Noninvasive, infrared monitoring of cerebral and myocardial oxygen sufficiency and circulatory parameters.

The relatively good transparency of biological materials in the near infrared region of the spectrum permits sufficient photon transmission through organs in situ for the monitoring of cellular events. Observations by infrared transillumination in the exposed heart and in the brain in cephalo without surgical intervention show that oxygen sufficiency for cytochrome a,a3, function, changes in tissue blood volume, and the average hemoglobin-oxyhemoglobin equilibrium can be recorded effectively and in continuous fashion for research and clinical purposes. The copper atom associated with heme a3 did not respond to anoxia and may be reduced under normoxic conditions, whereas the heme-a copper was at least partially reducible.

Force, shortening, and work in muscular contraction: relative contributions to overall energy utilization.

The amounts of energy used during muscular contraction under different loads have been compared by measurement of the total amount of disturbance of the concentration of reduced nicotinamide-adenine dinucleotide during the period of oxidative recovery. The results are in quantitative agreement with the concept that three parameters determine the energy utilization: namely the time-integral of the force development, the time-integral of the shortening process, and the mechanical work.

Cyanide-induced cytochrome a,a3 oxidation-reduction responses in rat brain in vivo.

The sensitivity of the brain to cyanide-induced histotoxic hypoxia and the protective effects of known cyanide antagonists, have been assessed in vivo by reflectance spectrophotometry. Cyanide-related changes in cytochrome a,a3 (cytochrome c oxidase) oxidation-reduction (redox) state, tissue hemoglobin saturation, and local blood volume were continuously monitored in cerebral cortex of rats. Noncumulative, dose-dependent inhibition of the in situ mitochondrial respiratory chain was evaluated directly by measuring increases in reduction levels of the terminal oxidase. These transient cytochrome a,a3 reductions were accompanied by increases in regional cerebral hemoglobin saturation and blood volume. Cytochrome redox responses were not altered either in magnitude or kinetics by hyperoxia; however, the cyanide-cytochrome dose-response curve was greatly shifted to the right by pretreatment with sodium nitrite, and the recovery rate of cytochrome a,a3 from cyanide-induced reduction was enhanced fourfold by pretreatment with sodium thiosulfate.

Cytochrome a,a3 reoxidation. Early indicator of metabolic recovery from hemorrhagic shock in rats.

To assess the metabolic recovery of mitochondria after injury, we have monitored, in vivo and noninvasively, changes in the redox state of cytochrome (cyt) a,a3 in 35 rats after tissue hypoxia induced by rapid exsanguination to a mean arterial pressure of 30-35 mmHg. This level of mean arterial pressure was maintained for a shorter period of time in group I (n = 17) and a longer period of time in group II (n = 18), then the shed blood was returned by infusion. The surviving animals were observed for 2 more h before terminating the experiments. During exsanguination, reinfusion and recovery intervals brain tissue parameters of blood oxygenation, relative blood volume, and cyt a,a3 redox state were monitored continuously by spectrophotometry through the closed skull and intact skin. Group I had a high survival rate while group II had a very low survival rate. In both groups, with the onset of hypotension, there was a prompt rapid shift, followed by a slow continued progressive shift, of cyt a,a3 toward a more reduced state. The extent of recovery of cyt a,a3 following reinfusion was different in each group. In group I there was a rapid reoxidation of cyt a,a3 to a level above the base line (16 +/- 12%, mean +/- SEM). In contrast, the extent of reoxidation of cyt a,a3 in group II was significantly lower and stayed 31 +/- 6% below the base-line level. To further evaluate the mechanisms responsible for these observations, another related experiment was performed. 12 rats were subjected to shock and resuscitation as outlined for groups I and II. After death or killing of the animal, we measured, in vitro, oxygen consumption of cerebral cortical slices. Oxygen consumption of cortical tissue slices in subgroup I was significantly higher than in subgroup II. We conclude that, under these experimental conditions, the oxidative response of cyt a,a3 correlates closely with survival or death in the two groups. If in group I animals the greater oxidation of cyt a,a3, in vivo after resuscitation, reflects greater oxygen utilization, as is suggested by the in vitro observations in subgroup I, then we may be observing a useful adaptive response to tissue injury leading to preserved organ function and enhanced survival. Therefore, noninvasively measured cyt a,a3 redox state, reflecting intracellular metabolic activity, seems to indicate both the overall cerebral cellular response to injury and the likelihood of survival.

