William D. Currie

Brain Adenosine Triphosphate: Decreased Concentration Precedes Convulsions

The concentration of adenosine triphosphate in the brain decreased before the onset of generalized convulsions in unanesthetized rats subjected to acute hypoxia or treated with hydroxylamine or pentylenetetrazole (Metrazol). As the convulsive episode continued, adenosine triphosphate decreased further. Stimulation of adenosine triphosphate production forestalled its disappearance from the brain and delayed the development of seizure activity.

Ozone affects breathing and pulmonary surfactant function in mice

The effect on breathing of BALB/c mice immediately following ozone exposure (2 ppm) for 0, 2, 4, 6, and 8 h was studied with a whole body plethysmograph. Whether such exposure affected the normal function of pulmonary surfactant of maintaining airway patency was evaluated with a capillary surfactometer. Respiratory rate in mice that were not exposed was 358+/-16 (mean+/-S.E.) breaths/min and decreased to 202+/-10 after 6 h exposure. The mean pressure change caused by breathing diminished significantly, indicating a reduced tidal volume. BAL fluid from controls maintained patency for 88+/-2% of the study time, 120 s, implying a good surfactant function, but the ozone exposure caused the surfactant to lose its capability of maintaining patency (P < 0.0001). This decaying surfactant function of the BAL fluid coincided with an increasing protein concentration in the fluid of exposed animals (1.46+/-0.14 mg/ml in the 8-h group) as compared to controls (0.44+/-0.04 mg/ml, P < 0.0001). It is concluded that leakage of plasma proteins into the airway lumen was probably the main reason for the surfactant dysfunction, which may have contributed to the altered breathing pattern.

Breathing and pulmonary surfactant function in mice 24 h after ozone exposure

The aim of this study was to determine whether an acute ozone exposure affects breathing, and the ability of pulmonary surfactant to maintain the patency of terminal conducting airways. BALB/c mice were exposed to ozone (1 part per million (ppm)) for 2, 4, 6, and 8 h. They were examined with plethysmography and with bronchoalveolar lavage (BAL) 24 h later. The BAL fluid was analysed for the presence of inflammatory cells and concentrations of proteins and phospholipids. Surfactant in the remaining BAL fluid was concentrated five-times and examined with a capillary surfactometer (CS). The surfactant was then washed with a large volume of saline solution which was removed following centrifugation. Already, after a 2 h ozone exposure, the respiratory frequency increased from 297+/-6 to 386+/-11 breaths x min(-1) (p<0.0001). Pressure amplitude per breath diminished (p<0.001), indicating a reduced tidal volume. A highly significant surfactant dysfunction was observed with the CS (p<0.0001), although phospholipids increased. However, proteins also increased (p<0.0001) and they or other water-soluble inhibitors apparently caused the surfactant dysfunction since, when they were removed with a washing procedure, the surfactants normal ability to maintain patency was restored. The acute ozone exposure affected breathing and caused an airway inflammation. The inflammatory proteins or other water-soluble inhibitors reduced the surfactants ability to secure airway patency.

Response of the Pulmonary Surfactant System To Phosgene

Rats were exposed to 240 ppm·min phosgene (1.0 ppm for 4 hrs) in a Rochester-type chamber. At intervals thereafter over a 4 day period, lungs were removed for determination of wet weight; total, microsomal and surfactant protein concentrations; surfactant phospholipid concentrations; and 1-acyl-2-lyso-phosphatidylcholine: palmitoyl-CoA acyl transferase activity. Immediately upon termination of the phosgene exposure, microsomal protein and acyl transferase activity were reduced below, and lung wet weight was elevated above, control levels. From Day 1 through Day 3 after the exposure, all measured parameters, except for the phosphatidylinositol constituent of the surfactant fraction, were increased above the control values. In general, maximum levels were observed on Day 2; however, the acyl transferase activity and surfactant concentration continued to increase on Day 3. The results suggest (a) components of the pulmonary surfactant system may be involved in maintenance of pulmonary fluid balance and (b) the presence of excess water in the lungs as a result of phosgene exposure may represent a signal for increased synthesis of anti-edematogenic materials in order to promote removal of the inappropriate fluid.

Protection of brain metabolism with glutathione, glutamate, gamma-aminobutyrate and succinate.

Studies on protective agents in oxygen toxicity experiments led us to believe that a glutathione (GSH)-glutamate-γ-aminobutyrate (GABA)-succinate pathway may serve as a secondary support system in the maintenance of brain energy levels (adenosine triphosphate [ATP] concentration). This “shunt” is shown in Fig. 1. The glutamate-GABA-succinicsemialdehyde-succinate shunt is a well established pathway (1-8) to which no major physiological significance has been attached. The GABA-succinate shunt has been suggested as a means of metabolizing GABA (9, 10). It has also been reported to function as a means of bypassing inhibition of the alpha-ketoglutarate dehydrogenase system of the citric acid cycle by withdrawal of alpha-ketoglutarate from the cycle by transamination with GABA to yield glutamate and reentry of the carbon chain of GABA into the cycle at the succinate level (9, 10). The possible physiological importance of the shunt is seen if one recognizes that succinate markedly stimulates respiration and oxidative phosphorylation. Krebs et al. (11) reported that succinate oxidation can monopolize the respiratory-electron transport chain which is the major source of ATP production. Sanders et al. (12, 13) observed significantly higher respiration rates with succinate in brain, liver, and kidney of rats when compared with alpha-ketoglutarate and glutamate. Data shown later indicate that succinate stimulates brain respiration and oxidative phosphorylation in the mouse, rat, guinea pig, rabbit, cat and dog. The factor limiting the amount of succinate available for metabolism has generally been considered to be the rate at which alpha-ketoglutarate is converted to succinate by enzymes of the Krebs cycle. Roberts (8) showed that conversion of GABA to succinate by GABA transaminase is rapid, and that both glutamic acid decarboxylase and GABA transaminase have pH optima (6.5 and 8.2, respectively) such that small changes in intracellular pH—within the physiological range—could result in large changes in these enzyme activities in situ (8, 14, 15).

