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The history of the pulmonary diffusing capacity for nitric oxide DL,NO
Accepted Manuscript Title: The history of the pulmonary diffusing capacity for nitric oxide DL,NO Author: Colin DR Borland Herv´e Gu´enard PII: DOI: Reference:
S1569-9048(16)30299-3 http://dx.doi.org/doi:10.1016/j.resp.2016.11.014 RESPNB 2731
To appear in:
Respiratory Physiology & Neurobiology
Received date: Revised date: Accepted date:
9-10-2016 27-11-2016 28-11-2016
Please cite this article as: Borland, Colin DR, Gu´enard, Herv´e, The history of the pulmonary diffusing capacity for nitric oxide DL,NO.Respiratory Physiology and Neurobiology http://dx.doi.org/10.1016/j.resp.2016.11.014 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
The history of the pulmonary diffusing capacity for nitric oxide DL,NO
1
Colin DR Borland
2
Hervé Guénard
1
Department of Medicine University of Cambridge, UK and Hinchingbrooke Hospital ,
Huntingdon, UK [email protected] 2
Physiologie
et
EFR,
Université
Bordeaux
2
et
CHU
Bordeaux,
France
[email protected].
Highlights
The DLNO (TLNO) had unexpected origins Adoption of the technique has been slow We believe that its worldwide use will increase
1
Abstract The DL,NO (TL,NO) had its unexpected origins in the Paris “events” of 1968 and the unsuccessful efforts of the UK tobacco industry in the 1970’s to create a ” safer cigarette.” Adoption of the technique has been slow due to the instability of NO in air, lack of standardisation of the technique and lack of agreement as to whether DL,NO is equal to or merely reflects membrane diffusing capacity (DM.) With the availability of inexpensive analysers, standardisation of the technique and publication of reference equations we believe that its worldwide use will increase.
Keywords: Diffusing capacity,Nitric Oxide; Carbon monoxide; History
History Early research on Nitric Oxide (NO) in the United Kingdom (UK) was directed towards its toxicology rather than its physiology. In the late 1970’s “oxides of nitrogen” were suspected as a cause of lung disease from atmospheric pollution, from indoor air pollution and from cigarette smoke. High doses (over 100 parts per million (ppm)) of nitric oxide and lower doses of nitrogen dioxide (25 ppm) were known to cause lung damage in cases of accidental human exposure (Clutton Brock, 1967) or experimental animal exposure (Roe, 1985). Interestingly an emphysema like lesion had been described (Roe, 1985) fuelling speculation that “oxides of nitrogen” caused emphysema in smokers. At that time the tobacco industry was under pressure to produce a “safer cigarette”. To investigate nitric oxide in cigarette smoke scientists at British American Tobacco built a prototype NO gas analyser. It was based on Professor Brian Thrush’s description of chemiluminescence (Clyne et al, 1964) :the analyser generated ozone from atmospheric oxygen which was reacted with the smoke sample. Any nitric oxide was immediately converted to nitrogen dioxide in an excited state which on return to the ground state emitted photons of light. This highly sensitive and specific technique has underpinned most of the work on lung nitric oxide uptake. As with the infrared-carbon monoxide 2
analyser physiological measurement followed the technology. Tim Higenbottam at Cambridge collaborated with British American Tobacco who loaned him the analyser. He and colleagues had previously failed to show a relationship between smokers’ cigarette tar yields and prevalence of airflow obstruction in a large epidemiological study (Higenbottam et al, 1980). They concluded that “urgent attention be paid to the gas phase” of which carbon monoxide and nitric oxide were the major constituents (Higenbottam et al, 1980). CDR Borland’s doctorate project (Borland, 1988) was to compare NO and CO uptake using the chemiluminescent analyser for NO and a commercial infrared CO with a Helium analyser bolted on. He looked at 40parts per million which was what he calculated (erroneously as it happened!) to be the alveolar NO concentration when a smoker inhaled a popular UK brand of cigarette. Initially Borland et al investigated percent retention comparing volume of gas exhaled to inhaled (Borland and Higenbottam, 1985). He found that uptake only occurred when volumes in excess of dead space were inhaled. Their then technician Andrew Chamberlain (now Professor of Bioarchaeology at the University of Manchester, UK) who was spending a gap year as a respiratory physiology technician prior to his PhD suggested that they measure the (TL,NO) DL,NO. Borland, influenced by JMB Hughes’ teaching at the