Stability of a Novel PEGylation Site on a Putative Haemoglobin-Based Oxygen Carrier.

PEGylation of protein sulfhydryl residues is a common method used to create a stable drug conjugate to enhance vascular retention times. We recently created a putative haemoglobin-based oxygen carrier using maleimide-PEG to selectively modify a single engineered cysteine residue in the α subunit (αAla19Cys). However, maleimide-PEG adducts are subject to deconjugation via retro-Michael reactions, with consequent cross-conjugation to endogenous plasma thiols such as those found on human serum albumin or glutathione. In previous studies mono-sulfone-PEG adducts have been shown to be less susceptible to deconjugation. We therefore compared the stability of our maleimide-PEG Hb adduct with one created using a mono-sulfone PEG. The corresponding mono-sulfone-PEG adduct was significantly more stable when incubated at 37 °C for 7 days in the presence of 1 mM reduced glutathione, 20 mg/mL human serum albumin, or human serum. In all cases haemoglobin treated with mono-sulfone-PEG retained >90% of its conjugation whereas maleimide-PEG showed significant deconjugation, especially in the presence of 1 mM reduced glutathione where <70% of the maleimide-PEG conjugate remained intact. Although maleimide-PEGylation of Hb seems adequate for an oxygen therapeutic intended for acute use, if longer vascular retention is required reagents such as mono-sulfone-PEG may be more appropriate.

Effect of acute exposure to high ambient temperature on the thermal, metabolic and hygric physiology of a small desert bird.

The intensity and frequency of extreme weather events, such as heat waves, are increasing as a consequence of global warming. Acute periods of extreme heat can be more problematic for wildlife than a chronic increase in mean temperature, to which animals can potentially acclimatise. Predicting effects of heat exposure requires a clear understanding of the capacity of individuals to respond to heat waves, so we examined the physiological response of a small desert bird, the zebra finch (Taeniopygia guttata), after acute previous exposure to high ambient temperature, simulating heatwave-like conditions. The standard physiology of the zebra finches was unaffected by prior exposure to heatwave-type conditions, suggesting that periodic exposure to heatwaves is unlikely to impact their longer-term day-to-day energy and water requirements. When finches were thermally challenged, prior experience of heatwave-like conditions did not impact overall body temperature and evaporative water loss, but birds previously experiencing high temperatures did reduce their metabolic heat production, and the variance in water loss and metabolism between individuals was significantly lower. This suggests that some individuals are more likely to become dehydrated if they have not had prior experience of high temperatures, and do not prioritise water conservation over thermoregulation. However, our observations overall suggest that acute periods of heat exposure do little to modify the general physiology of small birds, supporting the hypothesis that periodic extreme heat events may be more problematic for them than chronic warming.

Near Infrared Spectroscopy (NIRS) Observation of Vastus Lateralis (Muscle) and Prefrontal Cortex (Brain) Tissue Oxygenation During Synchronised Swimming Routines in Elite Athletes.

The development of underwater Near-Infrared Spectroscopy (uNIRS) has enabled the measurement of tissue oxygenation within the swim environment. Unique physiological responses, such as the diving reflex, have been shown to occur during synchronized swimming and demonstrate an innate oxygen-conserving reflex. However, the prevalence of a sudden loss of consciousness ('hypoxic blackout') is an ongoing concern in this swim population. The purpose of this study was to investigate the reported low tissue oxygen conditions experienced in elite level synchronized swimmers (SyncS) during swim routines. Changes in peripheral muscle and brain oxygenation (Tissue Saturation Index (TSI %)) were continuously recorded during simulated synchronized swim routines. Six elite female synchronized swimmers were assessed; age 29.0 ± 4.4 years; height 168.4 ± 7.1 cm; weight 53.2 ± 3.2 kg; quadriceps skin fold; 10.2 ± 0.8 mm; ΔTSI (%) between the vastus lateralis (VL) and prefrontal cortex (PFC) were analyzed using paired (two-tailed) t-tests. The level of significance for analysis was set at p < 0.05. Significant difference (p = 0.001) was found in ΔTSI (%) between the VL and PFC. During dynamic leg kicking exercise, the initial effect of each leg kicking sequence is a rapid drop in TSI (%). This is consistent with an initial constriction (drop in blood flow in the muscle) accompanied by an increase in oxygen consumption. Cerebral oxygenation (PFC) remained largely unchanged during both maximal breath-hold and during vigorous exercise, presumably due to protective mechanisms in the brain in this population. We conclude that uNIRS is able to provide novel insights into SyncS hemodynamic responses and could be used to inform on the safety of new routines.

