该研究得到了斯文德·安徒生基金会的财政支持。体育活动研究中心由 TrygFonden 资助 ID 101390、ID 20045 和 ID 125132 提供支持。JPH 由 HelseFonden 和哥本哈根大学医院 Rigshospitalet 资助，而 HLH 由 Beckett 基金会资助。

丹麦首都地区科学伦理委员会此前已批准在我们机构的健康志愿者和慢性阻塞性肺病 （COPD） 患者中测量休息、运动期间和仰卧位的 DL、CO/NO （协议 H-20052659、H-21021723 和 H-21060230）。
注意：在运动期间测量 DL、CO/NO 之前，必须进行动态肺活量测定和心肺运动测试 （CPET）。动态肺活量测定法用于单个 DL，CO/NO 动作的质量控制，而 CPET 用于确定在运动期...

评估运动期间肺泡毛细血管储备

该协议提供了一种使用双测试气体单次呼吸技术在运动期间测量 DL、CO/NO 的标准化方法。由于获得的 DL、CO/NO 指标因肺毛细血管复张和扩张而增加，因此该方法提供了具有生理意义的肺泡-毛细血管储备测量。
协议中的关键步骤 该方法需要呼气至残余容积，然后吸气至总肺活量，此时进行 5 秒屏气并终止至 RV。这是一个关键的步骤，因为在运动...