Royal Postgraduate Medical school favoured the KNO believing K to be the fundamental parameter of alveolar gas transfer (Hughes and Pride, 2001). The concentration of NO used (40 parts per million ) was stable to oxidation in air with a half life of about an hour (Borland and Higenbottam, 1989). The NO was stored in nitrogen and the mixture was made up immediately before the manoeuvre. Allowance was made for oxidation to NO2 and analysis was performed before the sample passed through sodalime or Calcium Chloride as this was found to cause loss of signal (Borland and Higenbottam, 1989). Their initial findings on a group of healthy male volunteers were presented to the UK Medical Research Society in Spring 1983 (Borland et al, 1983) :KNO was highly correlated with but exceeded KCO four to five fold and like KCO increased with exercise. They used a 40ppm inhaled concentration but had to shorten the breathhold time to have sufficient (>1ppm) exhalate. In a second abstract they measured DL,NO and DL,CO and calculated DM assuming θNO to be infinity (Borland et al, 3
1984). However by a third abstract where they compared a group of patients with anaemia due to renal failure(Scott et al, 1987) to their normal volunteers they modified their conclusions believing the blood resistance to be significant; in retrospect they were right but for the wrong reasons as DL,NO does not alter with Hb concentrations encountered clinically. Finally the paper for their original work on volunteers was published in 1989 (Borland and Higenbottam, 1989).It contained larger numbers and with observations on varying
time indicating semilogarithmic uptake (as
suggested by Dr John Cotes), undetectable back tension within the sensitivity of the analyser (1ppm), greater volume dependence of DL,NO compared to DL,CO and independence of hyperoxia. Going back to the original reasons for the Cambridge group’s work it is seems very unlikely that NO in smokers causes emphysema. Histologically NO2 emphysema in rats is very different from smokers’ emphysema or tobacco lung damage in laboratory rats (Roe ,1985). There are very large international differences between cigarette NO yields which do not parallel emphysema incidence (Borland and Higenbottam, 1985). Finally, relevant to the present discussion, the half life of oxidation of NO to the far more lung irritant NO2 in air (Borland and Higenbottam, 1989) or tobacco smoke (Borland et al, 1985) is one hour compared to two seconds for alveolar lung uptake. Nevertheless, the work that the NO-emphysema hypothesis inspired has stimulated NO use as a lung function test and indeed was the phase 1 trial for its therapeutic use. The French connection occurred quite independently, one positive side-effect of the “events” of May 1968 , which was a period of deep social agitation mainly in Paris and led mainly to nothing. Professor Daniel Bargeton, Hervé Guenard’s mentor, was the source of the concept of DMCO and Vc calculation using two tracer gases. At that time Prof Bargeton was vice dean of the Paris medical faculty which was a heavy commitment. As the University had come to a standstill during this period he stopped his administrative duties and went back to research projects in his office at Rue des St Pères. As usual he wrote the theory with a black pencil on white sheets of paper where fine horizontal lines were previously drawn; in case of error he would rub it and he never threw a sheet
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in the garbage bin. The concept of the double tracer was sound but unfortunately he suggested using CO and Hydrogen Sulphide (H2S). This last gas had several “inconveniences”; quite apart from the odour, the analysis of this gas with a mass spectrometer led to deposition of sulphur in the ionisation chamber which drove the engineer in charge of the device mad. Furthermore H2S was too soluble in water and would disappeared rapidly in the airway walls. Hervé Guenard started working in his lab after the revolution. Though the smell of rotten egg had faded the episode was engraved in everybody’s mind. Professor Bargeton gave Hervé Guenard’s a copy of his initial text written with his pencil. Hervé sought a gas less soluble, reactive with Hb, not toxic and easily analysable. Years later, in 1979, after arriving in Bordeaux Hervé Guenard looked at a list of gases in a chemistry book and made a selection of candidates. Sulphur compounds had for the most part the same drawbacks: too soluble, smelly and not easy to analyse.. The nitrogen oxides list was shorter and the choice of nitrogen monoxide was easy as others oxides N2O2 and