Novel Redox Active Tyrosine Mutations Enhance the Regeneration of Functional Oxyhemoglobin from Methemoglobin: Implications for Design of Blood Substitutes.

Heme mediated oxidative toxicity has been linked to adverse side effects in Hemoglobin Based Oxygen Carriers (HBOC), initiated by reactive ferryl (FeIV) iron and globin based free radical species. We recently showed that the addition of a redox active tyrosine residue in the beta subunit (ßF41Y) of recombinant hemoglobin had the capability to decrease lipid peroxidation by facilitating the reduction of FeIV iron by plasma antioxidants such as ascorbate. In order to explore this functionality further we created a suite of tyrosine mutants designed to be accessible for both reductant access at the protein surface, yet close enough to the heme cofactor to enable efficient electron transfer to the FeIV. The residues chosen were: ßF41Y; ßK66Y; ßF71Y; ßT84Y; ßF85Y; and ßL96Y. As with ßF41Y, all mutants significantly enhanced the rate of ferryl (FeIV) to ferric (FeIII) reduction by ascorbate. However, surprisingly a subset of these mutations (ßT84Y, and ßF85Y) also enhanced the further reduction of ferric (FeIII) to ferrous (FeII) heme, regenerating functional oxyhemoglobin. The largest increase was seen in ßT84Y with the percentage of oxyhemoglobin formed from ferric hemoglobin in the presence of 100 µM ascorbate over a time period of 60 min increasing from 10% in ßF41Y to over 50% in ßT84Y. This increase was accompanied by an increased rate of ascorbate consumption. We conclude that the insertion of novel redox active tyrosine residues may be a useful component of any recombinant HBOC designed for longer functional activity without oxidative side effects.

Can birds do it too? Evidence for convergence in evaporative water loss regulation for birds and mammals.

Birds have many physiological characteristics that are convergent with mammals. In the light of recent evidence that mammals can maintain a constant insensible evaporative water loss (EWL) over a range of perturbing environmental conditions, we hypothesized that birds might also regulate insensible EWL, reflecting this convergence. We found that budgerigars (Melopsittacus undulatus) maintain EWL constant over a range of relative humidities at three ambient temperatures. EWL, expressed as a function of water vapour pressure deficit, differed from a physical model where the water vapour pressure deficit between the animal and the ambient air is the driver of evaporation, indicating physiological control of EWL. Regulating EWL avoids thermoregulatory impacts of varied evaporative heat loss; changes in relative humidity had no effect on body temperature, metabolic rate or thermal conductance. Our findings that a small bird can regulate EWL are evidence that this is a common feature of convergently endothermic birds and mammals, and may therefore be a fundamental characteristic of endothermy.

Reexamining Echidna Physiology: The Big Picture for Tachyglossus aculeatus acanthion.

The early divergence of monotremes and therian mammals has resulted in considerable interest in the comparative physiology of the short-beaked echidna (Tachyglossus aculeatus), the most common and widespread living monotreme. However, there are many and varied interpretations of its physiology, reflecting the many and varied studies, limitations and uncertainties of aspects of some previous studies, and potential differences between the various subspecies. Consequently, we thoroughly examine here the standardized physiology of the most widely distributed subspecies of short-beaked echidna (T. aculeatus acanthion) over a wide range of ambient temperatures to definitively assess its physiology in a comparative context. We conclude that the low and variable body temperature of the short-beaked echidna is physiologically "primitive," but it also reflects adaptation to its myrmecophagous niche. Other aspects of its physiology are more typically mammalian. A low metabolic rate reflects its low body temperature, and ventilatory variables are matched to accommodate a modest gas exchange requirement. Thermal conductance is typical for a mammal of equivalent mass. In contrast to previous studies, we demonstrate that short-beaked echidnas can enhance evaporative water loss above thermoneutrality, like other mammals, with a similar capacity for evaporative heat loss. Cooling of their nasal blood sinus with nasal mucous may contribute to this enhanced evaporative cooling. Their capacity to evaporatively cool explains how their distribution can include habitats where ambient temperature, even in shelters, exceeds their supposed critical thermal limit.

