The pulmonary diffusing capacity for nitric oxide (DL,NO), also called the transfer factor of the lung for nitric oxide (TL,NO), is a pulmonary function measurement that quantifies the rate of uptake of inhaled nitric oxide (NO) from the alveolar gas into the pulmonary capillary blood. It is expressed in units of mL·min−1·mmHg−1 (traditional) or mmol·min−1·kPa−1 (SI), with the conversion factor being division by 2.985. Because NO reacts with haemoglobin roughly 1,500 times faster than carbon monoxide (CO), DL,NO is weighted toward the alveolar–capillary membrane component of gas transfer (DM), in contrast to DL,CO, which is dominated by the red cell or blood component (θCO·VC). When measured simultaneously with DL,CO, the combined single-breath DL,NO–DL,CO technique allows partitioning of total pulmonary gas transfer resistance into its membrane (DM) and microvascular (θ·VC) components using the Roughton–Forster equation.
The test was first performed in 1983 and has been the subject of a 2017 European Respiratory Society (ERS) technical standards document that standardised its measurement methodology, equipment specifications, quality control requirements, and reference equations for clinical use.
History
Origins in Cambridge
Early research on nitric oxide in the United Kingdom was directed toward its toxicology rather than its physiology. In the late 1970s, "oxides of nitrogen" were suspected as a cause of lung disease from atmospheric pollution, indoor air pollution, and cigarette smoke. High concentrations of NO (over 100 parts per million) and lower doses of nitrogen dioxide (NO2; 25 ppm) were known to cause lung damage in cases of accidental human exposure or experimental animal exposure. An emphysema-like lesion had been described in laboratory rats exposed to nitrogen oxides, fuelling speculation that these compounds caused emphysema in smokers.
At that time, the tobacco industry was under pressure to produce a "safer cigarette." To investigate NO in cigarette smoke, scientists at British American Tobacco constructed a prototype NO gas analyser based on chemiluminescence, a technique described by Brian Thrush and colleagues in 1964. The analyser generated ozone from atmospheric oxygen and reacted it with the gas sample; any NO was immediately converted to NO2 in an excited state, which emitted photons of light upon returning to the ground state. This highly sensitive and specific method has underpinned most subsequent work on pulmonary NO uptake.
Tim Higenbottam at Cambridge collaborated with British American Tobacco, who loaned him their analyser. Higenbottam and colleagues had previously failed to demonstrate a relationship between smokers' cigarette tar yields and airflow obstruction prevalence, and concluded that attention should be paid to the gas-phase constituents of smoke, principally CO and NO. Colin Borland's doctoral project was to compare NO and CO uptake using the chemiluminescent analyser for NO alongside a commercial infrared CO analyser with a helium analyser. He initially examined 40 ppm—a concentration he had (erroneously) calculated to be the alveolar NO level during inhalation of a popular UK cigarette brand. Borland found that NO uptake only occurred when volumes exceeding the dead space were inhaled.
The first measurements of DL,NO (reported as TL,NO) were presented by Borland, Chamberlain and Higenbottam to the UK Medical Research Society in Spring 1983. The suggestion to measure the transfer factor came from their technician, Andrew Chamberlain (subsequently Professor of Bioarchaeology at the University of Manchester). They found that KNO (the rate constant for alveolar NO uptake) was highly correlated with but exceeded KCO four- to five-fold, and like KCO, increased with exercise. In a subsequent abstract, they calculated DM assuming θNO to be infinite. The full paper, containing observations on varying breathhold time, undetectable back tension, greater volume dependence of DL,NO than DL,CO, and independence from hyperoxia, was published in 1989.
Regarding the original hypothesis, it is now considered very unlikely that NO in cigarette smoke causes emphysema: the half-life of oxidation of NO in air is approximately one hour, compared to roughly two seconds for alveolar uptake. Furthermore, histological NO2-induced emphysema in rats differs markedly from smokers' emphysema, and large international differences in cigarette NO yields do not parallel emphysema incidence.
The French connection
The Bordeaux contribution arose independently, a positive side-effect of the May 1968 events in Paris. Professor Daniel Bargeton, Hervé Guénard's mentor, was vice-dean of the Paris medical faculty. When the university came to a standstill during the social upheaval, he returned to research and conceived the idea of calculating DM,CO and VC using two transfer gases in a single manoeuvre. He initially proposed using CO and hydrogen sulphide (H2S), but this gas proved highly problematic: its odour was objectionable, it deposited sulphur in mass spectrometer ionisation chambers, and it was too soluble in water, disappearing rapidly into the airway walls.
After arriving in Bordeaux in 1979, Guénard systematically reviewed a list of candidate gases from a chemistry textbook. Sulphur compounds largely shared the same drawbacks; the list of nitrogen oxides was shorter, and the choice of NO was straightforward since other oxides such as N2O2 and NO2 did not meet specifications. Guénard reasoned that the equation 1/DL = 1/DM + 1/(θ·VC) could be solved by a single-breath manoeuvre using CO and NO simultaneously. The Bordeaux group published their formula for DM and VC from a combined single-breath DL,NO and DL,CO in 1987. Importantly, among their references was a 1958 paper by Carlsen and Comroe who had measured the rate constant for the reaction of NO with red cells in the absence of oxygen using the same rapid-reaction apparatus that Forster had used for CO.