NO2 did not fit the specifications. Hervé Guenard therefore reasoned that 1/DL = 1/DM + 1/θVc could be solved by a single breath manoeuvre using CO and NO. The Bordeaux group published their formula for DM and Vc from a combined single breath DL,NO and DL,COin 1987 (Guenard et al, 1987).The similarity of DM and Vc calculated their way and using Roughton and Forster’s formula and CO at two or more oxygen tensions (Roughton and Forster ,1957) was striking. Importantly among the Bordeaux group’s references was a long forgotten paper from 1958 where Carlsen and Comroe had measured the rate constant for the reaction of NO with red cells in the absence of oxygen (Carlsen and Comroe, 1958).This used the same continuous flow rapid reaction apparatus that Forster had used to measure the rate constant for CO reacting with red cells and hence θCO. It was therefore a relatively simple matter to calculate θNO =1500 mmol min-1 kpA-1 L-1 =4.5 min-1 mmHg-1 (Borland et al 2001, Borland and Cox, 1991). Two other significant events happened in 1987.Two groups proved that NO was identical to Endothelial Derived Relaxing Factor (Ignarro et al, 1987)(Palmer et al, 1987). This enormously increased interest in NO. Secondly Forster made further measurements of θCO, this time
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at physiological pH on a small group of human volunteers and obtained different values to 1957 yielding slightly lower values for Vc and rather larger values for DM (Forster, 1987). By the late 1980’s analysers became available which could detect down to 1ppb.This allowed detection of back tension (endogenous respiratory tract production) as c 10ppb (Borland et al, 1993).They also allowed longer breathhold times than 7.5 seconds. Rapidly responding analysers became available that potentially allowed intrabreath and alveolar profile measurements for steady state methods(Borland et al, 2001). Commercial gas transfer carts incorporating NO analysers also became available using a cheaper but less sensitive fuel cell analyser for NO and thus requiring a shorter breathhold time .Unfortunately as a result of therapeutic use of NO a patent was taken out which greatly increased the cost of clinical use of NO (Pierce et al, 2002). A number of studies appeared over the next twenty-five years measured combined DL,CO and DL,NO in volunteers in normal a variety of different situations and also patients with a variety of different diseases. DL,NO increased more with lung volume than DL,CO (Borland and Higenbottam, 1989,Tsoukias et al ,2000).DL,NO increased linearly on exercise (Borland et al 1989,Zavorsky et al, 2004).At altitude DL,NO along with DL,CO increased in newcomers by day 2/3 returning towards baseline by day 7/8 (Bisschop et al 2010,Martinot et al, 2013).Likewise it was reduced on diving when breathing oxygen (Linnarsson et al 2013, van Ooij et al, 2014) and with increased atmospheric pressure, perhaps by increased gas phase resistance (Linnarsson et al, 2013). In disease DL,NO in each study has generally been related to a control group. As recommended by Hughes and van der Lee (Hughes and van der Lee, 2013) it is helpful to examine changes in DL,NO in relation to the simultaneously measured DL,CO, as the DL,NO / DL,CO ratio. DL,NO is weighted by the membrane component (DM,NO ~ 2.DM,CO), and as DL,CO is weighted towards the red cell contribution (θVc), the DL,NO / DL,CO ratio will reflect the DM,CO/Vc ratio. An increase in DL,NO /
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DL,CO (or DM,CO/Vc) signifies a reduction in Vc which outweighs the reduction in DM, i.e. a predominantly microvascular pathology (Borland et al 1993) (Fraha et al, 2013). Likewise since DL,NO is insensitive to changes in haematocrit, the DL,NO / DL,CO ratio should rise in anaemia. As predicted, van der Lee et al (van der Lee et al, 2007) found an increased DL,NO / DL,CO ratio(5.7) which fell to 4.8) after red cell transfusion associated with an increased [Hb] from 7.8 to 10.4 g‧dL-1. A decrease in DL,NO / DL,CO suggests disorganization of alveolar membrane structures greater than a microvascular change. A recent European Society Task Force report documents studies published to date (Zavorsky et al, in press).Many of the studies have been small. Pathological change may impact on the membrane and capillary at different states of the disease. Heterogeneity may have an unpredictable effect.It may be too early, therefore, to draw firm conclusions. As yet there are no studies in asthma. In most studies the single breath method with Jones-Meade correction was used (Jones and Meade, 1961).