Underwater Near-Infrared Spectroscopy: Muscle Oxygen Changes in the Upper and Lower Extremities in Club Level Swimmers and Triathletes.

To date, measurements of oxygen status during swim exercise have focused upon systemic aerobic capacity. The development of a portable, waterproof NIRS device makes possible a local measurement of muscle hemodynamics and oxygenation that could provide a novel insight into the physiological changes that occur during swim exercise. The purpose of this study was to observe changes in muscle oxygenation in the vastus lateralis (VL) and latissimus dorsi (LD) of club level swimmers and triathletes. Ten subjects, five club level swimmers and five club level triathletes (three men and seven women) were used for assessment. Swim group; mean±SD=age 21.2±1.6 years; height 170.6±7.5 cm; weight 62.8±6.9 kg; vastus lateralis skin fold 13.8±5.6 mm; latissimus dorsi skin fold 12.6±3.7. Triathlete group; mean±SD=age 44.0±10.5 years; height 171.6±7.0 cm; weight 68.6±12.7 kg; vastus lateralis skin fold 11.8±3.5 mm; latissimus dorsi skin fold 11.2±3.1. All subjects completed a maximal 200 m freestyle swim, with the PortaMon, a portable NIR device, attached to the subject's dominant side musculature. ΔTSI% between the vastus lateralis and latissimus dorsi were analysed using either paired (2-tailed) t-tests or Wilcoxon signed rank test. The level of significance for analysis was set at p<0.05. No significant difference (p=0.686) was found in ΔTSI (%) between the VL and LD in club level swimmers. A significant difference (p=0.043) was found in ΔTSI (%) between the VL and LD in club level triathletes. Club level swimmers completed the 200 m freestyle swim significantly faster (p=0.04) than club level triathletes. Club level swimmers use both the upper and lower muscles to a similar extent during a maximal 200 m swim. Club level triathletes predominately use the upper body for propulsion during the same exercise. The data produced by NIRS in this study are the first of their kind and provide insight into muscle oxygenation changes during swim exercise which can indicate the contribution of one muscle compared to another. This also enables a greater understanding of the differences in swimming techniques seen between different cohorts of swimmers and potentially within individual swimmers.

The ßLys66Tyr Variant of Human Hemoglobin as a Component of a Blood Substitute.

It has been proposed that introducing tyrosine residues into human hemoglobin (e.g. ßPhe41Tyr) may be able to reduce the toxicity of the ferryl heme species in extracellular hemoglobin-based oxygen carriers (HBOC) by facilitating long-range electron transfer from endogenous and exogenous antioxidants. Surface-exposed residues lying close to the solvent exposed heme edge may be good candidates for mutations. We therefore studied the properties of the ßLys66Tyr mutation. Hydrogen peroxide (H2O2) was added to generate the ferryl protein. The ferryl state in ßLys66Tyr was more rapidly reduced to ferric (met) by ascorbate than recombinant wild type (rwt) or ßPhe41Tyr. However, ßLys66Tyr suffered more heme and globin damage following H2O2 addition as measured by UV/visible spectroscopy and HPLC analysis. ßLys66Tyr differed notably from the rwt protein in other ways. In the ferrous state the ßLys66Tyr forms oxy, CO, and NO bound heme complexes similar to rwt. However, the kinetics of CO binding to the mutant was faster than rwt, suggesting a more open heme crevice. In the ferric (met) form the typical met Hb acid-alkaline transition (H2O to -OH) appeared absent in the mutant protein. A biphasicity of cyanide binding was also evident. Expression in E. coli of the ßLys66Tyr mutant was lower than the rwt protein, and purification included significant protein heterogeneity. Whilst, ßLys66Tyr and rwt autoxidised (oxy to met) at similar rates, the oxygen p50 for ßLys66Tyr was very low. Therefore, despite the apparent introduction of a new electron transfer pathway in the ßLys66Tyr mutant, the heterogeneity, and susceptibility to oxidative damage argue against this mutant as a suitable starting material for a HBOC.

