Getting the most from venous occlusion plethysmography: proposed methods for the analysis of data with a rest/exercise protocol

In this methodological manuscript, we suggest a more detailed approach to processing venous occlusion plethysmography data, one which could provide additional physiological information. Six parameters are described, all of which are easily derived from a simple and reproducible experimental rest/exercise venous occlusion plethysmography protoco

A comparison of the quality of image acquisition between the incident dark field and sidestream dark field video-microscopes

BACKGROUND: The 'Cytocam' is a third generation video-microscope, which enables real time visualisation of the in vivo microcirculation. Based upon the principle of incident dark field (IDF) illumination, this hand held computer-controlled device was designed to address the technical limitations of its predecessors, orthogonal polarization spectroscopy and sidestream dark field (SDF) imaging. In this manuscript, we aimed to compare the quality of sublingual microcirculatory image acquisition between the IDF and SDF devices. METHODS: Using the microcirculatory image quality scoring (MIQS) system, (six categories scored as either 0 = optimal, 1 = acceptable, or 10 = unacceptable), two independent raters compared 30 films acquired using the Cytocam IDF video-microscope, to an equal number obtained with an SDF device. Blinded to the origin of the films, the raters were therefore able to score between 0 and 60 for each film analysed. The scores' distributions between the two techniques were compared. RESULTS: The median MIQS (95 % CI) given to the SDF camera was 7 (1.5-12), as compared to 1 (0.5-1.0) for the IDF device (p < 0.0001). Of the six categories assessed by the MIQS, nearly one fifth of the SDF videos were scored as unacceptable for pressure (20 %), content (20 %), and stability (17 %), with focus scoring deficiently 13 % of the time. High agreement between the two raters scoring values was evident, with an intra-class correlation coefficient (ICC) of 0.96 (95 % CI: 0.94, 0.98). CONCLUSIONS: These results demonstrate that the quality of sublingual microcirculatory image acquisition is superior in the Cytocam IDF video-microscope, as compared to the SDF video-microscope

Design and conduct of Xtreme Everest 2: an observational cohort study of Sherpa and lowlander responses to graduated hypobaric hypoxia

Objective: oxygen availability falls with ascent to altitude and also as a consequence of critical illness. Because cellular sequelae and adaptive processes may be shared in both circumstances, high altitude exposure (‘physiological hypoxia’) assists in the exploration of the response to pathological hypoxia. We therefore studied the response of healthy participants to progressive hypobaric hypoxia at altitude. The primary objective of the study was to identify differences between high altitude inhabitants (Sherpas) and lowland comparators.Methods: we performed an observational cohort study of human responses to progressive hypobaric hypoxia (during ascent) and subsequent normoxia (following descent) comparing Sherpas with lowlanders. Studies were conducted in London (35m), Kathmandu (1300m), Namche Bazaar (3500m) and Everest Base Camp (5300m). Of 180 healthy volunteers departing from Kathmandu, 64 were Sherpas and 116 were lowlanders. Physiological, biochemical, genetic and epigenetic data were collected. Core studies focused on nitric oxide metabolism, microcirculatory blood flow and exercise performance. Additional studies performed in nested subgroups examined mitochondrial and metabolic function, and ventilatory and cardiac variables. Of the 180 healthy participants who left Kathmandu, 178 (99%) completed the planned trek. Overall, more than 90% of planned testing was completed. Forty-four study protocols were successfully completed at altitudes up to and including 5300m. A subgroup of identical twins (all lowlanders) was also studied in detail.Conclusion: this programme of study (Xtreme Everest 2) will provide a rich dataset relating to human adaptation to hypoxia, and the responses seen on re-exposure to normoxia. It is the largest comprehensive high altitude study of Sherpas yet performed. Translational data generated from this study will be of relevance to diseases in which oxygenation is a major facto

Effects of dietary nitrate supplementation on symptoms of acute mountain sickness and basic physiological responses in a group of male adolescents during ascent to Mount Everest Base Camp