In most studies, also, the Bordeaux group’s assumption of θNO being infinity was adopted by others. The Cambridge group could not accept this hypothesis. It seemed odd to them to adopt θCO measurements calculated from rate constants made with the Philadelphia rapid reaction apparatus yet reject measurements made with NO using the same apparatus just a year later. Yet evidence appeared to support θNO being infinity: the Dutch group found no change in DL,NO before and after transfusion (van der Lee et al, 2007).This observation per se does not exclude θNO being finite: as the molar concentration of haemoglobin far exceeds the tissue NO molar concentration during a single breath DL,NO little Hb is used up in the chemical reaction. In other words the kinetics are pseudo first order and independent of haemoglobin concentration. What was needed was a way of altering the components of the Roughton and Forster equation whilst keeping others constant. The Cambridge group turned to in vitro methods initially looking at the gas/liquid interface in a laboratory flask. However the mass transfer conductance (DM) of the 7
interface was too low. What was needed was an apparatus for maximising blood gas contact. A membrane oxygenator is designed to achieve this function for oxygen and carbon monoxide for patients on heart-lung bypass so why not look at NO and CO? The critical experiment was to examine what happened with haemolysis. If there was a change then clearly the blood resistance in the oxygenator at least was significant. If there were no change the blood resistance must be negligible. An increase in oxygenator diffusing capacity
(D,NO) with haemolysis was indeed
observed (Borland et al, 2006). The difficulty now was how to extend this experiment to the in vivo situation. Obviously haemolysis would irrepairably damage the endothelial lining .In the mid 2000s great interest was aroused by cell-free haem based blood substitutes, the” holy grail” of trauma medicine. A bovine haem based product became available which was licenced for human use in South Africa which has a major problem of HIV blood contamination .It underwent US Navy trials but was withdrawn because of safety concerns. However it retained its US canine veterinary licence and that gave Hsia and colleagues at the Houston Group the opportunity to investigated its effect on DL,NO and DL,CO in three dogs and the Cambridge group to do a parallel in vitro study using horse blood in their oxygenator model. Almost identical increases with cell free haem were noted proving that the resistance of the blood to NO transfer was significant (Borland et al, 2010). This resistance could lie in plasma the red cell membrane or interior. The Cambridge group have attempted to alter each barrier in turn (Borland et al, 2014). They added a high molecular weight polysaccharide to the extracellular fluid to alter viscosity and they hoped diffusivity. They compared horse to (much larger) human red cells and altered the intracellular diffusion barrier by progressively converting oxyhaemoglobin to methaemoglobin by adding nitrite. Only altering the interior altered D,NO. Two bioengineering groups have pointed out that colloids do not obey the Stokes Einstein Law (Miller, 1924). In other words diffusivity is independent of viscosity. The case for or against a plasma barrier therefore needs further work. In 2010 the Pennsylvania group measured the rate of uptake of NO by human red cells in a Continuous Flow rapid reaction apparatus at pO2 75-350 torr and a
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1:40 suspension of human red cells and obtained a value for θNO of 2.8 ml ml-1 min-1 torr-1 (Botros et al unpublished observations.) US groups particularly studying DL,NO on exercise (Coffman et al 2016,Hsia, 2002, Tamhane et al , 2001) have consistently adopted the view that θNO is infinite and that α, the ratio of DMNO/DMCO exceeds 2. The basis for this is that their approach results in values for DM that are believable and increase on exercise. One intriguing explanation for α exceeding 2 might be a membrane carrier; Aquaporin-1 has been claimed as an NO carrier at physiological concentrations (Herrera et al, 2006).However if there were a carrier saturation should occur.This would give lower transfer rate constants at higher concentrations. In a single