Two other significant developments occurred in 1987: two independent groups proved that NO was identical to Endothelium-Derived Relaxing Factor (EDRF), enormously increasing scientific interest in NO; and Forster made further measurements of θCO at physiological pH, obtaining values that yielded slightly lower VC and rather larger DM compared to his 1957 data.
Evolution (1989–2016)
By the late 1980s, analysers capable of detecting NO concentrations down to 1 part per billion became available. This allowed detection of endogenous NO back tension at approximately 10 ppb, enabled longer breathhold times than 7.5 seconds, and permitted alveolar profile measurements using rapidly responding analysers for steady-state and intrabreath methods. Commercial gas transfer systems incorporating NO analysers also became available, using less expensive but less sensitive NO electrochemical cells that require shorter breathhold times.
Over the subsequent 25 years, numerous studies measured combined DL,NO and DL,CO in normal volunteers and in patients with a variety of cardiopulmonary diseases. Key findings included that DL,NO increases more with lung volume than DL,CO, increases linearly with exercise, increases at altitude in newcomers within 2–3 days (returning toward baseline by 7–8 days), and is reduced during diving while breathing oxygen.
The θNO debate: finite or infinite?
A central and ongoing debate concerns whether the specific blood conductance for NO (θNO) should be regarded as effectively infinite—meaning that DL,NO equals DM,NO directly—or finite with a meaningful blood resistance to NO transfer. The original Bordeaux assumption of infinite θNO was widely adopted by other investigators.
The Cambridge group challenged this view with a series of experiments. Using a membrane oxygenator model, they demonstrated that DL,NO increased upon haemolysis, proving that blood resistance to NO transfer was significant in the oxygenator. They subsequently showed near-identical increases in DL,NO with cell-free haemoglobin in both an in vivo canine model (using a bovine haemoglobin-based blood substitute) and their in vitro oxygenator, establishing that the resistance of blood to NO transfer was significant in the lung. Further work localised this resistance to the red cell interior specifically, rather than plasma or the red cell membrane.
The 2017 ERS Task Force established the current consensus that θNO should be taken as a finite value of 4.5 mL·min−1·mmHg−1·mL−1 of blood, as measured by Carlsen and Comroe in 1958.
Physiological basis
Roughton–Forster equation
The uptake of both NO and CO from the alveolus to the capillary blood is described by the Roughton–Forster equation:
where 1/DL is the total transfer resistance, 1/DM is the membrane resistance (from the alveolar epithelial surface to the red cell membrane), and 1/(θ·VC) is the red cell or blood resistance. DM is the alveolar–capillary membrane diffusing capacity, θ is the specific conductance of blood (the volume of gas taken up by red cells per mL of blood per minute per mmHg of partial pressure), and VC is the total volume of blood in the lung capillaries exposed to alveolar air.
The chief barrier to CO uptake is within the red cell (approximately 70–80% of total resistance), with approximately 25% of resistance located in the alveolar membrane. For NO, the main resistance lies between the alveolar and red blood cell membranes (approximately 60%), with the red cell resistance comprising approximately 40% of the total.
Reaction of NO and CO with capillary blood
The reaction of haemoglobin in solution with NO is extremely rapid—nearly 1,500 times faster than with CO. Crucially, the reaction of NO with haemoglobin solutions is 500–1,000 times faster than its reaction with intact erythrocytes from animal or human sources, demonstrating that θNO cannot be "infinite." This red cell resistance was localised to the cell interior by experiments progressively converting oxyhaemoglobin to methaemoglobin by adding nitrite, which altered DL,NO; by contrast, altering extracellular viscosity or comparing different erythrocyte sizes did not affect DL,NO.
Alveolar–capillary membrane diffusing capacity and the α-ratio
The membrane diffusing capacity DM encompasses the entire diffusion path from the surfactant lining layer through the alveolar epithelium, interstitium, capillary endothelium and plasma to the haemoglobin molecule. Its determinants are tissue diffusivity and the pressure gradient between alveolus and plasma. For a gas in tissue, diffusivity is the ratio of solubility to the square root of molecular weight. NO and CO have similar molecular weights (30 and 28 g·mol−1), but NO has approximately twice the solubility of CO in tissue. The resulting diffusivity ratio DM,NO/DM,CO is generally taken as α = 1.97.
Thus, DM,NO = α · DM,CO = 1.97 · DM,CO. This relationship, together with a finite θNO and an appropriate θCO equation, allows DM,CO and VC to be calculated from simultaneously measured DL,NO and DL,CO in a single manoeuvre.
NO in the gas phase
Airway uptake of inhaled NO in a single breathhold is negligible (approximately 0.02%). Within the acinus, the dominant mode of gas transport is molecular diffusion. Gas-phase diffusion coefficients are inversely proportional to the square root of molecular weight, so there is no significant difference between NO and CO. Gas-phase diffusion resistance as a proportion of total transfer resistance is greater for NO than for CO, but in normal lungs the effect is negligible (approximately 5% of total 1/DL,NO).
Diffusion-limited uptake