In vivo comparison of cerebral tissue PO2 and cytochrome aa3 reduction-oxidation state in cats during hemorrhagic shock.

To assess the adequacy of oxygen availability and utilization within the cerebral cortex in vivo, we have measured the partial pressure of oxygen in tissue (PtO2), as well as the reduction oxidation state of cytochrome c oxidase (cyt aa3) during shock induced by slow or rapid hemorrhage in anesthetized cats. PtO2 was measured with pyrenebutyric acid-generated fluorescence in cerebral cortical cells. Cyt aa3 redox state was measured by the absorption of monochromatic light at 605 nm absorption peak of the enzyme reflected from the same cortical field. The PtO2 remained within the normal range until either 30 +/- 1.5 ml blood/kg was removed or the mean arterial pressure fell by 70 +/- 5% of base line. Beyond either point, the PtO2 fell rapidly to a low value approximating zero. By contrast, the reduction of cyt aa3 began early when as little as 5 ml blood/kg was removed. Thereafter, the shift toward reduction was progressive and continuous with a slow rate at first and a rapid rate later. This accelerated rate of cyt aa3 reduction preceded the rapid fall of PtO2. We concluded that, under these experimental conditions, cyt aa3 reduction is a much earlier and more sensitive indicator of perturbed intracellular aerobic metabolism due to hemorrhage that is PtO2.

Characterization of the oxygen probe pyrenebutyric acid in rabbit heart mitochondria.

The fluorescence of pyrenebutyric acid is quenched by oxygen and the quenching is a linear function of the oxygen concentration. In addition, pyrenebutyric acid has been shown to be readily taken up by the cell with a partition coefficient of approx. 200. Results are presented on the effect of pyrenebutyric acid on oxidative metabolism in rabbit heart mitochondria. 1. Pyrenebutyric acid is readily taken up by heart mitochondria: 0.843 +/- 0.087 nmol pyrenebutyric acid taken up by Mr/mg dried weight. 2. Pyrenebutyric acid does not alter the steady-state redox levels (State 1, 2, 4 or 5) of any member of the respiratory chain. 3. Pyrenebutyric acid does not alter the response of cytochrome b, cytochrome c or NADH in the State 4-3-4 transition induced by the addition of ADP. Thus pyrenebutyric acid would appear to be non-toxic to oxidative metabolism.

Redox state of respiratory chain enzymes and potassium transport in silkworm mid-gut.

The midgut of Hyalophora cecropia actively transports potassium from hemolymph to lumen and the energy for this process appears to be intimately linked to oxidative metabolism. In the present investigation, we monitored concurrently the rate of active transport and the redox levels of the components of the respiratory chain in the intact tissue under a variety of experimental conditions. Approximately equal concentrations of cytochromes a3, a, c and b-557 were found. Other investigators (Pappenheimer, Jr, A.M. and Williams, C.M. (1954) J. Biol. Chem. 209, 915, Shappirio, D.G. and Williams, C.M. (1957) Proc. R. Soc. Lond. Ser. B 147, 233 and Chance, B. and Pappenheimer, Jr, A.M. (1957) J. Biol, Chem, 209, 931) have indentified cytochrome b-557 with b5 and found that it exists primarily in an extramitochondrial location. Steady-state experiments demonstrated that all these cytochromes were approximately 50% reduced while active transport proceeded at a high rate in regular cecropia Ringer containing 32 mM KCl. When the potassium concentration was reduced, the active transport decreased and all the cytochromes became more oxidized. Addition of 1 mM cyanide inhibited active transport by 90% and caused a 100% reduction of all cytochromes. Redox state and short circuit current (Isc) kinetics measured as the tissue was made anoxic showed that all the respiratory enzymes, except cytochrome b-557, became fully reduced at a faster rate than the rate of inhibition of the Isc. The rate of cytochrome b-557 reduction followed kinetically the Isc. These observations are interpreted in a scheme where cytochrome b-557 (possibly b5) branches off cytochrome c from the conventional resporatory chain, utilizing cytochrome a3 as the terminal oxidase for both branches. Cytochrome b-557 may be involved in providing a direct link between oxidative metabolism and active transport in the midgut of the silkworm.

Spectrophotometric studies on the pH of frog skeletal muscle. PH change during and after contractile activity.

