Increasing activity of H(2)-metabolizing microbes lowers decompression sickness risk in pigs during H(2) dives.

The risk of decompression sickness (DCS) was modulated by varying the biochemical activity used to eliminate some of the hydrogen (H(2)) stored in the tissues of pigs (19.4 +/- 0.2 kg) during hyperbaric exposures to H(2). Treated pigs (n = 16) received intestinal injections of Methanobrevibacter smithii, a microbe that metabolizes H(2) to water and CH(4). Surgical controls (n = 10) received intestinal injections of saline, and an additional control group (n = 10) was untreated. Pigs were placed in a chamber and compressed to 24 atm abs (20.6-22.9 atm H(2)). After 3 h, the pigs were decompressed and observed for symptoms of DCS for 1 h. Pigs with M. smithii had a significantly lower (P < 0.05) incidence of DCS (44%; 7/16) than all controls (80%; 16/20). The DCS risk decreased with increasing activity of microbes injected (logistic regression, P < 0.05). Thus the supplemental tissue washout of the diluent gas by microbial metabolism was inversely correlated with DCS risk in a dose-dependent manner in this pig model.

On the likelihood of decompression sickness during H(2) biochemical decompression in pigs.

A probabilistic model was used to predict decompression sickness (DCS) outcome in pigs during exposures to hyperbaric H(2) to quantify the effects of H(2) biochemical decompression, a process in which metabolism of H(2) by intestinal microbes facilitates decompression. The data set included 109 exposures to 22-26 atm, ca. 88% H(2), 9% He, 2% O(2), 1% N(2), for 0.5-24 h. Single exponential kinetics described the tissue partial pressures (Ptis) of H(2) and He at time t: Ptis = integral (Pamb - Ptis). tau(-1) dt, where Pamb is ambient pressure and tau is a time constant. The probability of DCS [P(DCS)] was predicted from the risk function: P(DCS) = 1 - e(-r), where r = integral (Ptis(H(2)) + Ptis(He) - Thr - Pamb). Pamb(-1) dt, and Thr is a threshold parameter. Inclusion of a parameter (A) to estimate the effect of H(2) metabolism on P(DCS): Ptis(H(2)) = integral (Pamb - A - Ptis(H(2))). tau(-1) dt, significantly improved the prediction of P(DCS). Thus lower P(DCS) was predicted by microbial H(2) metabolism during H(2) biochemical decompression.

Decompression sickness risk reduced by native intestinal flora in pigs after H2 dives.

Decompression sickness (DCS) risk following a simulated dive in H2 was lower in pigs with a native intestinal flora that metabolized H2. Pigs (n = 27; 19.4 +/- 0.2 kg body mass) were placed in a chamber that was pressurized to 22.2-25.5 atm (absolute; 2.2-2.6 MPa) with 84-93% H2 for 3 h. Chamber concentrations of O2, H2, He, N2, and CH4 were monitored by gas chromatography. Release of CH4 from the pigs indicated that intestinal microbes had metabolized H2 After decompressing to 11 atm, the pigs were observed for DCS. Animals with DCS released significantly less (P < 0.05) methane (0.53 +/- 0.37 ppm CH4; n = 5) than those without DCS (1.40 +/- 0.17 ppm CH4; n = 22). The DCS risk reduction was attributed to the loss of roughly 12% of the total volume of H2 that could be stored in the tissues of the pigs. Thus, H2 metabolism by the native intestinal flora of pigs may protect against DCS following a simulated H2 dive.

Calorimetry and respirometry in guinea pigs in hydrox and heliox at 10-60 atm.

We used direct calorimetry and respirometry to measure the total rate of heat loss (Qsigma) and of oxygen consumption (VO2) in guinea pigs in 1-atm (0.1 MPa) air and at 10-60 atm in either heliox (98% He, 2% O2) or hydrox (98% H2, 2% O2). Our objective was to determine if the physiological responses to these two gas mixtures were different and, if so, whether the differences were attributable to the thermal characteristics of the gases alone or were confounded by additional mechanisms. At 10-40 atm, Qsigma and VO2 were not significantly different in the two gas mixtures, whereas at 60 atm, Qsigma and VO2 were significantly higher in heliox than in hydrox. The VO2/Qsigma ratio suggested that the animals were not in thermal equilibrium in hyperbaria. Based solely on the differing thermal properties of the gas mixtures, a mathematical model predicted a Qsigma that was higher in hydrox than in heliox at all pressures. Two plausible explanations are suggested: one is an adaptive lowering of the surface temperature as a physiological response of the animal to the thermally more stressful hydrox environment, and the other is related to the narcotic suppression of the animal's activity by hydrox.