individual DL,NO at 0.7 ppm was 142 ml /min/mmHg whereas at 40ppm it was 158 ml/min/mmHg (C Borland unpublished observations.) This finding does not support a membrane carrier hypothesis. The Bordeaux group have recently published a value for θCO which uniquely yields believable (and similar) values for DM and Vc using both the multiple oxygen DLCO approach and via DL,NO (Guenard et al, 2016). Hughes and van der Lee have advocated the DL,NO / DL,CO ratio avoiding speculation about θNO and indeed about θCO about which there seems almost as much disagreement as to whether the 1987 or 1957 values should be used. If the 1987 values for θCO are used negative values for DM are often calculated. Forster himself (Forster, 1987) underplays DM regarding it as what gets left after calculating Vc. His interpretation of DM includes the entire diffusion path from alveolus across the alveolar capillary membrane through plasma and red cell membrane to the haemoglobin layer immediately adjacent to the membrane. Hughes and van der Lee suggest that the DL,NO / DL,CO ratio (Hughes and van der Lee, 2013) which will reflect DM/Vc is a better interpretation than calculating suspect numbers. However it is important to remember that anaemia and free haemoglobin from haemolysis will also increase the DL,NO / DL,CO ratio.
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One of the major strengths of DL,CO has been the development of prediction equations derived from measurements on normal individuals (Crapo and Morris 1981,Gulsvik et al 1992,Knudson et al 1987,Miller et al 1983,Neas et al 1996, Paoletti et al 1985, Pesola et al 2004,Robers et al 1991). However, in the past 10 years, several other groups have developed prediction equations for DL,NO; in children, for example (Thomas et al., 2014), and in Caucasian adults (Aguilianu 2008,van der Lee 2007, Zavorsky et al., 2008). The data in adults from those studies have recently been amalgamated in the first ERS Task Force Document on DL,NO so that a more representative prediction equation for DL,NO was made (Zavorsky et al., 2017). Over thirty years on from the original measurements of DL,NO on volunteers the test remains enigmatic and not in widespread use. By contrast the single breath DL,CO was in use in all respiratory function laboratories twenty five years after it was described.NO is a more unstable gas and because of controversial patent laws on its human use more expensive. Sensitive analysers are more expensive. Cheaper analysers demand a shorter breathhold time. The ERS task force has now achieved consensus on the optimal inspired concentration, breathhold time and sample collection together with reference equations. We are therefore confident that the test will gain widespread international acceptance and use.
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S1569-9048(16)30299-3 http://dx.doi.org/doi:10.1016/j.resp.2016.11.014 RESPNB 2731
To appear in:
Respiratory Physiology & Neurobiology
Received date: Revised date: Accepted date:
9-10-2016 27-11-2016 28-11-2016
Please cite this article as: Borland, Colin DR, Gu´enard, Herv´e, The history of the pulmonary diffusing capacity for nitric oxide DL,NO.Respiratory Physiology and Neurobiology http://dx.doi.org/10.1016/j.resp.2016.11.014 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
The history of the pulmonary diffusing capacity for nitric oxide DL,NO
1
Colin DR Borland
2
Hervé Guénard
1
Department of Medicine University of Cambridge, UK and Hinchingbrooke Hospital ,
Huntingdon, UK [email protected] 2
Physiologie
et
EFR,
Université
Bordeaux
2
et
CHU
Bordeaux,
France
[email protected].
Highlights
The DLNO (TLNO) had unexpected origins Adoption of the technique has been slow We believe that its worldwide use will increase
1
Abstract The DL,NO (TL,NO) had its unexpected origins in the Paris “events” of 1968 and the unsuccessful efforts of the UK tobacco industry in the 1970’s to create a ” safer cigarette.” Adoption of the technique has been slow due to the instability of NO in air, lack of standardisation of the technique and lack of agreement as to whether DL,NO is equal to or merely reflects membrane diffusing capacity (DM.) With the availability of inexpensive analysers, standardisation of the technique and publication of reference equations we believe that its worldwide use will increase.