How do measurement duration and timing interact to influence estimation of basal physiological variables of a nocturnal rodent?

Metabolic rate and evaporative water loss are two commonly measured physiological variables. It is therefore important, especially for comparative studies, that these variables (and others) are measured under standardised conditions, of which a resting state during the inactive phase is part of the accepted criteria. Here we show how measurement duration and timing affect these criteria and impact on the estimation of basal metabolic rate (oxygen consumption and carbon dioxide production) and standard evaporative water loss of a small nocturnal rodent. Oxygen consumption, carbon dioxide production and evaporative water loss all decreased over the duration of an experiment. Random assortment of hourly values indicated that this was an animal rather than a random effect for up to 11h. Experimental start time also had a significant effect on measurement of physiological variables. A longer time period was required to achieve minimal carbon dioxide consumption and evaporative water loss when experiments commenced earlier in the day; however, experiments with earlier start times had a lower overall estimates of minimal oxygen consumption and carbon dioxide production. For this species, measurement duration of at least 8h, ideally commencing between before the inactive phase at 03:00h and 05:00h, is required to obtain minimal standard values for physiological variables. Up to 80% of recently published studies measuring basal metabolic rate and/or evaporative water loss of small nocturnal mammals may overestimate basal values due to insufficiently long measurement duration.

Use of NIRS to assess effect of training on peripheral muscle oxygenation changes in elite rugby players performing repeated supramaximal cycling tests.

In most team sports, intermittent high intensity sprint efforts combined with short recovery periods have been identified as a key factor of physical performance; the ability to repeat these efforts at a sustained level is of great importance. Near-infrared spectroscopy (NIRS) has been proposed as a tool to monitor muscle oxygenation changes during such sprint efforts. The purpose of this study was to observe muscle reoxygenation rate (reoxy rate) (% s⁻¹) between sprint efforts in a repeat sprint cycle test. A two wavelength spatially resolved NIR spectrometer (Portamon, Artinis Inc.) was used to assess reoxy rate changes in the vastus lateralis of the dominant leg before and after a training stimulus. Eight UK premiership academy level rugby players were assessed (age 20.6 ± 0.9) years; height 187 ± 0.6 cm; weight 109.5 ± 8.6 kg; quadriceps skin fold 16.6 ± 4.5 mm); the subjects completed ten repeated 10-s cycle sprints interspersed with 40 s recovery, upon a Wattbike Pro cycle. Hemoglobin variables (ΔHHb, ΔtHb, ΔO2Hb, ΔTSI %) during the sprint and the post-sprint reoxygenation rate (%TSI s⁻¹) were measured. During both cycle tests all subjects experienced a drop in muscle oxygen saturation (Pre-Δ - 12.39 ± 6.01 %), Post-Δ - 14.83 ± 3.88 %). Post-training, there was an increase in the extent of desaturation (drop in TSI %) in the group means, both for the biggest single change and the average of all ten changes. Seven out of eight players showed an increase based on the maximum change and six based on the average of their ten tests. Additionally, seven out of eight players showed a significant increase in ΔHHb (Pre-Δ + 76.80 ± 61.92, Post-Δ + 121.28 ± 69.76) (p < 0.01) (including the one player who did not show a significant effect on the TSI measure). Players who exercised at the highest power tended to decrease their muscle oxygenation to a greater extent. The number of bike training sessions undertaken correlated with improvements in post-exercise recovery of oxygenation (R = 0.63). The simplest explanation for the increase in desaturation following training is an increase in muscle oxygen consumption due to an increase in mitochondrial content. This results in an increased extraction of delivered oxygen as confirmed by the HHb data. In conclusion, NIRS is able to measure positive training effects on muscle oxygen extraction, at the level of the individual elite athlete.