Decompression sickness risk in rats by microbial removal of dissolved gas.

We present a method for reducing the risk of decompression sickness (DCS) in rats exposed to high pressures of H2. Suspensions of the human colonic microbe Methanobrevibacter smithii were introduced via a colonic cannula into the large intestines of the rats. While the rats breathed H2 in a hyperbaric chamber, the microbe metabolized some of the H2 diffusing into the intestine, converting H2 and CO2 to methane and water. Rate of release of methane from the rats, which was monitored by gas chromatography, varied with chamber H2 pressure. This rate was higher during decompression than during compression, suggesting that during decompression the microbe was metabolizing H2 stored in the rats' tissues. Rats treated with M. smithii had a 25% (5 of 20) incidence of DCS, which was significantly lower (P < 0.01) than the 56% (28 of 50) incidence of untreated controls, brought on by a standardized compression and decompression sequence. Thus using a microbe in the intestine to remove an estimated 5% of the body burden of H2 reduced DCS risk by more than one-half. This method of biochemical decompression may potentially facilitate human diving.

Relationship between T-wave amplitude and oxygen pulse in guinea pigs in hyperbaric helium and hydrogen.

Diving is known to induce a change in the amplitude of the T wave (ATw) of electrocardiograms, but it is unknown whether this is linked to a change in cardiovascular performance. We analyzed ATw in guinea pigs at 10-60 atm and 25-36 degreesC, breathing 2% O2 in either helium (heliox; n = 10) or hydrogen (hydrox; n = 9) for 1 h at each pressure. Core temperature and electrocardiograms were detected by using implanted radiotelemeters. O2 consumption rate was measured by using gas chromatography. In a previous study (S. R. Kayar and E. C. Parker. J. Appl. Physiol. 82: 988-997, 1997), we analyzed the O2 pulse, i.e., the O2 consumption rate per heart beat, in the same animals. By multivariate regression analysis, we identified variables that were significant to O2 pulse: body surface area, chamber temperature, core temperature, and pressure. In this study, inclusion of ATw made a significantly better model with fewer variables. After normalizing for chamber temperature and pressure, the O2 pulse increased with increasing ATw in heliox (P = 0.001) but with decreasing ATw in hydrox (P < 0.001). Thus ATw is associated with the differences in O2 pulse for animals breathing heliox vs. hydrox.

Oxygen pulse in guinea pigs in hyperbaric helium and hydrogen.

We analyzed O2 pulse, the total volume of O2 consumed per heart beat, in guinea pigs at pressures from 10 to 60 atmospheres. Animals were placed in a hyperbaric chamber and breathed 2% O2 in either helium (heliox) or hydrogen (hydrox). Oxygen consumption rate (VO2) was measured by gas chromatographic analysis. Core temperature and heart rate were measured by using surgically implanted radiotelemeters. The VO2 was modulated over a fourfold range by varying chamber temperature from 25 to 36 degrees C. There was a direct correlation between VO2 and heart rate, which was significantly different for animals in heliox vs. hydrox (P = 0.003). By using multivariate regression analysis, we identified variables that were significant to O2 pulse: body surface area, chamber temperature, core temperature, and pressure. After normalizing for all nonpressure variables, the residual O2 pulse was found to decrease significantly (P = 0.02) with pressure for animals in heliox but did not decrease significantly (P = 0.38) with pressure for animals in hydrox over the range of pressures studied. This amounted to a roughly 25% lower O2 pulse for normothermic animals in 60 atmospheres heliox vs. hydrox. These results suggest that reduction of cardiovascular efficiency in a hyperbaric environment can be mitigated by the choice of breathing gas.

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.

Capillary blood transit time in muscles in relation to body size and aerobic capacity.

