Attenuation of brain hyperbaric oxygen toxicity by fasting is not related to ketosis.

The effect of 24 h of fasting and changes in blood glucose and beta-hydroxybutyrate (BHB) level on latency to seizures in hyperbaric oxygen (HBO2) was studied. Conscious, unrestrained rats implanted with cortical electroencephalogram electrodes were exposed to 0.5 MPa (gauge pressure) O2 until seizures were observed. Fasting for 24 h significantly (P < 0.01) decreased blood glucose (from 8.6 +/- 0.9 in fed to 6.9 +/- 0.7 mM in the fasted group), increased blood BHB (0.07 +/- 0.02 mM to 0.38 +/- 0.10 mM, respectively), and prolonged the latency to seizures compared with normally fed animals (21.0 +/- 9.8 vs. 34.6 +/- 17.7 min, P < 0.05). Injection of the ketone precursor 1,3-butanediol (BD) to the fed animals increased blood BHB level to 0.72 +/- 0.32; however, seizure latency remained the same as in fed animals. Restoration of blood glucose in fasted animals to the same level as in the fed group did not reverse the protection achieved by fast; instead it increased the latency to seizures. The results indicate that the protection against HBO2 seizures by fasting in short starvation is not related to the increase in circulating ketone bodies or decrease in blood glucose.

Effect of MK-801 on seizures induced by exposure to hyperbaric oxygen: comparison with AP-7.

The effect of the noncompetitive N-methyl-d-aspartate (NMDA)-receptor antagonist MK-801 on seizures induced by hyperbaric oxygen in relation to changes in cerebral blood flow (CBF) was investigated. Rats were injected with MK-801 (0.005-8 mg/kg) 30 min before exposure to 100% O2 at 5 atm (gauge pressure). MK-801 administration resulted in a biphasic response in seizure latency. Doses of 0.1-4 mg/kg significantly decreased time to EEG and motor seizures, while 8 mg/kg had no effect on seizure latency. MK-801 had no effect on seizure duration. In a dose range 0.1-8 mg/kg MK-801 increased CBF in awake animals, which might be responsible for the decreased seizure latency. The gradual increase in seizure latency with increasing MK-801 doses suggests involvement of an additional factor probably related to the drug's anticonvulsive effect. Unlike MK-801, a competitive NMDA receptor antagonist, AP-7, at a dose 250 mg/kg had no effect on latency to seizures or CBF.

Role of cerebral blood flow in seizures from hyperbaric oxygen exposure.

Hyperbaric O2 exposure causes seizures by an unknown mechanism. Cerebral blood flow (CBF) may affect seizure latency, although no studies have demonstrated a direct relationship. Awake rats (male, Sprague-Dawley, 350-450 g), instrumented for measuring electroencephalographic activity (EEG) and CBF (laser-Doppler flowmetry), were exposed to 100% O2 at 4 or 5 atm (gauge pressure) until EEG seizures. Compression with O2 caused vasoconstriction to about 70% of control flow that was maintained for various times. CBF then suddenly, but transiently, increased at a time that was reliably related to seizure latency (r=0.8, p<0.01). Additional animals were treated with agents that have diverse pharmacology and their effects on CBF and latency were measured. Glutamate receptor antagonists MK-801 (1 or 4 mg/kg) and ketamine (20-100 mg/kg) significantly increased CBF by 60-80% and decreased seizure latency from about 17+/-8 min (+/-S.D.) in controls to 5+/-1 and 6+/-2 min, respectively. In opposite, a nitric oxide synthase (NOS) inhibitor, N-nitro-L-arginine (NNA)(25 mg/kg) decreased CBF by about 25% and increased time to seizure to 60+/-16 min. If these effects occur in humans, non-invasive measurement of CBF could potentially improve the safety and reliability of hyperbaric O2 usage in clinical and diving applications. It also appears that the effect of drugs on seizure latency can be explained, at least in part, by their effect on CBF.

Lower decompression sickness risk in rats by intravenous injection of foreign protein.

