 700, Japan
Nitric oxide (NO) released from (Z)-1-]N-(2-aminoethyl)-N-(2 ammonioethyl)amino]diazen-1-ium-1,2-diolate (NOC 18) induces apoptosis in human leukemia HL60 cells. To investigate the mechanism by which NO mediates apoptosis, we have isolated HL60 clonarl variant HL-NR6 cells, which were significantly resistant to NOC 18 toxicity as assessed by DNA fragmentation, morphology, and colony forming ability. HL-NR6 cells possessed 2.0-fold increased activities of Cu,Zn superoxide dismutase and catalase but not of Mn-superoxide dismutase nor GSH peroxidase. Immunoblotting showed that endogenous levels of Cu,Zn-superoxide dismutase and catalase were obviously increased in HL-NR6 cells. Although catalase has been reported to be reversibly inhibited by NO, the enzyme activity in HL-NR6 cell extract was significantly high even in the presence of NO. Moreover, HL-NR6 cells were also resistant to other NO donors SNAP and nitroglycerin, hypoxanthine plus xanthine oxidase, and hydrogen peroxide. Taken together, these data indicated that reactive oxygen species are implicated in the induction of NO-mediated apoptosis and that Cu,Zn-superoxide dismutase and catalase contribute to cellular resistance to NO. HL-NR6 cells were not resistant to several antitumor agents such as etoposide, mitomycin C, and cisplatin, supporting the above notion. We also examined the involvement of the bcl-2 family proteins, and found that bcl-2 but not bcl-xL nor Bax was increased in HL-NR6 cells. Therefore, bcl-2 may also contribute to NO resistance in HL-NR6 cells. Furthermore, we examined the activities of cysteine proteases of caspases and found that caspase-3 but not caspase-1 was strongly activated in HL-60 cells compared to HL-NR6 cells after NOC 18 treatment. This result suggested that Cu,Zn superoxide dismutase and catalase functioned upstream in the pathway.
ESR study of UV-irradiated lipoic acid
A. Yamanaka*,Y. Noda¶, N. Azuma*, A. Mori¶, and L. Packer¶
*Department of Chemistry, Faculty of Science, Ehime University, ¶Membrane Bioenergetics Group, Lawrence Berkeley Laboratory, University of California, USA
Although an ESR study of uv-irradiated lipoic acid in aqueous medium was first reported by Smissman and Sorenson (E.E. Smissman and J.R.J. Sorenson, Org. Chem., 30, 4008 (1965)), the ESR parameters were not reported. Therefore, the ESR study of UV-irradi ated lipoic acid in several solvents has been carried out to re-examine and to obtain the fundamental data for the investigation of interaction between lipoic acid and [CrO(salen)] (salen = N,N'-ethylenebis(salicyl idenaminate)) oxochromium (V) complex.
The sample solution in a liquid-nitrogen Dewar flask was irradiated with a low-pressure mercury lamp for about two hours, and then the Dewar flask was put into X-band ESR cavity to record the spectrum. The solvents used were water (pH 7 - 11), methanol, benzene, toluene and m-xylene.
ESR signals of biradicals due to homolytic cleavage of the S-S bond was not obtained. Homolytic cleavage was postulated by Smissman and Sorenson. The ESR signal assignable to the S-centered monoradical was obtained only by using water and benzene as solvent. The other solvents have C-H and/or O-H bonds whose bonding energies are lower than those of water and benzene; the hydrogen abstraction of the S-centered radical from the solvent molecules is indicated. Interestingly, deuterated benzene also yields little S-cen tered radical. This might indicate that hydrogen atoms produced primarily by UV-irradiation play an important role to generate the S-centered radical. In fact the hydrogen signal was observed in all the irradiated samples. The ESR parameters of the S-centered radical was estimated with the computer simulation of the ESR spectra; g1 = 2.00, g2 = 2.02 and g3 = 2.06.
Min Yang1, Almas Rehman2, Clifford S. Collis1, George Paganga1, Barry Halliwell2, and Catherine Rice-Evans1
1International Antioxidant Research Centre at UMDS Guyís Hospital, London Bridge, London SE1 9RT, and 2International Antioxidant Research Centre at Kings College, Chelsea Campus, London SW3 6LX, United Kingdom
Studies have suggested that vitamin C may provide some protection against cancers. However, the benefits of vitamin C supplementation have been disputed and some studies are equivocal. Attention has been drawn to the well-known pro-oxidant effects of iron/ascobate mixtures in vitro, although no clear evidence for the physiological relevance of these effects in healthy subjects has been published to date.
To address this issue, we studied the effects of co-supplementation of iron with vitamin C in healthy volunteers. 20 well-nourished healthy subjects with initial plasma ascorbate levels of 71.9±14.0 µmol/l were applied iron (14 mg/d) and either 60 mg/d (Group A) or 260 mg /d (Group B) vitamin C for 12 wk. Blood samples were taken before supplementation, at 6 wk and 12 wk and analysed for iron levels, vitamin C status, platelet function and oxidative DNA damage in white blood cells.