Energy and water use by invasive goats (Capra hircus) in an Australian rangeland, and a caution against using broad-scale allometry to predict species-specific requirements.

Feral goats (Capra hircus) are ubiquitous across much of Australia's arid and semi-arid rangelands, where they compete with domestic stock, contribute to grazing pressure on fragile ecosystems, and have been implicated in the decline of several native marsupial herbivores. Understanding the success of feral goats in Australia may provide insights into management strategies for this and other invasive herbivores. It has been suggested that frugal use of energy and water contributes to the success of feral goats in Australia, but data on the energy and water use of free-ranging animals are lacking. We measured the field metabolic rate and water turnover rate of pregnant and non-pregnant feral goats in an Australian rangeland during late summer (dry season). Field metabolic rate of pregnant goats (601 ± 37 kJ kg(-0.73)d(-1)) was 1.3 times that of non-pregnant goats (456 ± 24 kJ kg(-0.73)d(-1)). The water turnover rate of pregnant goats (228 ± 18 mL kg(-0.79)d(-1)) was also 1.3 times that of non-pregnant goats (173 ± 18 kg(-0.79)d(-1)), but the difference was not significant (P=0.07). There was no significant difference in estimated dry matter digestibility between pregnant and non-pregnant goats (mean ca. 58%), blood or urine osmolality, or urine electrolyte concentrations, indicating they were probably eating similar diets and were able to maintain osmohomeostasis. Overall, the metabolic and hygric physiology of non-pregnant goats conformed statistically to the predictions for non-marine, non-reproductive placental mammals according to both conventional and phylogenetically independent analyses. That was despite the field metabolic rate and estimated dry matter intake of non-pregnant goats being only 60% of the predicted level. We suggest that general allometric analyses predict the range of adaptive possibilities for mammals, but that specific adaptations, as present in goats, result in ecologically significant departures from the average allometric curve. In the case of goats in the arid Australian rangelands, predictions from the allometric regression would overestimate their grazing pressure by about 40% with implications for the predicted impact on their local ecology.

Environmental physiology of a small marsupial inhabiting arid floodplains.

Giles' planigale (Planigale gilesi) is among the smallest extant marsupials and inhabits deep soil cracks in arid floodplains. We examined whether its physiology shows specific adaptations to its extreme habitat. Metabolic rate, body temperature, evaporative water loss and thermal conductance were measured for eight planigales (average mass 9 g) exposed to four different ambient temperatures ranging from 10 degrees C to 32 degrees C. Water economy and respiratory variables were measured for the first time in this species. All of these standard physiological variables conformed to allometrically-predicted values for a marsupial. All variables were significantly affected by ambient temperature, except tidal volume and dry thermal conductance. Metabolic rate increased substantially at low ambient temperatures, as required to maintain a relatively constant body temperature of about 32-34 degrees C. This increased oxygen demand was accommodated by increased ventilation rather than increased oxygen extraction. Planigales had a comparatively high point of relative water economy of 19.1 degrees C, consistent with their small body size and arid habitat. Torpor reduced energy expenditure by 79% and evaporative water loss by 62%. Our study suggests that torpor use, along with behavioural adaptations, suffice for P. gilesi to live underground in arid habitats without further physiological adaptations.

Effect of sampling regime on estimation of basal metabolic rate and standard evaporative water loss using flow-through respirometry.