We have identified a novel means of reducing the risk of decompression sickness (DCS) in rats. A substantial reduction in DCS, from 55% in untreated animals to 24% in animals injected intravenously with a hydrogenase of bacterial origin, was documented for animals breathing a mixture of oxygen and hydrogen. However, this reduction was clearly not a function of metabolic elimination of H2; injections of proteins lacking hydrogenase activity also elicited a lower DCS incidence, and animals breathing hyperbaric helium had the same protective advantage as animals breathing H2. The reduction in DCS risk was shown to be caused by intravenous injection of a foreign protein. The magnitude of the effect varied: two foreign proteins tested did not induce a statistically significant response. We speculated that the foreign protein elicited an immune reaction pre-dive, which diminished the subsequent response of the immune system in DCS. Identifying the underlying mechanism may be important to understanding the pathophysiology of this malady, and may ultimately lead to a therapy applied pre-decompression for reducing DCS risk in human diving.

Hydrogenase encapsulation into red blood cells and regeneration of electron acceptor.

Biochemical decompression has been proposed as a method for reducing the amount of time required for deep-sea divers to return to the surface. Divers breathing H2/O2 mixtures would be presented with hydrogenase enzyme, and decompression would be accelerated by means of the enzymic removal of excess H2 from the tissues. We have studied FAD as a hydrogenase electron acceptor that is capable of transferring electrons derived from H2 oxidation directly to O2. Kinetic activity constants for the soluble hydrogenase from the bacterium Alcaligenes eutrophus H16 were determined with FAD, FMN and riboflavin as electron acceptors, and these values were compared with those obtained with the physiological electron acceptor NAD+. The Michaelis constants (K(m)) were similar for FAD, FMN and NAD. However, the maximal catalytic-centre activity (Kcat) was much lower for the flavins, and the catalytic efficiency (Kcat/K(m)) with FAD was 1/20th the value for NAD+. After enzyme-catalysed FAD reduction to FADH2, the FAD could be regenerated by addition of O2 and reduced again by the enzyme in the presence of H2. Thus FAD served as a regenerable electron shuttle between H2 and O2. H2O2, a by-product of FADH2 oxidation by O2, inhibited the enzyme. Much greater inhibition was observed with the reduced form of the enzyme. Active hydrogenase was efficiently encapsulated into human and pig red blood cells. Hydrogen consumption was seen with lysed carrier cells, but was demonstrated with unlysed carrier cells only when FAD was co-encapsulated along with enzyme. These results demonstrate that red blood cells encapsulating hydrogenase and FAD act as a system for continuous H2 consumption in a mammalian tissue without addition of exogenous factors, and such cells may provide a biotherapeutic method for reducing the risk and treatment of decompression sickness.

Regional lipid peroxidation and protein oxidation in rat brain after hyperbaric oxygen exposure.

Reactive oxygen species may participate in development of neurological toxicity resulting from hyperbaric oxygen exposure. To explore the possibility that increased reactive O2 metabolite generation may result in oxidative modification of lipids and proteins, rats were exposed to five atmospheres (gauge pressure) of O2 until development of an electroencephalographic seizure. Lipid peroxidation (as thiobarbituric acid-reactive substances) and protein oxidation (as 2,4-dinitrophenyl-hydrazones) were measured in five brain regions. Oxidized and reduced glutathione were also determined because of their role in regulating lipid peroxidation. Lipid peroxidation was confined to the frontal cortex and hippocampus, while protein oxidation (in both cytoplasmic and membranous fractions) and increased oxidized glutathione was evident throughout the brain. These results support a role for formation of reactive O2 metabolites from hyperbaric O2 exposure and suggest that protein oxidation, especially in soluble proteins, may be one of the most sensitive measures.

A model for predicting central nervous system oxygen toxicity from hyperbaric oxygen exposures in humans.