The mean minimal transit time for blood in muscle capillaries (tc) was estimated in six species, spanning two orders of magnitude in body mass and aerobic capacity: horse, steer, dog, goat, fox and agouti. Arterial (CaO2) and mixed venous (CvO2) blood O2 concentrations, blood hemoglobin concentrations ([Hb]) and oxygen uptake rates were measured while the animals ran on a treadmill at a speed that elicited the maximal oxygen consumption rate (VO2max) from each animal. Blood flow to the muscles (Qm) was assumed to be 85% of cardiac output, which was calculated using the Fick relationship. Total muscle capillary blood volume (Vc) and total muscle mitochondrial volume were estimated by morphometry, using a whole-body muscle sampling scheme. The tc was computed as Vc/Qm. The tc was 0.3-0.5 s in the 4 kg foxes and agoutis, 0.7-0.8 s in the 25 kg dogs and goats, and 0.8-1.0 s in the 400 kg horses and steers. The tc was positively correlated with body mass and negatively correlated with transcapillary O2 release rate per unit capillary length. Mitochondrial content was positively correlated with VO2max and with the product of Qm and [Hb]. These data suggested that Qm, Vc, maximal hemoglobin flux, and consequently tc, are co-adjusted to result in muscle O2 supply conditions that are matched to the O2 demands of the muscles at VO2max.

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.

O2 delivery at VO2max and oxidative capacity in muscles of standardbred horses.

The purpose of this study was to describe the relationships between 16 physiological, biochemical, and morphological variables presumed to relate to the oxidative capacity in quadriceps muscles or muscle parts in Standardbred horses. The variables included O2 delivery (blood flow) and mean capillary transit time (MTT) during treadmill locomotion at whole animal maximal O2 consumption (VO2max, 134 +/- 2 ml.min-1 x kg-1), capillary density and capillary-to-fiber ratio, myoglobin concentration, oxidative enzyme activities, glycolytic enzyme activities, fiber type populations, and fiber size. These components of muscle metabolic capacity were found to be interrelated to varying degrees using correlation matrix analysis, with lactate dehydrogenase activity showing the most significant correlations (n = 14) with other variables. Most of the "oxidative" variables occurred in the highest quantities in the deepest muscle of the group (vastus intermedius) and in the deepest parts of the other quadriceps muscles where the highest proportions of type I fibers were localized. The highest blood flow measured with microspheres in the muscle group during exercise was in vastus intermedius muscle (145 ml.min-1 x 100 g-1), and the lowest was in the superficial part of rectus femoris muscle (32 ml.min-1 x 100 g-1). Average muscle blood flow during exercise at whole animal VO2max was 116 ml.min-1 x 100 g-1. Because skeletal muscle comprised 43% of total body mass (453 +/- 34 kg), total muscle blood flow was estimated at 226 l/min, which was approximately 78% of total cardiac output (288 l/min).(ABSTRACT TRUNCATED AT 250 WORDS)

Diffusion distances, total capillary length and mitochondrial volume in pressure-overload myocardial hypertrophy.

We examined the relationships between blood pressure, coronary blood flow, cardiac output, myofiber growth, capillarity, mitochondrial content, and capillary and mitochondrial distributions in a pressure-overload model of myocardial hypertrophy. The Goldblatt one kidney-one clip (1K1C) procedure was performed on seven adult rabbits. After 1 month, mean blood pressure increased 50% and mean heart mass increased 30%. Coronary blood flow and cardiac output at rest were similar in control and 1K1C hearts; cardiac output fell 40% when 1K1C hearts were paced to 35% above basal heart rate. Capillary density in the left ventricular free wall (LV) decreased with increasing fiber size by as much as 30%. However, capillary-to-fiber ratio and total capillary length in the LV increased with heart size by up to 30% and 80%, respectively. This indicated that there was some proliferation of capillaries taking place, but not enough in comparison to fiber growth to prevent the lengthening of distances between capillaries. Mitochondrial volume density decreased by as much as 30% with increasing heart size, but total mitochondrial volume increased up to 80%. This indicated that there was some proliferation of mitochondria, but not enough to prevent dilution of mitochondria by the growing myofibrillar elements. Analysis of the distribution of mitochondria suggested that the new mitochondrial material was added to the center of myofibers, thereby further lengthening oxygen diffusion distances. There was a constant ratio of 10.4 +/- 0.3 km of capillaries per ml of mitochondria in 1K1C and control hearts, demonstrating that the structures for oxygen supply and consumption were remaining in fixed proportion to each other. There was no evidence that the decreased performance of paced 1K1C hearts was attributable to an oxygen diffusion limitation to mitochondria.