与静息状态相比，运动导致能量需求显着增加。心脏和肺的反应是增加心输出量和通气量，导致肺泡-毛细血管床扩张，主要是肺毛细血管的募集和扩张1.这确保了足够的肺气体交换，这可以通过肺弥散能力（DL）的增加来衡量2,3,4。在运动过程中测量 DL 的第一次尝试可以追溯到一个多世纪前 5,6,7。从静息状态增加 DL 的能力通常被称为肺泡毛细血管储备 8,9。
在实验上，肺泡-毛细血管膜弥散能力 （DM） 和肺毛细血管血容量 （VC） 对肺泡-毛细血管储备的相对贡献可以通过不同的方法进行评估，包括经典的吸入氧多重组分 （<img alt="Equation 1" src="/files/ftp_upload/65871/65871eq01.jpg" style="margin: auto;">）方法 10。在这种情况下可能有用的另一种技术是双重测试气体方法，其中同时测量 DL 到一氧化碳 （CO） 和一氧化氮 （NO） （DL，CO/NO）11。该技术是在 1980 年代开发的，它利用了 NO 与血红蛋白 （Hb） 的反应速率大大大于 CO 的反应速率这一事实，因此 CO 的肺扩散比 NO 更依赖于 VC 。 因此，对 CO 扩散的抵抗主要部位 （~75%） 位于红细胞内， 而对 NO 扩散的主要阻力 （~60%） 在肺泡毛细血管膜和肺血浆12。因此，同时测量 DL，CO 和 DL，NO 可以评估 DM 和 VC 对 DL12 的相对贡献，其中在运动过程中观察到的 DL，NO 的变化在很大程度上反映了肺泡-毛细血管膜的扩张。在运动期间获得测量值时，这种方法的另一个优点是，与经典技术相比，它涉及相对较短的屏气时间（~5 s）和更少的操作，在经典 <img alt="Equation 1" src="/files/ftp_upload/65871/65871eq01.jpg" style="margin: auto;"> 技术中，在不同的氧气水平下进行多次重复操作，标准化的 10 秒屏气。虽然 <img alt="Equation 1" src="/files/ftp_upload/65871/65871eq01.jpg" style="margin: auto;"> 最近应用了更短的屏气时间和更少的动作，但每种强度13.然而， <img alt="Equation 1" src="/files/ftp_upload/65871/65871eq01.jpg" style="margin: auto;"> 每次会话总共只允许 6 次 DL，CO 操作，而最多可以执行 12 次重复的 DL，CO/NO 操作，而不会对结果估计值产生任何可测量的影响14。在运动期间进行测量时，这些都是重要的考虑因素，因为在非常高的强度下或在出现呼吸困难的患者群体中，长时间屏气和多次操作都可能难以进行。
本文提供了一个详细的方案，包括关于运动期间测量 DL、CO/NO 及其用作肺泡-毛细血管储备指标的理论考虑和实践建议。这种方法很容易应用于实验环境，并允许评估肺部的扩散限制如何影响不同人群的摄氧量。
理论和测量原理 DL，CO/NO 方法涉及单次呼吸气体混合物，假设气体在吸入后均匀分布在通风的肺泡空间中。气体混合物由几种气体组成，包括惰性示踪气体。通风肺泡腔中示踪气体的稀释度，基于其在呼气末空气中的分数，可用于计算肺泡容积 （VA）15。气体混合物还包括测试气体 CO 和 NO，它们都在通风的肺泡空间中稀释并扩散到肺泡毛细血管膜上。根据它们的肺泡分数，可以计算出它们从肺泡空间中消失的个体速率 （k），也称为弥散常数。按照惯例，在单次呼吸操作期间测量的测试气体的 DL 由以下公式16 推导：
<img alt="Equation 2" src="/files/ftp_upload/65871/65871eq02v3.jpg" style="margin: auto;">
其中 FA0 是单个 DL 动作屏气开始时测试气体（CO 或 NO）的肺泡分数，而 FA 是屏气结束时测试气体的肺泡分数，tBH 是屏气时间。DL 在机械上相当于测试气体穿过肺泡毛细血管膜，通过血浆和红细胞内部到达血红蛋白的电导。因此，它既取决于 DM 的电导率，也取决于所谓的肺毛细血管血 （θ） 的比电导率，后者既取决于测试气体在血液中的电导率，也取决于其与血红蛋白10 的反应速率。鉴于电导的倒数是电阻，则测试气体转移的总电阻取决于串联 10 中的以下电阻：
<img alt="Equation 3" src="/files/ftp_upload/65871/65871eq03v2.jpg" style="margin: auto;">
这些分量可以通过同时测量 DL 到 CO 和 NO 来区分，因为它们具有不同的 θ 值，因此它们各自的 DL 值对 VC 的依赖程度不同。与 NO 相比，CO 的肺扩散更依赖于 VC，对 CO 扩散的主要抵抗部位 （~75%） 位于红细胞内 12。相反，NO扩散的主要阻力（~60%）在肺泡毛细血管膜和肺血浆，因为NO与血红蛋白的反应速率明显大于CO。 而后者对 VC 的依赖要小得多，因此可以对决定 DL 的因素进行综合评估。
DL、CO/NO 指标的报告可以使用不同的单位完成。因此，欧洲呼吸学会 （ERS） 使用 mmol/min/kPa，而美国胸科学会 （ATS） 使用 mL/min/mmHg。单位之间的转换系数为 2.987 mmol/min/kPa = mL/min/mmHg。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>HemoCue Hb 201+&#160;</td>
			<td>HemoCue, Br&#248;nsh&#248;j, Denmark</td>
			<td>Unkown</td>
			<td>For measurements of hemoglobin</td>
		</tr>
		<tr>
			<td>Jaeger MasterScreen PFT pro (Lung Function Equipment)</td>
			<td>CareFusion, H&#246;chberg, Germany</td>
			<td>Unkown</td>
			<td>For measurements of DLCO/NO</td>
		</tr>
		<tr>
			<td>Mouthpiece</td>
			<td>SpiroBac, Henrotech, Aartselaar, Belgium</td>
			<td>Unkown</td>
			<td>Used together with the Lung Fuction Equipment. (dead space 56 ml, resistance to flow at 12 L s&#8722;1 0.9 cmH2O)&#160;</td>
		</tr>
		<tr>
			<td>Nose-clip</td>
			<td>IntraMedic, Gentofte, Denmark</td>
			<td>JAE-892895</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenumotach</td>
			<td>IntraMedic, Gentofte, Denmark</td>
			<td>JAE-705048</td>
			<td>Used together with the Lung Fuction Equipment</td>
		</tr>
		<tr>
			<td>SentrySuite Software Solution</td>
			<td>Vyaire&#39;s Medical GmbH, Leibnizstr. 7, D-97204 Hoechberg Germany</td>
			<td>Unkown</td>
			<td></td>
		</tr>
		<tr>
			<td>Test gasses</td>
			<td>IntraMedic, Gentofte, Denmark</td>
			<td>Unkown</td>
			<td>Concentrations: 0.28% CO, 20.9% O2, 69.52% N2 and 9.3% He</td>
		</tr>
	</tbody>
</table>

dual test gas pulmonary diffusing capacity measurement during exercise in humans using the single-breath method

一氧化碳 （DL，CO） 和一氧化氮 （DL，NO） 的扩散能力的联合单次呼吸测量是测量健康和患者人群肺泡毛细血管储备的有用技术。该测量值提供了参与者募集和扩张肺毛细血管的能力的估计。最近有报道称，该方法在健康志愿者中表现出高重测可靠性，用于轻度至中等强度的运动。值得注意的是，这种技术最多允许 12 次重复操作，并且只需要一次呼吸，屏气时间相对较短，为 5 秒。提供了代表性数据，显示了 DL，NO 和 DL，CO 从休息到运动的逐渐变化，强度高达最大工作量的 60%。弥散能力的测量和肺泡毛细血管储备的评估是评估健康人群和患者群体（如慢性肺病患者）肺部对运动的反应能力的有用工具。