Under certain circumstances, Navy divers breathe 100% O2 when working underwater. Serious symptoms of central nervous system (CNS) O2 toxicity can develop from hyperbaric O2 exposure; immersion and exercise are also known to exacerbate toxicity. We developed risk models for quantitative prediction of the probability of developing symptoms using a large set of human data in which occupational exposure conditions were simulated. Exposures were 5 to 265 min at PO2 levels from 20 to 50 feet of sea water (fsw) (1 fsw = 3.06 kPa). Approximately half of the exposures were to a single PO2, while the remainder were more complicated consisting of exposures to multiple levels of hyperbaric O2. In 688 trials, there were 42 exposure-stopping symptoms. We used maximum likelihood to estimate parameters, likelihood ratios to compare model fits, and chi 2 tests to judge goodness-of-fit of model predictions to observations. The modeling shows that risk has a steep PO2 dependence. A model with autocatalytic features fits the data as well as a simpler model: when PO2 is elevated beyond 34 fsw, risk accumulates rapidly without bound while accumulating toward an asymptote at lower PO2 levels. This autocatalytic feature of risk accumulation implies a testable hypothesis that substantial protection from human CNS O2 toxicity can be obtained from intermittent exposure (periodic exposure to lower PO2). The models predict that the probability of O2 toxicity is less than 7% with current Navy limits while breathing 95% O2. Probability of symptoms is < 1% if FIO2 is maintained at the United States Navy recommended level of 75%.

Kinetic mechanism studies of the soluble hydrogenase from Alcaligenes eutrophus H16.

Purified soluble hydrogenase (H2:NAD+ oxidoreductase, EC 1.12.1.2) from Alcaligenes eutrophus was activated to high specific activities by flushing the enzyme consecutively with N2 and H2 and then adding substoichiometric quantities of NADH. H2-dependent NAD+ reduction activities > or = 110 mumol NADH formed/min/mg protein at pH 8.0 and 30 degrees C were obtained which were stable for several hours at 4 degrees C. Kinetic studies were conducted anaerobically using activated enzyme for the purpose of evaluating the potential of using hydrogenase to enhance decompression of mammals breathing H2/O2 mixtures under hyperbaric conditions (i.e., at ambient pressures greater than 1 atm). Using nonlinear curve fitting of the kinetic data, it was found that H2 and NAD+ bind hydrogenase via a ping pong bi bi mechanism with Km values (+/- SE) of 11 +/- 0.9 and 138 +/- 11 microM, respectively, at 30 degrees C and pH 8.0. Sodium ions were found to reversibly inhibit hydrogenase via a dead-end type of inhibition in which two catalytic forms of the enzyme bind Na+ with dissociation constants calculated to be 8.3 +/- 1.2 and 49.8 +/- 11.5 mM. In the absence of NaCl, maximum NAD+ reduction activity was measured at pH 8.3 at 30 and 37 degrees C. In the presence of 50 mM NaCl, inhibition was observed primarily at alkaline pH, and at assay pH values < or = 7.0, little or no difference was observed in activity in the presence or absence of 50 mM NaCl at a given temperature. Least squares analyses of the kinetic data indicated that substrate inhibition by H2 occurs at high substrate concentrations (Ki = 1.46 +/- 0.64 mM), which would become a significant influence on enzyme catalytic activity at hyperbaric levels of H2.

FMN cofactor dissociation from the soluble hydrogenase of Alcaligenes eutrophus H16.

The specific activity of purified soluble hydrogenase of Alcaligenes eutrophus H16 was found to vary with enzyme concentration. Specific activity as a function of concentration of purified enzyme could be fit to an equation describing the dissociation of a compound into two components. An association constant, kappa(a), was determined in this way to be 39.4 +/- 8.7 micrograms protein/ml. The concentration of the enzyme affected its kinetic parameters: a tenfold decrease in enzyme concentration caused by a reduction of the V(max) and Kappa(m) (NAD) values to 45% and 58%, respectively, of the values for undiluted (0.64 mg/ml) enzyme. Diaphorase (NAD-dependent reduction of benzyl viologen) specific activity of the hydrogenase was unaffected by dilution. The extent of dilution-induced activity loss was dependent on pH, with greater activity loss observed at higher pH values. The substrate NAD prevented loss of specific activity due to dilution, while the product NADH did not. Specific activity loss due to dilution as reversed with the addition of the cofactor FMN. Dilution of the hydrogenase caused an increase in the enzyme's specific flavin fluorescence. These results suggest that dilution of the soluble hydrogenase of Alcaligenes eutrophus causes dissociation of the cofactor FMN, and this activity loss should be taken into account as an important factor governing hydrogenase activity and kinetic properties.