Estimating transit time for capillary blood in selected muscles of exercising animals.

The mean minimal capillary transit time was estimated in muscles of various animals using a combination of physiological and morphometric methods. Radioactive microspheres were injected intravascularly in various animals running on a treadmill at maximum oxygen consumption rate (VO2,max) to label blood flow to individual muscles. The muscles were then removed and preserved by standard methods for electron microscopy. The volume density of mitochondria was measured to assess muscle oxidative capacity. Capillary densities in muscle cross-sections, capillary diameters and tortuosities were incorporated into an estimate of capillary volume per unit muscle mass. Mean capillary transit time (tc) in the exercising muscles was estimated by dividing mass-specific capillary volume by mass-specific blood flow. Estimates of tc ranged from values near 1 s in horse heart and thigh muscles to 0.2 s in duck gastrocnemius. The relationship between muscle blood flow and tc was hyperbolic. The experimental data indicate a limiting value of 0.2 s for transit times at very high blood flows. There was no correlation between tc and body-mass-specific VO2,max.

Capillary recruitment and heterogeneity of perfused capillary distribution in dog myocardium.

To estimate functional diffusion distances, the distribution of perfused capillaries was calculated in dog myocardium. A fluorescent dye was injected via a femoral vein in 6 anesthetized, open-chest dogs, and passed once through the coronary circulation (23 +/- 3 s). In 6 animals the dye circulated 4-20 min. In 6 animals the dye circulated for one pass following 2-3 min of asphyxia. The heart was then removed and frozen. Frozen sections from the left ventricule were cut, illuminated to excite the dye to fluoresce, and photographed. They were then stained by silver methenamine to mark all capillaries. The density of all capillaries was compared to that of capillaries containing fluorescent label. The distributions of capillaries were estimated by morphometry. In one pass of the normoxic coronary circulation, 66(+/- 4 SE)% of subepicardial and 60(+/- 4)% of subendocardial capillaries were detectably labeled. Their distribution approached a random pattern, and maximal distances to the nearest labeled capillary were lengthened by 50% compared to all capillaries. With multiple passes of the dye, or with asphyxia 76-79% of the capillaries were detectably labeled and their distribution approached the ordered pattern of the total capillary bed. We speculated that the unlabeled capillaries represented a spatially heterogeneous blood flow reserve.

Rat soleus muscle ultrastructure after hindlimb suspension.

The aim of the present investigation was to determine, by quantitative electron microscopy, the effects of a 5-wk tail-suspension period on rat soleus muscle ultrastructure. A marked decline (-60%) in muscle mass occurred. The mean fiber cross-sectional area decreased to a greater extent (-75%) than the capillary-to-fiber ratio (-37%), leading to a higher capillary density (+148%) after hypokinesia. The total mitochondrial volume density remained unchanged, whereas the volume density of myofibrils was slightly but significantly reduced (-6%). A shift from subsarcolemmal to interfibrillar mitochondria occurred. Interfibrillar mitochondrial volume density was highest near the fiber border and decreased toward the fiber center. An increase in volume density of satellite cells suggested muscle regenerative events. Soleus atrophy with tail suspension greatly decreases the muscular volume but leaves the ultrastructural composition of muscle fibers relatively unaffected.

[Muscle structure and aerobic performance capacity in recruit training school]. / Muskelstruktur und aerobe Leistungsfähigkeit im Verlaufe der Rekrutenschule.