Strict criteria have been established for measurement of basal metabolic rate and standard evaporative water loss to ensure that data can be compared intra- and interspecifically. However, data-sampling regimes vary, from essentially continuous sampling to interrupted (switching) systems with data recorded periodically at more widely spaced intervals. Here we compare one continuous and three interrupted sampling regimes to determine whether sampling regime has a significant effect on estimation of basal metabolic rate or standard evaporative water loss. Compared to continuous 20-s sampling averaged over 20 min, sampling every 6 min and averaging over 60 min overestimated basal metabolic rate and evaporative water loss, sampling every 3 min and averaging over 21 min underestimated basal metabolic rate, and sampling every 12 min and averaging over 36 min showed no difference in estimates. Increasing the period over which the minimum mean was calculated significantly increased estimates of physiological variables. Reducing the frequency of sampling from 20 s to a longer interval of 3, 6, or 12 min underestimated basal metabolic rate but not evaporative water loss. This indicates that sampling frequency per se influences estimates of basal metabolic rate and that differences are not just an artifact of differences in the period over which the mean is calculated. Sampling regime can have a highly significant influence on estimation of standard physiological variables, although the actual differences between sampling regimes were generally small (usually <5%). Although continuous sampling is the preferred sampling regime for open-flow respirometry studies, if time and cost are prohibitive, then use of an appropriate switching system will result in smaller errors than measuring individuals continuously for shorter periods.

Effects of measurement duration on the determination of basal metabolic rate and evaporative water loss of small marsupials: how long is long enough?

We examined the time course for measurement of basal metabolic rate (BMR; measured as O(2) consumption and CO(2) production) and standard evaporative water loss (EWL) for six species of small marsupial to determine the minimum time required to achieve basal/standard values. There was a highly significant effect of measurement duration on measured physiological variables with values for O(2) consumption, CO(2) production, and EWL decreasing with time for all species. The time required to attain values statistically indistinguishable from minimal differed significantly between species, but in general O(2) consumption rate reached basal values after 4.3 h, CO(2) production after 4.5 h, and evaporative water loss after 5.2 h. For 16 BMR measurements of small marsupial species in the literature, with experimental duration provided, 10 were for less than 4 h, suggesting that their BMR values might be overestimates. For EWL, three of the four published values for small marsupials may be overestimates. It is clear that appropriate experimental duration is an important component of the measurement protocol for both BMR and standardized water loss, which needs to be rigorously observed in future studies.

Metabolic, ventilatory and hygric physiology of the chuditch (Dasyurus geoffroii; Marsupialia, Dasyuridae).

The chuditch is a large carnivorous dasyurid marsupial. Historically it had one of the widest geographical distributions of all marsupials, encompassing much of arid Australia, but it is now restricted to the mesic south-west of Western Australia. It is therefore of interest to determine if its physiology better reflects adaptation to its historically arid or present mesic habitat. The basic physiological parameters of the chuditch conform to other marsupials. Body mass of males (1385 g) was >400% of that predicted by phylogeny and this may be related to its carnivorous diet. Body temperature was 33.9 degrees C at ambient temperatures < or = thermoneutrality, with hyperthermia occurring above thermoneutrality. Basal metabolic rate was 0.361 mL O(2) g(-1) h(-1) at an ambient temperature of 31 degrees C. Metabolic rate increased below the thermoneutral zone by 0.038 mL O(2) g(-1) h(-1) degrees C(-1), and above the thermoneutral zone to 0.444+/-0.059 mL O(2) g(-1) h(-1) at 33.3 degrees C. Standard evaporative water loss was 0.498+/-0.071 mg g(-1) h(-1) at an ambient temperature of 26.0 degrees C, and increased at higher ambient temperatures due to panting and licking. Changes in wet thermal conductance largely reflected changes in evaporative heat loss, and dry thermal conductance increased at high ambient temperature due in part to posture change. Ventilatory parameters were consistent with metabolic demands in and below thermoneutrality, and suggested augmented evaporative heat loss above the thermoneutral zone. Chuditch had a high point of relative water economy of 22.6 degrees C, indicating favourable water economy at even moderate ambient temperatures, due to its low evaporative water loss rather than high metabolic water production. Chuditch were physiologically more similar to marsupials from arid rather than mesic habitats, better reflecting their historical distribution than their current geographical range.