Plasma total ascorbate did not change significantly after supplementation of subjects in both Group A and Group B. Transferrin saturation increased slightly after 6 wk but remained within the normal range and returned approximately to the initial values after 12 wks. Platelet sensitivity to ADP-induced aggregation was significantly decreased (P<0.05) after 12 wk in Group A, whereas those receiving higher vitamin C supplements (Group B) showed a significant decrease (P<0.05) after 6 wk which subsequently increased towards pre-supplemental levels. Platelets from the latter subjects also showed a significant reduction in ADP-induced ATP secretion at both 6 wk and 12 wk. Total DNA base damage was markedly elevated at 6 wk in both supplementation groups but, interestingly, by 12 wk total DNA damage had returned approximately to the initial values. This may suggest improved sequestration of iron and/or up-regulation of DNA repair systems.
Clearly, in order to clarify the consequence of iron/vitamin C co-supplementation, a wide range of ëëbiomarkersíí must studied.
The authors are grateful to the Ministry of Agriculture, Fisheries and Food for funding
Alternate magnetic fields potentiate monoamine oxidase activity in the brain
Isao Yokoi, Hideaki Kabuto, Nihei Yamamoto, Norio Ogawa, and Akitane Mori*
Department of Neuroscience, Institute of Molecular & Cellular Medicine, Okayama University School of Medicine, Okayama, Japan, and *Department of Molecular and Cell Biology, University of California, Berkeley, California, USA
Catecholamines and serotonin, which act as neurotransmitters and regulate blood circulation, are degraded by monoamine oxidase (MAO)[EC 1.4.3.4.]. Monoamine oxidation by MAO is thought to involve spin-correlated radical pair intermediates, so there is a possibility that oxidation by MAO is a magnetically sensitive enzymatic reaction. In this study, the effects of magnetic fields on MAO activity in the rat brain were examined.
Fifty hertz of alternate isosceles triangular magnetic fields (AITMF) were generated using a Helmholtz coil operated by a programmable bipolar power supply controlled with a computer. All studies were performed in the electrostatic shielded room in where terrestrial magnetism was 28-32microT. MAO activity was measured using m-nitrobenzylamine as substrate.
In an AITMF of 100mT/s (peak to peak: 1mT), MAO activity was increased to 117% (p<0.025), though it was not changed in AITMF's of 10, 34, 340, or 800mT/s. In the AITMF at 100mT/s, Michaelis' constant of MAO decreased to 72% (p<0.01), suggesting that the affinity of the enzyme for the substrate also increased. However MAO activity was not changed in static magnetic fields (0.1-320mT).
These results indicate that the effectiveness of a magnetic field on MAO activity has a so called 'window' phenomenon, and that AITMF might affect neural activity and hemodynamics.
Shinichi Yoshida1,2, Katsuko Uno3, Nobuo Matsui2, and Takashi Okamoto1
Departments of 1Molecular Genetics and 2Orthopedics, Nagoya City University, Nagoya and 3Louis Pasteur Center of Medical Research, Kyoto, Japan
Involvement of oxidative stress in rheumatoid arthritis (RA) is well established. We have investigated the role of thioredoxin (Trx) in the pathophysiology of RA. The Trx concentration of 40 plasma samples and 36 synovial fluid (SF) samples from RA patients were measured by ELISA method. While Trx in the RA plasma was slightly elevated as compared from healthy individuals (27.2 ± 21.9 versus 11.8 ± 6.6 ng per ml), Trx in the RA SF was greatly increased as compared from patients with osteoarthritis (OA) (103.4 ± 53.3 versus 24.6 ± 17.4 ng per ml). Since Trx is known to be involved in the NF-kB activation process (Okamoto et al., Cur Top Cell Regul 35:149-161, 1997; Sakurada et al., Int Immunol 8:1483-1493, 1996), we have examined the effect of thioredoxin on the TNF-mediated cytokine induction from the cultured rheumatoid synovial fibroblasts (RSF). It was noted that addition of Trx appeared to acceralate the nuclear translocation of NF-kB upon TNF-stimulation as demonstrated by immunostaining using anti-NF-kB (p65) antibody. Furtheremore, IkB-alpha degradation in response to TNF stimulation of RSF was facilitated by Trx as observed by western blotting analysis. Accordingly, the extents of IL 6 and IL-8 induction in RSF were greatly augmented by Trx as compared from TNF alone. These findings indicate that the elevated TRX levels in synovial fluid of RA patients might be involved in the aggravation of rheumatoid inflammation by augumentation of cytokine production from synovial fibroblasts. Thus, redox regulatory mechanism may be a feasible therapeutic target of RA.