The generation of oxygen radicals after drinking of oxygenated water.

UNLABELLED: It has been speculated whether ingestion of oxygenated water can lead to an enhanced generation of oxygen radicals. The purpose of three prospective randomized blinded clinical studies was therefore to measure if, when and at which oxygen content in the water,drinking of oxygenated water induces the generation of radicals. Moreover in the fourth prospective,randomized, blinded study possible longterm effects of drinking oxygenated water were examined. METHODS: Altogether 66 volunteers were drinking 300 ml oxygenated or tap water within 15 minutes. Before drinking, altogether 15 ml of blood from the antecubital vein was collected for determination of ascorbyl radicals with ESR, routine laboratory data (hemoglobin, erythrocytes, hematocrit, leukocytes, thrombocytes, uric acid) and the vitamins A,C,E by HPLC. After drinking the ascorbyl radical measurements were repeated from blood of the antecubital vein. In the longterm study ( fourth study) the volunteers had to undergo the same procedure, as described above, at day 1 and day 21. In the meantime they were drinking per day three times 300 ml either oxygenated water or tap water. RESULTS: All subjects exhibited normal vitamin levels in all three studies. Concommitantly in the fourth study there was no statistically relevant alteration of vitamin concentrations during the observation period of three weeks in the verum and placebo-group. 30 minutes after drinking oxygenated water the concentration of ascorbyl radicals increased significantly by median 42 % from median 48 to 65 nmol/l. This increase of ascorbyl radicals after 30 minutes was reproducible in all studies. The levels of ascorbyl radicals remained elevated for 60 minutes after drinking and returned to normal after 120 minutes. This increase was independent of the oxygen concentration in the water, beginning at 30 mg oxygen/l. Water containing 15 mg oxygen/l did not lead to an enhanced radical formation. Longterm consumption of oxygenated water attenuated the ascorbyl radical increase normally observed, thus the initial increase of ascorbyl radicals at day 1 could not be observed after day 21, if the subjects were drinking oxygenated water regularly during the observation period. CONCLUSION: Drinking of oxygenated water possibly leads to a time-limited, yet very moderate, systemic generation of radicals. Regular consumption of oxygenated water over a longer period of time seems to attenuate this effect. The mechanisms leading to this effect and adaptation are unknown.

Non-protein-bound iron is elevated in cerebrospinal fluid from preterm infants with posthemorrhagic ventricular dilatation.

Posthemorrhagic ventricular dilatation (PHVD) is closely associated with white matter injury and neurologic disability in the preterm infant. An important factor in periventricular white matter damage may be the specific vulnerability of iron-rich immature oligodendroglia to reactive oxygen species toxicity. Non-protein-bound iron (NPBI) is a potent catalyst in the generation of hydroxyl radicals (Fenton reaction). Our objective was to determine whether NPBI is increased in cerebrospinal fluid (CSF) from preterm infants with PHVD compared with preterm control infants. Samples of CSF were obtained from 20 infants with PHVD and 10 control subjects. The level of NPBI was determined by a new spectrophotometric method using bathophenanthroline as a chelator. To evaluate the effect of hemolysis, CSF and blood were mixed in different proportions, spun, frozen and thawed, and then analyzed for NPBI. NPBI was found in 75% (15 of 20) of infants with PHVD and in 0% (0 of 10) of control infants (p = 0.0002). Hemolysis induced in vitro did not result in any significant levels of NPBI. Within the group with PHVD, NPBI concentrations in CSF did not correlate with disability, parenchymal brain lesions, or the need for shunt surgery. NPBI was increased in CSF from preterm infants with PHVD, and the increase could not be explained by hemolysis alone. Free iron may help to explain the association between intraventricular hemorrhage and white matter damage.

Free radicals are formed in the brain of fetal sheep during reperfusion after cerebral ischemia.