Metabolic, ventilatory, and hygric physiology of the gracile mouse opossum (Gracilinanus agilis).

We present the first complete study of basic laboratory-measured physiological variables (metabolism, thermoregulation, evaporative water loss, and ventilation) for a South American marsupial, the gracile mouse opossum (Gracilinanus agilis). Body temperature (T(b)) was thermolabile below thermoneutrality (T(b) = 33.5 degrees C), but a substantial gradient between T(b) and ambient temperature (T(a)) was sustained even at T(a) = 12 degrees C (T(b) = 30.6 degrees C). Basal metabolic rate of 1.00 mL O2 g(-1) h(-1) at T(a) = 30 degrees C conformed to the general allometric relationship for marsupials, as did wet thermal conductance (5.7 mL O2 g(-1) h(-1) degrees C(-1)). Respiratory rate, tidal volume, and minute volume at thermoneutrality matched metabolic demand such that O2 extraction was 12.4%, and ventilation increased in proportion to metabolic rate at low T(a). Ventilatory accommodation of increased metabolic rate at low T(a) was by an increase in respiratory rate rather than by tidal volume or O2 extraction. Evaporative water loss at the lower limit of thermoneutrality conformed to that of other marsupials. Relative water economy was negative at thermoneutrality but positive below T(a) = 12 degrees C. Interestingly, the Neotropical gracile mouse opossums have a more positive water economy at low T(a) than an Australian arid-zone marsupial, perhaps reflecting seasonal variation in water availability for the mouse opossum. Torpor occurred at low T(a), with spontaneous arousal when T(b) > 20 degrees C. Torpor resulted in absolute energy and water savings but lower relative water economy. We found no evidence that gracile mouse opossums differ physiologically from other marsupials, despite their Neotropical distribution, sympatry with placental mammals, and long period of separation from Australian marsupials.

Allometry of evaporative water loss in marsupials: implications of the effect of ambient relative humidity on the physiology of brushtail possums (Trichosurus vulpecula).

To better understand the effects of ambient relative humidity (RH) on physiological variables and the implications of RH-correcting evaporative water loss (EWL) data for marsupials, we examined the effect of RH on EWL, body temperature (Tb), metabolic rate (MR) and thermal conductance (C) of the brushtail possum (Trichosurus vulpecula), a medium-sized marsupial. Correcting EWL data for 27 species of marsupial for water vapour pressure deficit (DeltaWVP) in the chamber during measurement significantly increased, rather than decreased, the variability of the allometric relationship for EWL. For the brushtail possum, both ambient temperature (Ta) and RH significantly affected EWL. At Ta=25 degrees C, EWL was independent of RH at "63% RH, but decreased linearly at higher RH values. At Ta=30 degrees C, EWL was significantly related to RH from 26% to 92% RH. There was a significant effect of Ta on Tb and dry thermal conductance (Cdry; higher at 30 degrees C), but no effect of RH. For MR and wet thermal conductance (Cwet) there was a significant effect of Ta (MR higher and Cwet lower at 25 degrees C), and RH at Ta=30 degrees C (MR higher and Cwet lower at the lowest RH) but not at 25 degrees C. Our results indicate that brushtail possums do not necessarily show the linear relationship between ambient RH and EWL expected for an endotherm, possibly because of behavioural modification of their immediate microclimate. This may account for the failure of WVP deficit correction to improve the allometric EWL relationship for marsupials. Chamber RH is an important environmental factor to be considered when measuring standard physiological variables such as MR and Cwet.

Investigation of in vivo measurement of cerebral cytochrome-c-oxidase redox changes using near-infrared spectroscopy in patients with orthostatic hypotension.