Response of pulmonary energy metabolism to phosgene.

Rats were exposed to phosgene at a concentration of 1.0 ppm for 4 hours in a Rochester-type chamber. At intervals thereafter over a 4 day period, lungs were obtained for histological and biochemical assessments. Edema was estimated by histological examination and by measurement of lung wet and dry weights. In parallel studies, pulmonary mitochondrial respiratory activity was measured using Clark oxygen electrodes. The significant reduction in respiratory control index (State 3 respiration/State 4 respiration)found immediately following phosgene exposure coincided with the highest level of % lung water. There was a concomitant decrease of A TP concentration that persisted on the third day after exposure. Na-K-A TPase activity was reduced 1 day after exposure, thus a lowered A TP level preceded a reduction in Na-K-ATPase or sodium pump activity. The reduction in A TP level and Na-K-A TPase activity may play a major role in damage to lung tissue following exposure to phosgene.

Effects of Hyperbaric Oxygenation on Metabolism V. Comparison of Protective Agents at 5 Atmospheres 100% Oxygen

Summary During 5 ATA oxygen exposures: The GSH was the best single compound for protecting against oxygen toxicity. This appears to be a combined SH protection and metabolite (glutamate → GABA → succinate) protection. The SH group protection, as evidenced by the cysteine experiments, was not as effective as the metabolites: succinate, glutamate, and GABA. The acid-base buffer Tris was less effective than the SH group protectors, or GABA, glutamate, or succinate. Glucose and malate gave no protection against oxygen toxicity. We thank Marvin and Julia Nunn for valuable technical assistance.

Pulmonary O2 Toxicity: Energy Metabolism and Data Analysis

Summary Comparison of homogenates vs mitochondrial preparations in evaluating in vivo respiration and oxidative phosphorylation in lung from rats exposed to 1.5 ATA O2 for 24, 27, and 30 hr showed that homogenates from experimentally exposed rats may contain extramitochondrial factors inhibitory to respiration which are washed out in mitochondrial isolation procedures. Thus, the use of homogenates is preferable to isolated mitochondria, since they more nearly reflect in vivo oxidative phosphorylation in pulmonary oxygen toxicity studies. Intercomparison of methods of expressing lung respiration rates and ATP concentration were made in lung of rat subjected to 1.0 and 1.5 ATA O2 exposures. Care should be exercised in selecting the means for expressing data. Our results indicate that the preferred method for expressing respiration rates is microliters O2/milligram mitochondrial protein per minute, and for expressing ATP levels is micromoles ATP/milligram mitochondrial protein, in pulmonary O2 toxicity studies. Evaluation of metabolic activity requires that accurate determinations of the total lung (or individual lung) weight, and some unit of the lung (preferably the organelle responsible for the activity being studied) be determined, e.g., milligram mitochondrial protein per gram lung.

CHEMICAL PROTECTION AGAINST OXYGEN TOXICITY

Publisher Summary This chapter focuses on the efficacy of several chemical compounds in counteracting O2 toxicity and the O2 pressure above which no protection is afforded by a specific chemical agent. Various investigators have reported the use of SH compounds, acid–base buffers, and metabolic agents in O2 toxicity studies. The chapter presents an experiment in which male Sprague–Dawley rats—weighing between 150 and 200 gm—were fasted from 16 to 18 h and were given intraperitoneal injections of a selected compound 50 min prior to being exposed to HPO. The dosage of all compounds except cysteine, glucose, and malate was 12 mmoles/kg. At 5 atm abs, GSH proved to be the best single protective agent against O2 toxicity.

Effects of Hyperbaric Oxygenation on Metabolism VII. Succinate Protection Against Oxygen Toxicity in Large Animals

Significant protection was demonstrated against the development of convulsive activity in small animals using 0.4 M sodium succinate (1-4) injected intraperitoneally in a dosage of 12 mM/kg of body weight, Succinate protection against hyperbaric oxygen (HPO) at 5, 7, 9, and 11 ATA of 100% oxygen (5) was shown not to be due to the hyperosmolarity of the solution infused and led to our proposal that maintenance of normal ATP concentrations in brain and other tissues is of prime importance in protecting animals subjected to HPO. No significant delay of onset of convulsive activity was observed with hyperosmolar solutions of NaCl, sodium malate, an NAD-linked TCA cycle intermediate, or glucose. These results are consonant with the observation of Chance et al. (6) that HPO adversely affects NAD-linked substrates of oxidative phosphorylation, and have stimulated further studies using the FAD-linked substrates, succinate and alpha-glycerophosphate. We report here the results of experiments using sodium succinate as a protective agent against the toxic effects of HPO in large animals. These studies were carried out to determine the feasibility of using succinate in humans as a protective agent when HPO is used in the treatment of certain clinical conditions such as gas gangrene. Dogs (12-18 kg) were infused intravenously with 0.4 M sodium succinate, pH 6.4, in a dosage of 8 mM/kg of body weight/hr, for 50 min prior to and during exposure to 100% oxygen at a pressure of 40 psig. Control experiments were conducted by subjecting the same animals, either untreated or infused with equivalent doses of saline or sodium malate, to identical conditions of HPO 48-72 hr prior to the succinate experiments. Thus, each dog served as its own control, and time to convulsions could be compared directly (Table I).