Free radical production in the brain of acutely anesthetized, exteriorized lamb fetuses (n = 11, gestational age = 135 d) was measured using spin trap methodology. Communications between the vertebral and carotid circulations were tied, producing a two-vessel supply to the brain. Flow probes and occlusion slings were placed around each carotid. The spin trap 2-ethyl-3-hydroxy-2,4,4-trimethyloxazolidine (OXANOH) was infused intermittently into one carotid at a constant rate, and blood samples were taken at intervals from the sagittal sinus. These samples were analyzed for the stable radical OXANO. using electron spin resonance spectrometry. Six animals were subjected to 30 min of complete cerebral ischemia, and five fetuses served as sham-operated control animals. During postischemic reperfusion radical formation increased 2-fold during the first 20 min. However, the elevation of OXANO. in the venous effluent from the brain did not start until the transient hyperemia had passed. It is thus concluded that the increase of OXANO. observed is caused by an augmentation of free radical production during reperfusion. Because the spin trap agent was infused directly into the arterial supply and recovered directly from the venous effluent of the brain, the site of production could be the brain tissue, the endothelial cells of the cerebral circulation, and activated leukocytes. This is the first demonstration of increased radical production from the fetal brain. It is noteworthy that it takes place despite oxygen tension of the reperfusing blood of only 3-3.5 kPa.

Formation of free radicals in hypoxic ischemic brain damage in the neonatal rat, assessed by an endogenous spin trap and lipid peroxidation.

The formation of free radicals and lipid peroxidation in the brain after hypoxic ischemia was investigated. Seven-day-old rats were subjected to unilateral (left) carotid artery ligation followed by 70 min of hypoxia with 8% oxygen at 36 degrees C. The animals were randomized into six groups as follows: control animals (no anesthesia, ligation or hypoxia) and animals decapitated at 0, 15, 30, 60 and 180 min into the reoxygenation period. Lipid peroxidation was quantified in brain homogenates using the thiobarbituric acid assay (TBA). The TBA-malondialdehyde (MDA) complex was measured with HPLC. The semi-dehydroascorbate radical was measured using electron spin resonance (ESR) spectroscopy. The semi-dehydroascorbate radical levels increased more than 3-fold in the left HI hemisphere compared to the left control hemisphere 15 min posthypoxic ischemia. The amount of MDA was significantly increased in the hypoxic ischemic (HI) hemisphere ipsilateral to the carotid ligation compared with contralateral hypoxic hemisphere. The MDA level in the left HI hemisphere was also significantly elevated at 0, 15, 30 and 60 min, but not at 180 min into the reoxygenation period. Reoxygenation after hypoxic ischemia thus induced formation of semi-dehydroascorbate radicals and lipid peroxidation.

Free radicals and pathogenesis during ischemia and reperfusion of the cat small intestine.

BACKGROUND/AIM: In spite of the interest in free radicals as mediators of ischemic damage, most information on these species in biological systems is derived from indirect measurements. Our aim was to obtain more direct information concerning sources of free radical production during ischemia and reperfusion. METHODS: We have performed simultaneous measurement of radical generation, purine metabolites, reduced glutathione, neutrophil infiltration and morphological appearance in the cat small intestine in vivo during 60 minutes of ischemia followed by 60 minutes of reperfusion. RESULTS: Radical formation increased abruptly on reperfusion and remained elevated in untreated animals. Inhibition by a monoclonal antibody (IB4) against the neutrophil and by allopurinol treatment was paralleled by improvement of biochemical and morphological parameters. The radicals detected during reperfusion could be divided into one component arising directly from the neutrophils, one due to the xanthine oxidase reaction, and one unknown source. CONCLUSIONS: Neutrophils are a major source of radical production during reperfusion after ischemia. Radicals formed in the xanthine oxidase reaction seem to function as a primer for the neutrophils. The nonsignificant linear correlation between radical formation and morphological appearance suggests that factors other than free radicals are important for the development of intestinal damage after a period of ischemia.

ESR-measurement of oxygen radicals in vivo after renal ischaemia in the rabbit. Effects of pre-treatment with superoxide dismutase and heparin.