We have previously used a continuous four-wavelength near-infrared spectrometer to measure changes in the cerebral concentrations of oxy-haemoglobin (Delta[HbO(2)] and deoxy-haemoglobin (Delta[HHb]) during head-up tilt in patients with primary autonomic failure. The measured changes in light attenuation also allow calculation of changes in the concentration of oxidized cytochrome-c-oxidase (Delta[(ox)CCO]), and this paper analyses the Delta[(ox)CCO] during the severe episodes of orthostatic hypotension produced by this experimental protocol. We studied 12 patients during a passive change in position from supine to a 60 degrees head-up tilt. The challenge caused a reduction in mean blood pressure of 59.93 (+/-26.12) mmHg (Mean (+/-SD), p < 0.0001), which was associated with a reduction in the total concentration of haemoglobin (Delta[HbT] = Delta[HbO(2)] + Delta[HHb]) of 5.02 (+/-3.81) microM (p < 0.0001) and a reduction in the haemoglobin difference concentration (Delta[Hb(diff)] = Delta[HbO(2)] - Delta[HHb]) of 14.4 (+/-6.73) microM (p < 0.0001). We observed a wide range of responses in Delta[(ox)CCO]. Six patients demonstrated a drop in Delta[(ox)CCO] (0.17 +/- 0.15 microM); four patients demonstrated no change (0.01 +/- 0.12 microM) and two patients showed an increase in Delta[(ox)CCO] (0.21 +/- 0.01 microM). Investigation of the association between the changes in concentrations of haemoglobin species and the Delta[(ox)CCO] for each patient show a range of relationships. This suggests that a simple mechanism for crosstalk, which might produce artefactual changes in [(ox)CCO], is not present between the haemoglobin and the (ox)CCO NIRS signals. Further investigation is required to determine the clinical significance of the changes in [(ox)CCO].

Environmental correlates of physiological variables in marsupials.

We analyzed body temperature (T(b)), basal metabolic rate (BMR), wet thermal conductance (C(wet)), and evaporative water loss (EWL) of marsupials by conventional and phylogenetically corrected regression. Allometric effects were substantial for BMR, C(wet), and EWL but not T(b). There was a strong phylogenetic signal for mass and all physiological traits. A significant phylogenetic signal remained for BMR, C(wet), and EWL even after accounting for the highly significant phylogenetic signal of mass. T(b), BMR, C(wet), and EWL allometric residuals were correlated with some diet, distribution, and climatic variables before and after correction for phylogeny. T(b) residuals were higher for marsupials from arid environments (high T(a) and more variable rainfall). The fossorial marsupial mole had a lower-than-expected T(b) residual. The allometric slope for BMR was 0.72-0.75. Residuals were consistently related to distribution aridity and rainfall variability, with species from arid and variable rainfall habitats having a low BMR, presumably to conserve energy in a low-productivity environment. The nectarivorous honey possum had a higher-than-expected BMR. For C(wet), the allometric slope was 0.55-0.62; residuals were related to diet, with folivores having low and insectivores high C(wet) residuals. The allometric slope for EWL was 0.68-0.73. EWL residuals were consistently correlated with rainfall variability, presumably facilitating maintenance of water balance during dry periods.

Numbats and aardwolves--how low is low? A re-affirmation of the need for statistical rigour in evaluating regression predictions.

Many comparative physiological studies aim to determine if a particular species differs from a prediction based on a linear allometric regression for other species. However, the judgment as to whether the species in question conforms to this allometric relationship is often not based on any formal statistical analysis. An appropriate statistical method is to compare the new species' value with the 95% confidence limits for predicting an additional datum from the relationship for the other species. We examine the basal metabolic rate (BMR) of the termitivorous numbat (Myrmecobius fasciatus) and aardwolf (Proteles cristatus) to demonstrate the use of the 95% prediction limits to determine statistically if they have a lower-than-expected BMR compared to related species. The numbat's BMR was 83.6% of expected from mass, but fell inside the 95% prediction limits for a further datum; a BMR < 72.5% of predicted was required to fall below the one-tail 95% prediction limits. The aardwolf had a BMR that was only 74.2% of predicted from the allometric equation, but it also fell well within the 95% prediction limits; a BMR of only 41.8% of predicted was necessary to fall below the one-tail 95% prediction limits. We conclude that a formal statistical approach is essential, although it is difficult to demonstrate that a single species statistically differs from a regression relationship for other species.