The aerobic performance capacity (VO2 max) and muscle ultrastructural composition was analyzed in 18 subjects undergoing basic training in the Swiss army. Three groups were selected based on their sports activity. Group S contained subjects that had a previous systematic background in sports activities and trained regularly at least three times per week. A second group consisted of subjects that had no previous training and were subjected to an additional, individually adjusted endurance exercise (three times per week) for the first 8 weeks of their service period (group T). The control group (C) had no previous training and followed only the regular military duties. VO2 max was found to be significantly higher in group S at the beginning of the military service. However, VO2 max did not change significantly during the service period in any of the groups, Muscle mitochondria showed a significant change (+19%) only in group T. Heart rate recordings indicated that despite "strenuous" military activity, heart rates rarely reached levels sufficient for an increase in aerobic performance capacity.

Morphological adaptations of human skeletal muscle to chronic hypoxia.

Muscle structural changes during typical mountaineering expeditions to the Himalayas were assessed by taking muscle biopsies from 14 mountaineers before and after their sojourn at high altitude (greater than 5000 m for over 8 weeks). M. vastus lateralis samples were analyzed morphometrically from electron micrographs. A significant reduction (-10%) of muscle cross-sectional area was found on CT scans of the thigh. Morphologically this loss in muscle mass appeared as a decrease in muscle fiber size mainly due to a loss of myofibrillar proteins. A loss of muscle oxidative capacity was also evident, as indicated by a decrease in the volume of muscle mitochondria (-25%). In contrast, the capillary network was mostly spared from catabolism. It is therefore concluded that oxygen availability to muscle mitochondria after prolonged high-altitude exposure in humans is improved due to an unchanged capillary network, supplying a reduced muscle oxidative capacity.

Oxygen transport during exercise in large mammals. I. Adaptive variation in oxygen demand.

This study investigated mechanisms used by horses and steers to increase O2 uptake and delivery (VO2) from resting to maximal rates and identified the mechanisms that enable horses to achieve higher maximal rates of O2 consumption (VO2max) than steers. VO2 and circulatory variables were measured while Standardbred trotting horses and steers (450-kg body mass) stood quietly and ran on a treadmill at speeds up to those eliciting VO2max. As VO2 increased in both species, heart rate and circulating hemoglobin (Hb) concentration increased, thereby increasing O2 delivery by the circulation, while cardiac stroke volume remained unchanged. At VO2max arterial PCO2 increased from its resting value in horses but was unchanged in steers, and arterial PO2 decreased in both species. Although the horses hypoventilated and were hypoxemic at VO2max, no significant decrease in arterial Hb saturation occurred. VO2max of the horses was 2.6 times higher than that of the steers and was associated with a 100% larger cardiac output, 100% larger stroke volume, and 40% higher Hb concentration, whereas heart rates at VO2max were identical in the two species. The higher cardiac output of the horses at VO2max resulted from a 1.2-fold higher mean arterial pressure and 1.6-fold lower peripheral tissue resistance (associated with a larger skeletal muscle capillary bed). Both the magnitude of the difference in VO2max between horses and steers and the mechanisms used to achieve it are the same as observed in smaller pairs of mammalian species with large variation in aerobic capacity.

Oxidative capacity of muscle and mitochondria: correlation of physiological, biochemical, and morphometric characteristics.

The oxidative capacity of cat skeletal muscles (soleus, gracilis, and gracilis chronically stimulated for 28 days) was derived from the total mitochondrial content in the muscle, the surface area of mitochondrial inner membranes, and respiratory activities of isolated mitochondria. Mitochondrial content was estimated by standard morphometry. The surface area of mitochondrial inner membranes per unit volume of mitochondria was estimated by a stereological method. The respiratory activities of isolated mitochondria were measured biochemically, using pyruvate/malate, glutamate/malate, succinate, or cytochrome c as substrate. Structurally and functionally, mitochondria from the three muscle types showed nearly identical characteristics. Oxidative activity was dependent on substrate; with succinate, 5.8 ml of O2 per min per ml of mitochondria was the rate most likely to represent physiological conditions. Oxidative activities of 3.1 ml.min-1.ml-1 with pyruvate/malate and 14.5 ml.min-1.ml-1 with cytochrome c as substrates were theoretical lower and upper bounds. The oxidative capacity of each of the three muscles was thus in direct proportion to the total volume of mitochondria in the muscle. The respiratory capacity of isolated mitochondria was very near to the maximal oxygen uptake rate of mitochondria that is commonly estimated in intact muscles of a wide variety of animals.