The purpose of this study was to investigate the effects of dietary nitrate supplementation, in the form of beetroot juice, on acute mountain sickness (AMS) symptoms and physiological responses, in a group of young males trekking to Mount Everest Base Camp (EBC). Forty healthy male students (mean age (SD): 16 (1) yrs) trekked to EBC over 11 days. Following an overnight fast, each morning participants completed the Lake Louise AMS questionnaire and underwent a series of physiological tests: resting blood pressure as well as resting and exercising heart rate, respiratory rate, and peripheral oxygen saturation. The exercise test consisted of a standardised 2-minute stepping protocol and measurements were taken in the last 10 seconds. Participants in the intervention arm of the study consumed 140 ml of concentrated beetroot juice daily, containing approximately 10 mmoles of nitrate, while those in the control arm consumed 140 ml of concentrated blackcurrant cordial with negligible nitrate content. Drinks were taken for the first seven days at high altitude (days 2 to 8), in two equal doses; one with breakfast, and one with the evening meal. Mixed modelling revealed no significant between-groups difference in the incidence of AMS (Odds Rationitrate vs. control: 1.16 (95% CI: 0.59; 2.29)). Physiological changes occurring during ascent to high altitude generally were not significantly different between the two groups (Model Coef (95% CI) – average difference nitrate vs. control: systolic blood pressure, 0.16 (-4.47; 4.79); peripheral oxygen saturation, 0.28 (-0.85; 1.41); heart rate, -0.48 (-8.47; 7.50) (Model Coef (95% CI) – relative difference nitrate vs. control: ventilatory rate, 0.95 (0.82; 1.08)). Modelling revealed that diastolic blood pressure was 3.37 mmHg (0.24; 6.49) higher for participants in the beetroot juice, however this difference was no larger than that found at baseline and no interaction effect was observed. Supplementation with dietary nitrate did not significantly change symptoms of AMS or alter key physiological variables, in a group of adolescent males during a high altitude trekking expedition. There was no evidence of harm from dietary nitrate supplementation in this context. Given the wide confidence intervals in all models, a larger sample size would be required to exclude a false negative result. Our data suggest that prolonged oral nitrate supplementation is safe and feasible at altitude but has little physiological or clinical effect

Design and conduct of 'Xtreme Alps' : a double-blind, randomised controlled study of the effects of dietary nitrate supplementation on acclimatisation to high altitude

The study of healthy human volunteers ascending to high altitude provides a robust model of the complex physiological interplay that emulates human adaptation to hypoxaemia in clinical conditions. Nitric oxide (NO) metabolism may play an important role in both adaptation to high altitude and response to hypoxaemia during critical illness at sea level. Circulating nitrate and nitrite concentrations can be augmented by dietary supplementation and this is associated with improved exercise performance and mitochondrial efficiency. We hypothesised that the administration of a dietary substance (beetroot juice) rich in nitrate would improve oxygen efficiency during exercise at high altitude by enhancing tissue microcirculatory blood flow and oxygenation. Furthermore, nitrate supplementation would lead to measurable increases in NO bioactivity throughout the body. This methodological manuscript describes the design and conduct of the ‘Xtreme Alps’ expedition, a double-blind randomised controlled trial investigating the effects of dietary nitrate supplementation on acclimatisation to hypobaric hypoxia at high altitude in healthy human volunteers. The primary outcome measure was the change in oxygen efficiency during exercise at high altitude between participants allocated to receive nitrate supplementation and those receiving a placebo. A number of secondary measures were recorded, including exercise capacity, peripheral and microcirculatory blood flow and tissue oxygenation. Results from this study will further elucidate the role of NO in adaption to hypoxaemia and guide clinical trials in critically ill patients. Improved understanding of hypoxaemia in critical illness may provide new therapeutic avenues for interventions that will improve survival in critically ill patients

Physiological responses during ascent to high altitude and the incidence of acute mountain sickness.

Acute mountain sickness (AMS) occurs when there is failure of acclimatisation to high altitude. The aim of this study was to describe the relationship between physiological variables and the incidence of AMS during ascent to 5300 m. A total of 332 lowland-dwelling volunteers followed an identical ascent profile on staggered treks. Self-reported symptoms of AMS were recorded daily using the Lake Louise score (mild 3-4; moderate-severe ≥5), alongside measurements of physiological variables (heart rate, respiratory rate (RR), peripheral oxygen saturation (SpO2 ) and blood pressure) before and after a standardised Xtreme Everest Step-Test (XEST). The overall occurrence of AMS among participants was 73.5% (23.2% mild, 50.3% moderate-severe). There was no difference in gender, age, previous AMS, weight or body mass index between participants who developed AMS and those who did not. Participants who had not previously ascended >5000 m were more likely to get moderate-to-severe AMS. Participants who suffered moderate-to-severe AMS had a lower resting SpO2 at 3500 m (88.5 vs. 89.6%, p = 0.02), while participants who suffered mild or moderate-to-severe AMS had a lower end-exercise SpO2 at 3500 m (82.2 vs. 83.8%, p = 0.027; 81.5 vs. 83.8%, p 5000 m (OR 2.740, p-value 0.003) predicted the development of moderate-to-severe AMS. The Xtreme Everest Step-Test offers a simple, reproducible field test to help predict AMS, albeit with relatively limited predictive precision

The Smell of Hypoxia: using an electronic nose at altitude and proof of concept of its role in the prediction and diagnosis of acute mountain sickness.