The spectral characteristics of the pH-sensitive dyes neutral red (NR) and bromcresol purple (BCP) were utilized for studies of the changing intracellular pH (pHi) of sartorius muscles from Rana pipiens, both during the course of an isometric twitch and during recovery metabolism subsequent to a train of twitches. The information from the two dissimilar dyes correlated to confirm the methodology. Neither the fast realkalinization observed during a twitch nor the slow alkalizing phase of recovery metabolism was affected in an obvious manner when phosphocreatine (PC) hydrolysis was blocked by 1-fluoro-2,4-dinitrobenzene (FDNB). Iodoacetic acid (IAA) did inhibit the slow acidic phase of recovery metabolism. The conclusion is made that alkalizing reactions other than PC breakdown must be considered as operative at these levels of activity. Hypertonic solutions altered twitch tension and time course without altering the pHi shifts observed until approximately 75% of the twitch amplitude was abolished. Multiple effects of hypertonic solutions as the muscle approach tonic equilibrium are proposed.

Oxidative and glycolytic recovery metabolism in muscle.

The average degree of reduction of mitochondrial NAD has been measured in the intact toad sartorius by a fluorometric technique. It has been shown that cytoplasmic NADH does not interfere materially with these measurements. The percentage reduction of this respiratory coenzyme has been determined in a number of physiological steady states which are well correlated with fluorometrically determined levels of NADH in suspensions of mitochondria from the hind leg musculature of the toad. In addition, these findings are closely comparable to similar, spectrophotometric measurements on mitochondria from other sources. In the presence of an adequate O(2) level a single twitch produces a decrease in fluorescence from the resting steady state which is followed by a slow return to the base line condition. This cycle indicates the intensity and the time course of the oxidative recovery metabolism. The area under this curve is directly related to the number of twitches up to three or four. Greater activity produces a curtailment of oxidative recovery due to glycolysis. In the presence of iodoacetate the linear relation holds for five to seven twitches. At still higher levels of activity a curtailment of the change in NAD level sets in, probably due to the removal of AMP by catabolic reactions.

Insufficient supply of reducing equivalents to the respiratory chain in cerebral cortex during severe insulin-induced hypoglycemia in cats.

The ability of endogenous substrates in brain to substitute for glucose as sources for energy metabolism during insulin-induced hypoglycemia was studied. The ratio of the arteriovenous difference of glucose to the arteriovenous difference of oxygen in the cerebral cortex was measured during progressive hypoglycemia in paralyzed, artificially ventilated cats that were anesthetized with pentobarbital sodium and nitrous oxide. The ratio did not change when blood glucose fell from a mean of 7.68 to approximately 2 mumol/ml. Below 2 mumol/ml the ratio decreased, indicating that substrates other than the glucose supplied by the blood were being utilized. In another series of experiments, changes in the redox state of respiratory chain NAD were monitored from the cerebral cortex using microfluorometry during the onset of hypoglycemia and the recovery. Hypoglycemia severe enough to produce isoelectric EEG was accompanied by an oxidation of NADH, demonstrating that the supply of reducing equivalents to the respiratory chain was decreased. Recovery from hypoglycemia, produced by intravenous glucose injections, was accompanied by an increase in blood glucose concentrations, the return of EEG activity, and a decrease in the NAD/NADH ratio. When blood glucose concentration reached 2.23 during the recovery, further increases in blood glucose had no effect on the redox state of NAD. Although alternative substrates appear to be utilized for energy metabolism during severe hypoglycemia, they cannot fully replace glucose as the source of reducing equivalent to the respiratory chain.

Response of toad brain respiratory chain enzymes to ouabain, elevated potassium, and electrical stimulus.