A test for variations in individual sensitivity to hyperbaric oxygen toxicity.

It has been suggested that some individuals have above-average sensitivity to hyperbaric oxygen toxicity. An extensive human study completed at the Naval Experimental Diving Unit (NEDU) tested human tolerance to HBO and raised the possibility of assessing this hypothesis. In a group of 113 subjects given multiple exposures, some developed no symptoms of O2 toxicity while others developed symptoms on several occasions. The subjects in this study received unequal numbers of exposures of different depths and durations however, and it was not obvious how to determine unusual sensitivity. To assess the influences of chance vs. differences in sensitivity on the outcome of this experimental series, we performed a Monte Carlo simulation in which the experimental design was duplicated and the sensitivity hypothesis was evaluated statistically. The number of subjects giving rise to any symptoms and the distribution of individuals having symptoms on multiple occasions were evaluated. The simulation showed that the NEDU results were not unusual: nearly one quarter of the time the observed pattern of multiple symptoms could have been expected due to chance alone. The power of this simulation would have permitted detection of sensitivity factors 10 times (or greater) normal in 20% of the subjects at least half of the time.

Hydrogen gas is not oxidized by mammalian tissues under hyperbaric conditions.

Mammalian tissues, including heart, lung, liver, kidney, spleen, and skeletal muscle of guinea pig, rat, or pig, were exposed to tritium (T2) and high pressures of H2. Incorporation of the tritium label was measured to test for a latent capacity by mammalian tissues to oxidize H2 under conditions such as those experienced by deep divers breathing H2. Tissues were removed aseptically, and either minced, homogenized, or prepared as live cell cultures. The tissues were placed in a chamber to which 8 mCi T2, 1 MPa He, and either 1 or 5 MPa H2 were added. After 1 h the chamber was decompressed. The tissues were spun briefly in a vortex mixer to facilitate elimination of T2 in the gas phase. Samples were analyzed by scintillation counting for tritium incorporation in the liquid phase or in the tissues. Saline and distilled water were used as negative controls. Palladium (Pd) beads immersed in water, and cultures of the H2-metabolizing bacterium Alcaligenes eutrophus were used as positive controls. The tissues incorporated on the order of 10 nCi T2.ml-1, which implied a H2 incorporation of 10-50 nmol H2.g-1.min-1. However this incorporation was not different from that found in the water controls and was attributed to radioisotope effects. The Pd and bacterial samples incorporated over 1,000-fold more T2 than the mammalian tissues. We concluded that the mammalian tissues did not oxidize H2 under hyperbaric conditions, with a limit of detection of 100 nmol H2.g-1.min-1.

Increased red cell osmotic fragility and hematocrit after hyperbaric O2 exposure are related to acidosis.

Exposure to hyperbaric O2 (0.6 MPa) until convulsion occurred resulted in increased red blood cell osmotic fragility, increased hematocrit, and acidosis. Correction of the mixed metabolic and respiratory acidosis in arterial blood samples completely restored osmotic fragility and lowered hematocrit by 20%. Rats exposed to intermittent hyperbaric O2 (repeated cycles of 7 minutes of O2 and 7 minutes of air) tolerated significantly more O2 time than those exposed continuously. Intermittently exposed animals had smaller increases in osmotic fragility and less-severe acidosis. We verified the influence of pH on osmotic fragility and hematocrit in rats made acutely acidotic and corrected in vivo. Acidosis caused by CO2 inhalation and lactic and hydrochloric acid infusion raised osmotic fragility and hematocrit; these effects were completely reversed in the animal when we restored normal acid-base status. These studies demonstrate that conditions causing acidosis, including hyperbaric O2 exposure, increase red cell fragility and size and increase hematocrit.

Response of antioxidant enzymes to intermittent and continuous hyperbaric oxygen.