Electronic nose (e-nose) devices may be used to identify volatile organic compounds (VOCs) in exhaled breath. VOCs generated via metabolic processes are candidate biomarkers of (patho)physiological pathways. We explored the feasibility of using an e-nose to generate human "breathprints" at high altitude. Furthermore, we explored the hypothesis that pathophysiological processes involved in the development of acute mountain sickness (AMS) would manifest as altered VOC profiles. Breath analysis was performed on Sherpa and lowlander trekkers at high altitude (3500 m). The Lake Louise Scoring (LLS) system was used to diagnose AMS. Raw data were reduced by principal component (PC) analysis (PCA). Cross validated linear discriminant analysis (CV-LDA) and receiver-operating characteristic area under curve (ROC-AUC) assessed discriminative function. Breathprints suitable for analysis were obtained from 58% (37/64) of samples. PCA showed significant differences between breathprints from participants with, and without, AMS; CV-LDA showed correct classification of 83.8%, ROC-AUC 0.86; PC 1 correlated with AMS severity. There were significant differences between breathprints of participants who remained AMS negative and those whom later developed AMS (CV-LDA 68.8%, ROC-AUC 0.76). PCA demonstrated discrimination between Sherpas and lowlanders (CV-LDA 89.2%, ROC-AUC 0.936). This study demonstrated the feasibility of breath analysis for VOCs using an e-nose at high altitude. Furthermore, it provided proof-of-concept data supporting e-nose utility as an objective tool in the prediction and diagnosis of AMS. E-nose technology may have substantial utility both in altitude medicine and under other circumstances where (mal)adaptation to hypoxia may be important (e.g., critically ill patients)

Effects of dietary nitrate supplementation on microvascular physiology at 4559 m altitude – a randomised controlled trial (Xtreme Alps)

Native highlanders (e.g. Sherpa) demonstrate remarkable hypoxic tolerance, possibly secondary to higher levels of circulating nitric oxide (NO) and increased microcirculatory blood flow. As part of the Xtreme Alps study (a randomised placebo-controlled trial of dietary nitrate supplementation under field conditions of hypobaric hypoxia), we investigated whether dietary supplementation with nitrate could improve NO availability and microvascular blood flow in lowlanders. Plasma measurements of nitrate, nitrite and nitroso species were performed together with measurements of sublingual (sidestream dark-field camera) and forearm blood flow (venous occlusion plethysmography) in 28 healthy adult volunteers resident at 4559 m for 1 week; half receiving a beetroot-based high-nitrate supplement and half receiving an identically-tasting low nitrate 'placebo'. Dietary supplementation increased plasma nitrate concentrations 4-fold compared to the placebo group, both at sea level (SL; 19.2 vs 76.9 μM) and at day 5 (D5) of high altitude (22.9 vs 84.3 μM,

Metabolic basis to Sherpa altitude adaptation.

The Himalayan Sherpas, a human population of Tibetan descent, are highly adapted to life in the hypobaric hypoxia of high altitude. Mechanisms involving enhanced tissue oxygen delivery in comparison to Lowlander populations have been postulated to play a role in such adaptation. Whether differences in tissue oxygen utilization (i.e., metabolic adaptation) underpin this adaptation is not known, however. We sought to address this issue, applying parallel molecular, biochemical, physiological, and genetic approaches to the study of Sherpas and native Lowlanders, studied before and during exposure to hypobaric hypoxia on a gradual ascent to Mount Everest Base Camp (5,300 m). Compared with Lowlanders, Sherpas demonstrated a lower capacity for fatty acid oxidation in skeletal muscle biopsies, along with enhanced efficiency of oxygen utilization, improved muscle energetics, and protection against oxidative stress. This adaptation appeared to be related, in part, to a putatively advantageous allele for the peroxisome proliferator-activated receptor A (PPARA) gene, which was enriched in the Sherpas compared with the Lowlanders. Our findings suggest that metabolic adaptations underpin human evolution to life at high altitude, and could have an impact upon our understanding of human diseases in which hypoxia is a feature.The work was supported by PhD studentships from the BBSRC to JH (BB/F016581/1) and British Heart Foundation to AK (FS/09/050), an Academic Fellowship to AM from the Research Councils UK (EP/E500552/1), a Physiological Society grant and support from Oroboros Instruments. JG thanks the MRC (MC UP A90 1006) and AB Sciex. MF thanks the MRC and Faculty of Medicine, Southampton University. For full acknowledgements see SI

King of the mountains: Tibetan and Sherpa physiological adaptations for life at high altitude

Anecdotal evidence surrounding Tibetans' and Sherpas' exceptional tolerance to hypobaric hypoxia has been recorded since the beginning of high-altitude exploration. These populations have successfully lived and reproduced at high altitude for hundreds of generations with hypoxia as a constant evolutionary pressure. Consequently, they are likely to have undergone natural selection toward a genotype (and phenotype) tending to offer beneficial adaptation to sustained hypoxia. With the advent of translational human hypoxic research, in which genotype/phenotype studies of healthy individuals at high altitude may be of benefit to hypoxemic critically ill patients in a hospital setting, high-altitude natives may provide a valuable and intriguing model. The aim of this review is to provide a comprehensive summary of the scientific literature encompassing Tibetan and Sherpa physiological adaptations to a high-altitude residence. The review demonstrates the extent to which evolutionary pressure has refined the physiology of this high-altitude population. Furthermore, although many physiological differences between highlanders and lowlanders have been found, it also suggests many more potential avenues of investigation