Exercise leads to a considerable increase in energy demand compared to the resting state. The heart and lungs respond by increasing cardiac output and ventilation resulting in an expansion of the alveolar-capillary bed, mainly the recruitment and distention of pulmonary capillaries1. This ensures a sufficient pulmonary gas exchange, which can be measured by an increase in pulmonary diffusing capacity (DL)2,3,4. The first attempts to measure DL during exercise date back more than a century5,6,7.&#160;The ability to increase&#160;DL from the resting state is often referred to as the alveolar-capillary reserve8, 9.
Experimentally, the relative contributions of alveolar-capillary membrane diffusing capacity (DM) and pulmonary capillary blood volume (VC) to alveolar-capillary reserve can be assessed by different methods, including the classical multiple fractions of inspired oxygen (<img alt="Equation 1" src="/files/ftp_upload/65871/65871eq01.jpg" style="margin: auto;" />) method10. An alternative technique that may be useful in this context is the dual-test gas method, in which the DL to carbon monoxide (CO) and nitric oxide (NO) (DL,CO/NO) are concurrently measured11. This technique was developed in the 1980s, and takes advantage of the fact that the reaction rate of NO with hemoglobin (Hb) is substantially greater than that of CO, such that the pulmonary diffusion of CO depends more on VC than does NO. Hence, the main site of resistance (~75%) to CO diffusion is located within the red blood cell, while the main resistance (~60%) to NO diffusion is at the alveolar-capillary membrane and pulmonary plasma12. The concurrent measurement of DL,CO and DL,NO thus permits the assessment of the relative contributions of DM and VC to DL12, where the change in DL,NO observed during exercise thus largely reflects the expansion of the alveolar-capillary membrane. An additional advantage of this method when obtaining measurements during exercise is that it involves a relatively short breath-hold time (~5 s) and fewer maneuvers compared to the classical <img alt="Equation 1" src="/files/ftp_upload/65871/65871eq01.jpg" style="margin: auto;" /> technique, where multiple repeated maneuvers with a standardized 10 s breath-hold are performed at different oxygen levels. Although <img alt="Equation 1" src="/files/ftp_upload/65871/65871eq01.jpg" style="margin: auto;" /> has recently been applied with a shorter breath-hold time and fewer maneuvers at each intensity13. Nevertheless, <img alt="Equation 1" src="/files/ftp_upload/65871/65871eq01.jpg" style="margin: auto;" /> only permits a total of six DL,CO maneuvers per session, whereas up to 12 repeated DL,CO/NO maneuvers can be performed without any measurable effect on the resultant estimates14. These are important considerations when obtaining measurements during exercise since both a long breath-hold and multiple maneuvers may be difficult to perform at very high intensities or in patient populations who experience dyspnea.
The present paper provides a detailed protocol, including theoretical considerations and practical recommendations on the measurement of DL,CO/NO during exercise and its use as an index of the alveolar-capillary reserve. This method is easily applicable in the experimental setting and permits the assessment of how diffusion limitation in the lungs may affect oxygen uptake in different populations.
Theory and measurement principles 
The DL,CO/NO method involves a single breath of a gas mixture with the assumption that the gases distribute equally in the ventilated alveolar space after inhalation. The gas mixture consists of several gases including an inert tracer gas. The dilution of the tracer gas in the ventilated alveolar space, as based on its fraction in end-expiratory&#160;air, can be used to calculate the alveolar volume (VA)15. The gas mixture also includes the test gas CO and NO, both of which are diluted in the ventilated alveolar space and diffuse across the alveolar-capillary membrane. Based on their alveolar fractions, their individual rates of disappearance (k), also termed the diffusing constant, from the alveolar space can be calculated. By convention, the DL for a test gas measured during a single-breath maneuver, is derived by the following equation16:
<img alt="Equation 2" src="/files/ftp_upload/65871/65871eq02v3.jpg" style="margin: auto;" />
where FA0&#160;is the alveolar fraction of the test gas (CO or NO) at the onset of the breath-hold of the individual DL maneuver, while FA is the alveolar fraction of the test gas at the end of the breath-hold, and tBH is the breath-hold time. DL is mechanically equivalent to the conductance of the test gas across the alveolar-capillary membrane, through plasma and the red blood cell interior to hemoglobin. It thus depends both on the conductance of DM&#160;and the so-called specific conductance of pulmonary capillary blood (&#952;), of which the latter depends both on the conductance of the test gas in blood and on its reaction rate with hemoglobin10. Given that the reciprocal of conductance is resistance, the total resistance to the transfer of a test gas depends on the following resistances in series10:
<img alt="Equation 3" src="/files/ftp_upload/65871/65871eq03v2.jpg" style="margin: auto;" />
These components may be distinguished by concurrently measuring the DL to CO and NO, because these have different &#952;-values, and their respective DL values&#160;thus depend differently on VC. The pulmonary diffusion of CO depends more heavily on VC than does NO, with the main site of resistance (~75%) to CO diffusion being located within the red blood cell12. In contrast, the main resistance (~60%) to NO diffusion is at the alveolar-capillary membrane and pulmonary plasma, because the reaction rate of NO with hemoglobin&#160;is substantially greater than that of CO. Hence, by concurrently measuring DL,CO and DL,NO, changes in both DM and VC will markedly affect the former, while the latter will depend much less on VC, thus permitting an integrative assessment of the factors that determine DL.
The reporting of DL,CO/NO metrics may be done using different units. Hence, the European respiratory society (ERS) uses mmol/min/kPa, whereas the American Thoracic society (ATS) uses mL/min/mmHg. The conversion factor between the units is 2.987 mmol/min/kPa = mL/min/mmHg.

