Assessment of Pulmonary Capillary Blood Volume, Membrane Diffusing Capacity, and Intrapulmonary Arteriovenous Anastomoses During Exercise
February 20th, 2017
To assess the pulmonary diffusion and vasculature responses to exercise, we describe the multiple-inspired oxygen diffusion capacity technique to determine capillary blood volume and membrane diffusing capacity, as well as agitated saline contrast echocardiography to assess the recruitment of intrapulmonary arteriovenous anastomoses.
The overall goal of this procedure is to assess the pulmonary vascular response to exercise using multiple fraction of inspired oxygen, or FiO2, diffusion capacities and contrast-enhanced echocardiography. This method helps us better understand how the pulmonary circulation responds to the stress of exercise. The main advantage of this technique is that it can be applied to a variety of experimental, clinical, and pharmacological conditions, including during exercise in participants with heart or lung disease.
Demonstrating the breath hold technique will be Wade Michaelchuk and Sophie Collins, graduate students in my lab. Performing the contrast echocardiography technique will be Doctor Sean van Diepen, cardiologist. Before beginning the procedure, use an air oxygen blender system to fill one 60-liter non-diffusing Douglas bag with 40%oxygen, and one with 60%oxygen.
Attach two large bore three-way stopcock valves for modulation of the inhaled gases, and use flexible non-compressing tubing to connect the Douglas bags to the valve system. Then, connect the valve system to a two-way T-shaped non-rebreathing valve connected to the test gas intake assembly of the mass flow sensor. Switch the pre-breathe valve and the carbon monoxide gas selector to the appropriate orientation for the selected FiO2, and instruct the subject to begin cycling at the intensity of interest while maintaining a heart rate of between plus or minus three beats per minute.
Once the steady state has been achieved, affix the nose clips and mouthpiece onto the subject, and have the subject breathe normally for five breaths from the 40%oxygen Douglas bag. After the fifth breath, instruct the subject to inhale to the total lung capacity, and then exhale to the residual volume. When the lung volume begins to plateau, press F1 on the valve system keyboard to switch to the carbon monoxide gas, and instruct the subject to continue exhaling for three seconds, followed by inhalation of the carbon monoxide gas to the total lung capacity.
Take a big breath in, blow it all the way out, keep pushing, three, two, one, and big breath in, up, up, up, up, up, up, up, six, five, four-Count down from six seconds. Three, two, one, blow it out-And instruct the subject to exhale again to the residual volume. Good, breathe normally.
Monitoring the methane tracing during the exhalation to ensure that the slope is horizontal. The success of this step relies on the skill of the tester and the ability of the subject to follow commands and remain calm during the breath hold. We suggest that you rehearse the timing of the breath holds with the subject before attempting this procedure.
Now, remove the mouthpiece, and have the subject cycle for at least two more minutes to allow the residual carbon monoxide to be washed out. Then, repeat the breath hold maneuvers for the remaining FiO2s. To ensure a high-quality maneuver, confirm that the alveolar volume is within 5%of the previous trials, and that the breath hold time is approximately six seconds.
For intrapulmonary arteriovenous anastomosis, or IPAVA, recruitment, first insert a 20-gage intravenous catheter into one of the subject's antecubital veins. Then, use a six-inch piece of extension tubing to attach the catheter to a three-way stopcock, and connect two 10-milliliter syringes to the stopcock. To create the intravenous contrast, combine 10 milliliters of sterile saline with zero point five milliliters of air, and rapidly agitate the two syringes back and forth through the stopcock until the bubbles are finally suspended.
Instruct the subject to begin the cycling exercise at 100 watts of intensity. Once the steady state has been reached, have an experienced cardiologist obtain a standard apical four chamber view of the heart. Continue agitating the saline until the cardiologist is ready to record.
Then, inject the contrast while the cardiologist maintains the four chamber view, and record 15 cardiac cycles following the detection of contrast in the right ventricle. The quality of the ultrasound image depends on the technical ability of the sonographer to maintain an acceptable image of all four chambers of the heart during exercise. Then, have an echocardiographer who is blinded to experimental conditions interpret the echocardiograms for the presence of IPAVAs.
The capillary blood volume and diffusing capacity increase as the exercise intensity increases, consistent with the need for an expanded diffusion capacity for a greater oxygen consumption. Indeed, in this representative experiment, the echocardiographic data demonstrate no IPAVA recruitment at rest, but with an IPAVA score that increases as the exercise intensity increases. In this recording of a subject's heart at rest, the contrast agent can clearly be visualized entering the right ventricle.
Even after five cardiac cycles, no contrast can be observed in the left ventricle, giving this participant a resting IPAVA score of zero. Here, representative tracings of four chamber contrast echocardiographs are shown. Note that as the exercise intensity increases, the intrapulmonary arteriovenous anastomosis score increases from zero at rest to three at the highest exercise intensity.
Once mastered, this technique can be completed within two hours. While attempting this procedure, it's important to remember to coach the volunteer in the proper breathing technique and timing. Following this procedure, other interventions, such as a drug infusion, can be used to answer questions about the pulmonary circulation.
After its development, this technique paved the way for researchers in the field of pulmonary physiology, to explore the determinants of pulmonary diffusing capacity in humans. After watching this video, you should have a good understanding of how to assess the pulmonary vasculature at rest and during exercise. Don't forget that working with carbon monoxide can be extremely hazardous, and that precautions, such as proper room ventilation, should always be taken when performing this procedure.