Keywords: Diffusing capacity,Nitric Oxide; Carbon monoxide; History
History Early research on Nitric Oxide (NO) in the United Kingdom (UK) was directed towards its toxicology rather than its physiology. In the late 1970’s “oxides of nitrogen” were suspected as a cause of lung disease from atmospheric pollution, from indoor air pollution and from cigarette smoke. High doses (over 100 parts per million (ppm)) of nitric oxide and lower doses of nitrogen dioxide (25 ppm) were known to cause lung damage in cases of accidental human exposure (Clutton Brock, 1967) or experimental animal exposure (Roe, 1985). Interestingly an emphysema like lesion had been described (Roe, 1985) fuelling speculation that “oxides of nitrogen” caused emphysema in smokers. At that time the tobacco industry was under pressure to produce a “safer cigarette”. To investigate nitric oxide in cigarette smoke scientists at British American Tobacco built a prototype NO gas analyser. It was based on Professor Brian Thrush’s description of chemiluminescence (Clyne et al, 1964) :the analyser generated ozone from atmospheric oxygen which was reacted with the smoke sample. Any nitric oxide was immediately converted to nitrogen dioxide in an excited state which on return to the ground state emitted photons of light. This highly sensitive and specific technique has underpinned most of the work on lung nitric oxide uptake. As with the infrared-carbon monoxide 2
analyser physiological measurement followed the technology. Tim Higenbottam at Cambridge collaborated with British American Tobacco who loaned him the analyser. He and colleagues had previously failed to show a relationship between smokers’ cigarette tar yields and prevalence of airflow obstruction in a large epidemiological study (Higenbottam et al, 1980). They concluded that “urgent attention be paid to the gas phase” of which carbon monoxide and nitric oxide were the major constituents (Higenbottam et al, 1980). CDR Borland’s doctorate project (Borland, 1988) was to compare NO and CO uptake using the chemiluminescent analyser for NO and a commercial infrared CO with a Helium analyser bolted on. He looked at 40parts per million which was what he calculated (erroneously as it happened!) to be the alveolar NO concentration when a smoker inhaled a popular UK brand of cigarette. Initially Borland et al investigated percent retention comparing volume of gas exhaled to inhaled (Borland and Higenbottam, 1985). He found that uptake only occurred when volumes in excess of dead space were inhaled. Their then technician Andrew Chamberlain (now Professor of Bioarchaeology at the University of Manchester, UK) who was spending a gap year as a respiratory physiology technician prior to his PhD suggested that they measure the (TL,NO) DL,NO. Borland, influenced by JMB Hughes’ teaching at the Royal Postgraduate Medical school favoured the KNO believing K to be the fundamental parameter of alveolar gas transfer (Hughes and Pride, 2001). The concentration of NO used (40 parts per million ) was stable to oxidation in air with a half life of about an hour (Borland and Higenbottam, 1989). The NO was stored in nitrogen and the mixture was made up immediately before the manoeuvre. Allowance was made for oxidation to NO2 and analysis was performed before the sample passed through sodalime or Calcium Chloride as this was found to cause loss of signal (Borland and Higenbottam, 1989). Their initial findings on a group of healthy male volunteers were presented to the UK Medical Research Society in Spring 1983 (Borland et al, 1983) :KNO was highly correlated with but exceeded KCO four to five fold and like KCO increased with exercise. They used a 40ppm inhaled concentration but had to shorten the breathhold time to have sufficient (>1ppm) exhalate. In a second abstract they measured DL,NO and DL,CO and calculated DM assuming θNO to be infinity (Borland et al, 3
1984). However by a third abstract where they compared a group of patients with anaemia due to renal failure(Scott et al, 1987) to their normal volunteers they modified their conclusions believing the blood resistance to be significant; in retrospect they were right but for the wrong reasons as DL,NO does not alter with Hb concentrations encountered clinically. Finally the paper for their original work on volunteers was published in 1989 (Borland and Higenbottam, 1989).It contained larger numbers and with observations on varying
time indicating semilogarithmic uptake (as