The effects of intracellular and extracellular superoxide dismutase and heparin administration on oxygen radical formation after ischaemia in the rabbit kidney were studied. Radicals were measured with ESR and spin trapping. At reperfusion after 60 min of renal ischaemia there was a significant increase in the production of free radicals in the venous effluent from the kidney. Administration of either intracellular superoxide dismutase or extracellular superoxide dismutase before ischaemia and before reperfusion prevented approximately 85% of the radical formation seen in the untreated control group. Administration of heparin 5 min before recirculation resulted in a 65% decrease in radical production compared to the control group.

Feline intestinal ischemia and reperfusion: relation between radical formation and tissue damage.

Experimental ischemia of 15, 30, or 60 min length, followed by 30 min of reperfusion, was produced in situ in the cat small intestine by means of an adjustable arterial clamp. Arterial perfusion pressure was lowered to such an extent that intestinal blood flow decreased from about 25 to 3.5 ml x min-1 x 100 g-1. The rate of free radical formation was followed intermittently with ESR and a modified spin trapping technique in the control period prior to ischemia and at various times during reperfusion. Upon reperfusion radical formation increased above the preischemic control value in all three series of experiments. Cumulative radical production during the 30 min reperfusion period rose from about 3 mumol x 100 g-1 after 15 min ischemia to approximately 4.5 mumol x 100 g-1 after 30 min and 8-10 mumol x 100 g-1 after 60 or 120 min ischemia. At the same time mucosal damage became more pronounced, suggesting a causal connection between tissue damage and radical formation. More specifically, radical production was strongly correlated to histological damage occurring during reperfusion as seen when comparing radical production in animals not experiencing reperfusion damage to those who did. Radical formation in these two groups were 0.35 and 9.0 mumol x 100 g-1, respectively (p < 0.0005).

Cerebral lipid peroxidation in the growth retarded rat fetus under normoxia and hypoxia.

To detect a possible relationship between the increased vulnerability of the brain of the growth retarded fetus and lipid peroxidation, the activity of the latter was investigated in the forebrain of rat fetuses on day 22 of gestation (term = 23). Growth retardation was induced by uterine artery ligation on day 18. The susceptibility of the forebrain to lipid peroxidation was analysed using the thiobarbituric acid technique after exposure of the awake dams to room air or hypoxia (10% O2) for 58 minutes. Hemodynamics and blood gas status in the dams were stable under conditions of normoxia. When exposed to hypoxia, oxygen availability decreased and blood pressure fell slightly. When normalised for litter, lipid peroxidation covaried with fetal weight in fetuses from normoxic dams, only when Fe2+ (a promotor of lipid peroxidation) was added in the incubation. In fetuses from hypoxic dams, this relationship existed irrespective of whether Fe2+ was present during incubation. In all of these associations, smaller fetuses exhibited more intense lipid peroxidation. In the perinatal period, an increased vulnerability of the central nervous system prevails in growth retarded individuals. An increased tendency to undergo lipid peroxidation and/or a weak defence against this process may be part of the pathophysiologic background.

The hydroxylamine OXANOH and its reaction product, the nitroxide OXANO., act as complementary inhibitors of lipid peroxidation.

The effects of the nitroxide 2-ethyl-2,5,5-trimethyl-3-oxazolidinoxyl (OXANO.) and the corresponding hydroxylamine 2-ethyl-1-hydroxy-2,5,5-trimethyl-3-oxazolidine (OXANOH) on in vitro lipid peroxidation in rat liver microsomes and reconstituted lipid vesicles were investigated, and compared with those of some commonly used spin trapping agents. OXANO. and OXANOH (10-100 microM) inhibited iron-dependent lipid peroxidation, as did the spin trapping agents (10-100 mM). OXANO. mainly inhibited the rate of peroxidation, but caused only a small delay in the time of onset. OXANOH exerted its effect by delaying the onset of peroxidation in an antioxidant fashion, and also by inhibiting the rate. Higher concentrations of both substances were required to inhibit t-butylhydroperoxide-dependent lipid peroxidation. OXANO. was found to oxidize the ferrous-ADP complex required for initiation of peroxidation, and this is probably the basis of the inhibitory effect of this compound. Since the reaction of OXANO. tends to produce OXANOH and vice versa, either one could inhibit all reactions of lipid peroxidation.