Overview
This article demonstrates two key techniques for assessing pulmonary vascular responses to exercise: multiple-fraction of inspired oxygen (FiO2) diffusion capacity measurements and contrast-enhanced echocardiography. These methods enable researchers to evaluate changes in pulmonary capillary blood volume, membrane diffusing capacity, and the recruitment of intrapulmonary arteriovenous anastomoses (IPAVAs) during exercise, providing insights into how the lung vasculature adapts to increased physiological stress.
Key Study Components
Area of Science
- Pulmonary physiology
- Exercise physiology
- Cardiopulmonary assessment
Background
- Exercise increases the demand for oxygen, requiring the pulmonary diffusing capacity (DLCO) to rise accordingly.
- DLCO increases through elevated capillary blood volume (Vc) and membrane diffusing capacity (Dm), facilitated by recruitment and distension of pulmonary capillaries.
- Recruitment of IPAVAs during exercise may impact gas exchange and pulmonary artery pressure.
- Understanding these adaptations is important for both healthy individuals and those with cardiopulmonary diseases.
Purpose of Study
- To describe and demonstrate techniques for measuring pulmonary diffusion and circulation at rest and during exercise.
- To quantify changes in DLCO, Vc, and Dm with exercise intensity.
- To assess IPAVA recruitment using echocardiography with agitated saline contrast.
Methods Used
- Multiple-fraction of inspired oxygen (FiO2) DLCO breath hold maneuvers to determine Vc and Dm at rest and during exercise.
- Preparation of Douglas bags with different oxygen concentrations for controlled gas delivery.
- Contrast-enhanced echocardiography using intravenous agitated saline to visualize IPAVA recruitment.
- Blinded interpretation of echocardiograms for IPAVA scoring.
Main Results
- DLCO, Vc, and Dm all increased with higher exercise intensity.
- No IPAVA recruitment was observed at rest; however, contrast bubbles appeared in the left ventricle during exercise, indicating exercise-induced IPAVA recruitment.
- IPAVA scores increased progressively with exercise intensity.
- The techniques were feasible and could be completed within two hours.
Conclusions
- These methods effectively characterize pulmonary vascular adaptation to exercise stress.
- They are applicable in both healthy subjects and those with pulmonary or cardiac diseases.
- Proper coaching and safety precautions, especially regarding carbon monoxide use, are essential for successful implementation.
Frequently Asked Questions
Executive Industry Relevance
Quantitative assessment of pulmonary capillary blood volume, membrane diffusing capacity, and IPAVA recruitment during exercise provides critical insight into vascular adaptation under physiological stress. These measurements enable biopharma teams to evaluate pulmonary function in both healthy and disease-relevant populations, supporting translational research and mechanistic de-risking for cardiopulmonary drug development. Integrating these techniques into early discovery and preclinical workflows enhances predictive confidence for portfolio advancement decisions.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables interrogation of pulmonary vascular response to stress for target validation in cardiopulmonary indications.
- Supports mechanistic de-risking by quantifying capillary recruitment and membrane diffusion changes during exercise.
- Facilitates identification of disease-relevant physiological endpoints for early-stage compound evaluation.
Screening & Assay Development
- Provides validated physiological readouts for screening compounds affecting pulmonary vascular function.
- Standardizes measurement of DLCO, Vc, and Dm across experimental conditions for reproducibility.
- Enables quantitative comparison of intervention effects on pulmonary diffusion and vascular adaptation.
Translational & Preclinical Research
- Aligns preclinical models with human physiological endpoints relevant to pulmonary hypertension and COPD.
- Supports continuity from discovery through preclinical validation by tracking vascular adaptation metrics.
- De-risks translational progression by linking mechanistic outputs to disease-relevant functional changes.
Pipeline & Workflow Integration
This method bridges early discovery, target validation, and preclinical research by providing quantitative, reproducible measures of pulmonary vascular adaptation to exercise.
- Discovery Biology: Quantifies vascular and membrane responses to physiological stress for hypothesis testing and pathway clarification.
- Screening: Delivers standardized, reproducible outputs (DLCO, Vc, Dm, IPAVA scores) for compound evaluation.
- Analytics: Enables statistical comparison of intervention effects on pulmonary function across conditions.
- Translational Research: Connects preclinical findings to human disease endpoints for risk-adjusted advancement.
- Enterprise Reuse: Establishes a reusable platform for cardiopulmonary functional assessment in diverse R&D programs.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence and reduces mechanistic ambiguity in pulmonary target validation.
- Operational Value: Standardizes physiological measurements and supports reproducibility across studies.
- Strategic Value: Informs go/no-go decisions and reduces late-stage biological risk in cardiopulmonary portfolios.
- Portfolio Impact: Enables risk-adjusted prioritization and advancement of assets targeting pulmonary vascular adaptation.
Implementation Considerations
- Requires expertise in pulmonary physiology, echocardiography, and gas diffusion measurement.
- Needs specialized instrumentation for gas delivery, mass flow sensing, and contrast echocardiography.
- Demands rigorous cross-team standardization of protocols and data interpretation.
- Adaptation across model systems may require protocol optimization for species or disease context.
- Safety precautions are essential when handling carbon monoxide and intravenous contrast agents.