Spectrophotometric and fluorometric techniques were used to monitor the proportion of reduced to oxidized cytochrome (cyt) and levels of reduced pyridine nucleotide in preparations of whole toad brain in vitro. In resting, well-oxygenated brain, levels of reduction for cyt a3 ranged between 5% and 23%; for cyt a, 17-23%; for cyt c, 18-32%, and for cyt b, 25-42%. These levels of reduction cannot be due to functional hypoxia since hemoglobin in resting brains is 100% oxygenated. In brains treated with 10(-4) M ouabain, stimulant of brain respiration, the cytochromes first become more oxidized, then more reduced; ultimately there is a tendency to return to the initial levels of reduction. In brains bathed with solutions containing 30 mM potassium, also a stimulant of brain respiration, the response is an immediate pulse of reduction in all cytochromes, followed by a tendency to return to the initial levels. Short trains of pulses of electrical field stimulation result in a biphasic change in the level of reduction of cyt a3, an initial slight reduction being followed by a transient of increased oxidation. This response can be abolished by low-sodium bathing solution but not by ouabain. Cytochromes a, b and c show a simple oxidative response to electrical stimulation; the kinetics of this oxidative response are similar to those of the oxidative transient of the cyt a3 response. Pyridine nucleotides, as measured by their fluorescence, respond to electrical stimulation with a transient oxidation which exhibits slower kinetics than the response of the cytochromes. The high resting levels of reduction of cyt a and cyt a3, the reductive response to ouabain and potassium, and the oxidative response of all cytochromes to electrical stimulation suggest a tighter coupling between oxygen utilization and neuronal function than would be expected if mitochondrial redox states simply reflected changes in phosphate acceptor potential resulting from activity of Na+-K+ ATPase.

The cerebral redox state in cats during severe insulin induced hypoglycemia.

The cerebral metabolic state was studied in cats during insulin-induced hypoglycemia and the recovery after glucose infusion. Changes in the redox state of nicotinamide adenine dinucleotide (NAD) were monitored from the surface of the exposed cerebral cortex using microfluorometry. After insulin injection blood glucose fell from 6.85 mumol/ml to 0.45 mumol/ml at EEG isoelectricity and was accompanied by an oxidation of NADH+. Upon intravenous glucose infusion EEG activity rapidly returned and NAD became more reduced. The oxidation of NADH+ during severe hypoglycemia demonstrated that the in vivo redox state of mitochondria behave in a similar manner as isolated mitochondria when reducing equivalents become limiting to the respiratory chain.

Effects of respiratory gases on cytochrome A in intact cerebral cortex: is there a critical Po2?

Changes in the redox level of cytochrome a and in the amount of oxygenated hemoglobin were measured by dual wavelength reflectance spectrophotometry in the intact cerebral cortex of cats (cerveau isolé preparation) and in unanesthetized rabbits with chronically implanted cranial windows. Increases in inspired oxygen were accompanied by an increase in the oxidation level of cytochrome a and an increase in the amount of oxygenated hemoglobin in the optical field. These changes were larger in the presence of 5% CO2. Reduction of the inspired oxygen concentration produced a decrease in the oxidation/reduction ratio of cytochrome a and a disoxygenation of hemoglobin. The presence of CO2 at these lower oxygen levels diminished the reduction of cytochrome a and the disoxygenation of hemoglobin. These data indicate that, in the resting subject, the reduction levels of cytochrome a are well above the low values seen in isolated mitochondria. They also indicate that the blood supply to the cerebral cortex is regulated at a level of slight hypoxia.

Infra-red technique for cerebral blood flow: comparison with 133Xenon clearance.

A rapid infra-red optical technique has been developed for the measurement of cerebral blood flow. The method measures optical density changes across the intact skull during the passage of a bolus of the dye. Cardio-Green (CG). The clearance curves obtained for CG boluses are very short (less than 30 sec) in comparison with those obtained with tracers such as 133Xenon (10-30 min) that distribute into cerebral tissue. The volume of distribution of CG is totally intravascular, and the dye is relatively slowly cleared from the body. The important advantages of this spectrophotometric technique are its speed, versatility, and the avoidance of radioactive materials. The differential spectrophotometer used in this study, with trivial modifications, has been used to monitor changes in brain blood volume, oxygen saturation of hemoglobin, and cortical mitochondrial respiratory function, which illustrate the versatility of the technique for neurological assessments.

Pyrenebutyric acid as an optical oxygen probe in the intact cerebral cortex.

The application of the fluorochrome pyrenebutyric acid (PBA) as an in vivo quantitative probe for intracellular oxygen tension (iPO2) is reported. PBA is nontoxic, noninvasive, nonconsumptive, and allows optical monitoring of iPO2 on line. Cats were anesthetized with ether and lesioned midcollicularly to produce cerveau isolé preparations with optical monitoring via a compensated microfluorometer. The "normal" iPO2 was determined to be 42.8 /- 8.6 (SD) mmHg. Decrease in cerebral perfusion results in observing a decreased iPO2. This method of monitoring iPO2 was found to overcome drawbacks inherent in the polarographic technique and to posess additional advantages.