Radical production during in vivo intestinal ischemia and reperfusion in the cat.

Free radical formation was studied with electron spin resonance during 2 h of intestinal ischemia in the cat, at a blood flow less than 5 ml.min-1.100 g-1, followed by 30-min reperfusion. A modification of the spin trapping technique was used to stabilize highly reactive free radicals. The rate of secondary radical formation was 0.32 +/- 0.06 mumol.min-1.100 g intestine-1 before ischemia and increased to a maximum of 0.66 +/- 0.09 mumol.min-1.100 g-1 during the first minutes of reperfusion (mean +/- SE, n = 5). This could be prevented either by maintaining intestinal blood flow at 8-15 ml.min-1.100 g-1, by administering allopurinol before and during ischemia, or by perfusing the intestinal lumen with an O2-saturated buffer solution during ischemia, resulting in maximum rates of radical production during reperfusion of 0.37 +/- 0.04 (n = 6), 0.33 +/- 0.04 (n = 5), and 0.39 +/- 0.13 mumol.min-1.100 g-1 (n = 5), respectively. The results demonstrate that free radicals are produced in the intestine during reperfusion after a period of reduced blood flow below a certain critical level, and that inhibition of xanthine oxidase and prevention of hypoxia will eliminate this radical production.

Inhibition of lipid peroxidation by spin labels. Relationships between structure and function.

Inhibition of lipid peroxidation by nitroxide radicals and their corresponding hydroxylamines was investigated. The nitroxides were either oxazolidines or piperidines, differing in substitution of the backbone of the molecule (a five or six-membered ring structure, respectively). Concentration requirements for 50% inhibition of microsomal lipid peroxidation varied from 340 to 6 microM for the nitroxides, and from 120 to 3 microM for the hydroxylamines, correlating with lipophilicity and chemical structure. Intramembrane concentrations required for 50% inhibition was independent of lipophilicity when peroxidation was initiated with ADP-Fe2+ but increased with lipophilicity when peroxidation was initiated with t-butylhydroperoxide. During studies of the kinetics of the inhibition, two modes were seen: a delay or a decreased rate of the process. The former mode was seen with the more lipophilic inhibitors. The mechanism of inhibition was similar for all nitroxides and consisted of the following three major components: blocking of primary initiation, prevention of secondary (peroxide-dependent) initiation, and scavenging of various lipoid radicals in the membrane, the major mode of action of the hydroxylamines. Inhibitory efficiency was interpreted in terms of steric hindrance, diffusibility, regeneration of inhibitor, and ability to interact with hydrophilic sites in a hydrophobic environment.

Detection of oxygen radicals during reperfusion of intestinal cells in vitro.

The nitroxide OXANO. (2-Ethyl-2,5,5-trimethyl-3-oxazolidinoxyl) which in its reduced form, OXANOH (2-Ethyl-1-hydroxy-2,5,5-trimethyl-3-oxazolidine), is capable of reacting with short-lived radicals, forming a secondary stable radical, was used for ESR-detection of radical production in isolated cells. The properties of OXANO. and OXANOH in terms of stability in cellular and subcellular systems, membrane permeability and effects on cellular viability were evaluated. Ischemia and reperfusion was simulated in vitro in a preparation of cells from rat intestinal mucosa by incubation at high density (4 X 10(8) cells/ml) under an atmosphere of nitrogen for 25 min and resuspended with fresh oxygenated buffer containing 5 mM OXANOH. A significant increase in radical formation during the 15 min reperfusion period studied was obtained in cells exposed to ischemia compared to control cells incubated at normal density under an atmosphere of oxygen. The addition of 5 microM of the scavenging enzyme superoxide dismutase reduced the radical formation by 50%. The time sequence of the superoxide formation was calculated as the difference in radical production in the presence and absence of superoxide dismutase.