suggested by Dr John Cotes), undetectable back tension within the sensitivity of the analyser (1ppm), greater volume dependence of DL,NO compared to DL,CO and independence of hyperoxia. Going back to the original reasons for the Cambridge group’s work it is seems very unlikely that NO in smokers causes emphysema. Histologically NO2 emphysema in rats is very different from smokers’ emphysema or tobacco lung damage in laboratory rats (Roe ,1985). There are very large international differences between cigarette NO yields which do not parallel emphysema incidence (Borland and Higenbottam, 1985). Finally, relevant to the present discussion, the half life of oxidation of NO to the far more lung irritant NO2 in air (Borland and Higenbottam, 1989) or tobacco smoke (Borland et al, 1985) is one hour compared to two seconds for alveolar lung uptake. Nevertheless, the work that the NO-emphysema hypothesis inspired has stimulated NO use as a lung function test and indeed was the phase 1 trial for its therapeutic use. The French connection occurred quite independently, one positive side-effect of the “events” of May 1968 , which was a period of deep social agitation mainly in Paris and led mainly to nothing. Professor Daniel Bargeton, Hervé Guenard’s mentor, was the source of the concept of DMCO and Vc calculation using two tracer gases. At that time Prof Bargeton was vice dean of the Paris medical faculty which was a heavy commitment. As the University had come to a standstill during this period he stopped his administrative duties and went back to research projects in his office at Rue des St Pères. As usual he wrote the theory with a black pencil on white sheets of paper where fine horizontal lines were previously drawn; in case of error he would rub it and he never threw a sheet
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in the garbage bin. The concept of the double tracer was sound but unfortunately he suggested using CO and Hydrogen Sulphide (H2S). This last gas had several “inconveniences”; quite apart from the odour, the analysis of this gas with a mass spectrometer led to deposition of sulphur in the ionisation chamber which drove the engineer in charge of the device mad. Furthermore H2S was too soluble in water and would disappeared rapidly in the airway walls. Hervé Guenard started working in his lab after the revolution. Though the smell of rotten egg had faded the episode was engraved in everybody’s mind. Professor Bargeton gave Hervé Guenard’s a copy of his initial text written with his pencil. Hervé sought a gas less soluble, reactive with Hb, not toxic and easily analysable. Years later, in 1979, after arriving in Bordeaux Hervé Guenard looked at a list of gases in a chemistry book and made a selection of candidates. Sulphur compounds had for the most part the same drawbacks: too soluble, smelly and not easy to analyse.. The nitrogen oxides list was shorter and the choice of nitrogen monoxide was easy as others oxides N2O2 and NO2 did not fit the specifications. Hervé Guenard therefore reasoned that 1/DL = 1/DM + 1/θVc could be solved by a single breath manoeuvre using CO and NO. The Bordeaux group published their formula for DM and Vc from a combined single breath DL,NO and DL,COin 1987 (Guenard et al, 1987).The similarity of DM and Vc calculated their way and using Roughton and Forster’s formula and CO at two or more oxygen tensions (Roughton and Forster ,1957) was striking. Importantly among the Bordeaux group’s references was a long forgotten paper from 1958 where Carlsen and Comroe had measured the rate constant for the reaction of NO with red cells in the absence of oxygen (Carlsen and Comroe, 1958).This used the same continuous flow rapid reaction apparatus that Forster had used to measure the rate constant for CO reacting with red cells and hence θCO. It was therefore a relatively simple matter to calculate θNO =1500 mmol min-1 kpA-1 L-1 =4.5 min-1 mmHg-1 (Borland et al 2001, Borland and Cox, 1991). Two other significant events happened in 1987.Two groups proved that NO was identical to Endothelial Derived Relaxing Factor (Ignarro et al, 1987)(Palmer et al, 1987). This enormously increased interest in NO. Secondly Forster made further measurements of θCO, this time