作者聚焦：将肺泡毛细血管储备测量整合到运动适应和治疗策略中

使用单次呼吸法测量人体运动期间的双重测试气体肺弥散能力

Our research focuses on mechanism of impaired pulmonary gas exchange in various disease states and how knowledge of these may be used for diagnostic and target treatments. We aim to refine the understanding of acute exercise adaptation by assessing alveolar capillary reserve through diffusion capacity measurements during exercise. Conventional lung function testing, such as dynamic spirometry, and diffusion capacity measurements during rest provides limited information about pulmonary limitations and adaptations to acute exercise.Our protocol addresses this gap by introducing a more comprehensive and physiologically-relevant measurement method that assesses the alveolar capillary reserve. By providing a more detailed analysis of lung function during exercise, this research contributes to the development of target diagnostic and therapeutic strategies for pulmonary diseases, enhancing clinical management and treatment efficacy. By this technique, our studies will specifically elucidate how pulmonary gas exchange limitations contributes to the pathophysiology of chronic lung disease and how this may be affected by exercise training.Our research group intends to extend this research to explore the impact of various therapeutic and training interventions on pulmonary function during exercise, and to investigate the specific responses in patients with chronic obstructive pulmonary disease. This will further elucidate the lungs adaptability and resilience on distress.

该协议于 2021 年实施，在撰写本文时，在运动期间共进行了 124 次测量（即健康志愿者 51 次，不同严重程度的 COPD 患者 73 次）。 表 3中提供了操作，以及有关满足可接受性和可重复性标准的数据，以及故障率。
计算 例如，这里根据健康组中以 20% Wmax 进行第一次操作的数据提供单个 DL、CO/NO 操作的计算，作为下面描述?...

Watch this Scientific Journal Video about Author Spotlight: Integrating Alveolar-Capillary Reserve Measurements in Exercise Adaptation and Therapeutic Strategies at JoVE.com

该方案提出了一种评估肺泡毛细血管储备的方法，该方法通过联合单次呼吸测量运动期间对一氧化碳（DL，CO）和一氧化氮（DL，NO）的扩散能力进行测量。在锻炼过程中使用该技术的假设和建议构成了本文的基础。

The combined single-breath measurement of the diffusing capacity of carbon monoxide (DL,CO) and nitric oxide (DL,NO) is a useful technique to measure ...

我们的研究重点是各种疾病状态下肺气体交换受损的机制，以及如何将这些知识用于诊断和靶向治疗。我们的目标是通过运动期间的弥散能力测量来评估肺泡毛细血管储备，从而完善对急性运动适应的理解。常规肺功能测试（如动态肺活量测定）和休息期间的弥散量测量，只能提供有关肺功能限制和对急性运动的适应的有限信息。我们的方案通过引入一种更全面且与生理相关的测量方法来评估肺泡毛细血管储备，从而解决了这一差距。通过对运动过程中的肺功能进行更详细的分析，本研究有助于开发肺部疾病的靶向诊断和治疗策略，提高临床管理和治疗效果。通过这种技术，我们的研究将具体阐明肺气体交换限制如何导致慢性肺病的病理生理学，以及运动训练如何影响慢性肺病。我们的研究小组打算扩展这项研究，以探索各种治疗和训练干预措施对运动期间肺功能的影响，并调查慢性阻塞性肺疾病患者的具体反应。这将进一步阐明肺部对痛苦的适应性和弹性。

使用单次呼吸法测量人体运动期间的双重测试气体肺弥散能力

Abstract