Rats and guinea pigs were exposed to O2 at 2.8 ATA (HBO) delivered either continuously or intermittently (repeated cycles of 10 min of 100% O2 followed by 2.5 min of air). The O2 time required to produce convulsions and death was increased significantly in both species by intermittency. To determine whether changes in brain and lung superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSHPx) correlated with the observed tolerance, enzyme activities were measured after short or long HBO exposures. For each exposure duration, one group received continuous and one intermittent HBO; O2 times were matched. HBO had marked effects on these enzymes: lung SOD increased (guinea pigs 47%, rats 88%) and CAT and GSHPx activities decreased (33%) in brain and lung. No differences were seen in lung GSHPx or brain CAT in rats or brain SOD in either species. In guinea pigs, but less so in rats, the observed changes in activity were usually modulated by intermittency. Increases in hematocrit, organ protein, and lung DNA, which may also reflect ongoing oxidative damage, were also slowed with intermittency in guinea pigs. Intermittency benefited both species by postponing gross symptoms of toxicity, but its modulation of changes in enzyme activities and other biochemical variables was more pronounced in guinea pigs than in rats, suggesting that there are additional mechanisms for tolerance.

The modulation of oxygen toxicity by intermittent exposure.

Intermittent delivery of hyperbaric O2 protects animals from pulmonary and central nervous system toxicity: more total O2 time can be tolerated if interrupted by short periods of low O2. Little is known about the mechanisms or optimization of systematically varied intermittency. Survival time was recorded in groups of 16 awake guinea pigs (239 +/- 23(SD) g) exposed to continuous O2 at 2.8 ATA or to one of six different schedules of O2 delivered with periodic air (PO2 = 0.588 ATA) interruptions. The survival curves had a lag time (11-14 hr of O2 time depending on the intermittency schedule) with a rapid loss of animals thereafter. Data were analyzed with risk models linking the probability of death to the accumulation of a putative toxic substance, X1. A model in which X1 accumulated in proportion to the PO2 and disappeared by first-order decay during periods of low O2 exposure was modified to include an effective rate constant for changes in X1: dX1/dt = a.PO2 + K1.(PO2 - Os).X1. First-order kinetics operated when PO2 was below the oxygen set point (Os), but the rate constant reversed sign to become a self-amplifying system when PO2 was above Os. This model achieved an excellent fit as judged by goodness-of-fit statistics while a simpler one did not. Our analysis suggests that the accumulation of toxicity does not correspond to a stable linear toxic process, but requires one in which a toxic process grows autocatalytically.

An analysis of decrements in vital capacity as an index of pulmonary oxygen toxicity.

Decrements in vital capacity (% delta VC) were proposed by the Pennsylvania group in the early 1970s as an index of O2-induced lung damage. These workers used the combined effects of PO2 and time of exposure to develop recommendations to limit expected % delta VC. Adopting this general approach, we fitted human pulmonary O2 toxicity data to the hyperbolic equation % delta VC = Bs.(PO2 - B1).(time)B3 using a nonlinear least squares analysis. In addition to the data considered in 1970, our analysis included new data available from the literature. The best fit was obtained when 1) an individual slope parameter, Bs, was estimated for each subject instead of an average slope; 2) PO2 asymptote B1 = 0.38 ATA; and 3) exponent B3 = 1.0. Wide individual variation imposed large uncertainty on any % delta VC prediction. A 12-h exposure to a PO2 of 1 ATA would be expected to yield a median VC decrement of 4%. The 80% confidence limits, however, included changes from +1.0 and -12% delta VC. Until an improved index of pulmonary O2 toxicity is developed, a simplified expression % delta VC = -0.011.(PO2 - 0.5).time (PO2 in ATA and time in min) can be used to predict a median response with little loss in predictability. The limitations of changes in VC as an index are discussed.

Failure of heparin, superoxide dismutase, and catalase to protect against decompression sickness.

The effects of heparin (HEP), superoxide dismutase (SOD), and catalase (CAT) on the course of decompression sickness (DCS) were studied in anesthetized dogs (Canis familiaris). Animals were divided into 4 groups: a drug assay group (n = 4) received HEP + SOD or HEP + SOD + CAT but were not dived; a control group (n = 14) was dived without drug treatment; a HEPSOD group (n = 11) received HEP + SOD predive and postdive; and a HEPSODCAT group (n = 15) received HEP + SOD + CAT before diving. All dived animals were subjected to repetitive air dives to 10 ATA until pulmonary artery pressure at least doubled within 10 min postdive. Physiologic variables were measured for 3 h postdive or until death. Animals were not recompressed. More early deaths occurred in the HEPSOD (7/11) and HEPSODCAT (8/15) groups than in the control group (5/14). All dived animals developed pulmonary hypertension, systemic hypotension, hemoconcentration, acidosis, hypoxemia, and interstitial pulmonary edema postdive. Drug therapy did not alter these responses to decompression. We conclude that without recompression, treatment with either HEP + SOD OR HEP + SOD + CAT does not improve the outcome of severe DCS in this animal model.

Blood-gas transport in awake rabbits exposed to normobaric hyperoxia.

The time course and terminal effects of normobaric oxygen exposure on the gas transport chain were studied in awake, catheterized rabbits exposed to air (n = 8) for 96 h or 100% O2 (n = 10) until death. O2-breathing animals survived 60.2 (+/- 13.5 SD) h. Pao2 increased and was maintained until within 4.9 (+/- 1.4) h of death. Mixed venous O2 tension rose sharply but transiently upon O2 exposure. In most animals, death followed a precipitous fall in PaCO2, a moderate rise in PaCO2, and a drop in pHa. The terminal acidosis was largely metabolic, nearly half due to lactic acidemia. There were transient appearances of metabolic acids early in the exposures before the PaCO2 decreased. Furthermore, in the last hours, fixed acids appeared when PaCO2 was unchanged or slightly decreased, but before the animal became hypoxemic. Metabolic acidosis without arterial hypoxemia could result from cardiac insufficiency, or alterations in metabolism, or in patterns of distribution of blood flow within peripheral beds. Thus, normobaric O2 exposure has precipitous and terminal effects on pulmonary gas exchange, but arterial hypoxemia is not necessarily the cause of death.

Pulmonary oxygen toxicity in awake dogs: metabolic and physiological effects.

Because the pulmonary endothelium is sensitive to O2-induced damage, we studied in vivo angiotensin-converting enzyme (ACE) activity in the lungs of 14 catheterized unanesthetized dogs exposed either to air or continuous 100% O2 at 1 ATA. For 5 days, or until the dog died, we measured physiological variables and lung ACE activity. The metabolic data were analyzed with a model that accounted for the effect of changes in cardiac output. Nine dogs breathing O2 lived 88 +/- 21.8 (SD) h and except for blood O2 tensions were indistinguishible from controls until development of a terminal response lasting only a few hours. Hemodynamic instability preceded a precipitous terminal change in blood gas tensions which resulted in impairment of arterial oxygenation, hypercapnia, and acidosis. Plasma renin activity increased. The metabolic capacity of the pulmonary endothelium of O2-exposed animals decreased with time so that after 96 h it was 50% of the control. That of five control animals did not change with time. Thus changes in lung ACE activity preceded alterations in hemodynamics or gas exchange, and the contributions of each are discussed.

Steady-state vascular responses to graded hypoxia in isolated lungs of five species.

In the isolated pig lung exposed to graded levels of hypoxia, steady-state pulmonary vasomotor tone is maximum at an O2 tension (PO2) of 50 Torr. Below 50-Torr decreases in PO2 cause steady-state tone to fall below this maximum. To determine whether this stimulus-response relation was peculiar to pigs, we measured the steady-state relation between PO2 and vasomotor tone in the isolated lungs of dogs, rabbits, cats, and ferrets, by using identical techniques in each species. Marked species differences were apparent in both the level of PO2 required to elicit responses and the amplitude of the responses. The ferret and the pig had the largest vasoconstrictor responses to hypoxia. No significant responses were obtained in the dog. The cat and rabbit were intermediate responders. In the ferret, cat, and rabbit, the stimulus-response relationship was biphasic, as in the pig. On the average, maximal constriction occurred at an PO2 of 25 Torr. When PO2 was lowered below 25 Torr, steady-state tone fell. Thus pulmonary vasodilation at low PO2 occurs in the isolated lungs of several species.