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at physiological pH on a small group of human volunteers and obtained different values to 1957 yielding slightly lower values for Vc and rather larger values for DM (Forster, 1987). By the late 1980’s analysers became available which could detect down to 1ppb.This allowed detection of back tension (endogenous respiratory tract production) as c 10ppb (Borland et al, 1993).They also allowed longer breathhold times than 7.5 seconds. Rapidly responding analysers became available that potentially allowed intrabreath and alveolar profile measurements for steady state methods(Borland et al, 2001). Commercial gas transfer carts incorporating NO analysers also became available using a cheaper but less sensitive fuel cell analyser for NO and thus requiring a shorter breathhold time .Unfortunately as a result of therapeutic use of NO a patent was taken out which greatly increased the cost of clinical use of NO (Pierce et al, 2002). A number of studies appeared over the next twenty-five years measured combined DL,CO and DL,NO in volunteers in normal a variety of different situations and also patients with a variety of different diseases. DL,NO increased more with lung volume than DL,CO (Borland and Higenbottam, 1989,Tsoukias et al ,2000).DL,NO increased linearly on exercise (Borland et al 1989,Zavorsky et al, 2004).At altitude DL,NO along with DL,CO increased in newcomers by day 2/3 returning towards baseline by day 7/8 (Bisschop et al 2010,Martinot et al, 2013).Likewise it was reduced on diving when breathing oxygen (Linnarsson et al 2013, van Ooij et al, 2014) and with increased atmospheric pressure, perhaps by increased gas phase resistance (Linnarsson et al, 2013). In disease DL,NO in each study has generally been related to a control group. As recommended by Hughes and van der Lee (Hughes and van der Lee, 2013) it is helpful to examine changes in DL,NO in relation to the simultaneously measured DL,CO, as the DL,NO / DL,CO ratio. DL,NO is weighted by the membrane component (DM,NO ~ 2.DM,CO), and as DL,CO is weighted towards the red cell contribution (θVc), the DL,NO / DL,CO ratio will reflect the DM,CO/Vc ratio. An increase in DL,NO /
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DL,CO (or DM,CO/Vc) signifies a reduction in Vc which outweighs the reduction in DM, i.e. a predominantly microvascular pathology (Borland et al 1993) (Fraha et al, 2013). Likewise since DL,NO is insensitive to changes in haematocrit, the DL,NO / DL,CO ratio should rise in anaemia. As predicted, van der Lee et al (van der Lee et al, 2007) found an increased DL,NO / DL,CO ratio(5.7) which fell to 4.8) after red cell transfusion associated with an increased [Hb] from 7.8 to 10.4 g‧dL-1. A decrease in DL,NO / DL,CO suggests disorganization of alveolar membrane structures greater than a microvascular change. A recent European Society Task Force report documents studies published to date (Zavorsky et al, in press).Many of the studies have been small. Pathological change may impact on the membrane and capillary at different states of the disease. Heterogeneity may have an unpredictable effect.It may be too early, therefore, to draw firm conclusions. As yet there are no studies in asthma. In most studies the single breath method with Jones-Meade correction was used (Jones and Meade, 1961).
In most studies, also, the Bordeaux group’s assumption of θNO being infinity was adopted by others. The Cambridge group could not accept this hypothesis. It seemed odd to them to adopt θCO measurements calculated from rate constants made with the Philadelphia rapid reaction apparatus yet reject measurements made with NO using the same apparatus just a year later. Yet evidence appeared to support θNO being infinity: the Dutch group found no change in DL,NO before and after transfusion (van der Lee et al, 2007).This observation per se does not exclude θNO being finite: as the molar concentration of haemoglobin far exceeds the tissue NO molar concentration during a single breath DL,NO little Hb is used up in the chemical reaction. In other words the kinetics are pseudo first order and independent of haemoglobin concentration. What was needed was a way of altering the components of the Roughton and Forster equation whilst keeping others constant. The Cambridge group turned to in vitro methods initially looking at the gas/liquid interface in a laboratory flask. However the mass transfer conductance (DM) of the 7
interface was too low. What was needed was an apparatus for maximising blood gas contact. A membrane oxygenator is designed to achieve this function for oxygen and carbon monoxide for patients on heart-lung bypass so why not look at NO and CO? The critical experiment was to examine what happened with haemolysis. If there was a change then clearly the blood resistance in the oxygenator at least was significant. If there were no change the blood resistance must be negligible. An increase in oxygenator diffusing capacity
(D,NO) with haemolysis was indeed
observed (Borland et al, 2006). The difficulty now was how to extend this experiment to the in vivo situation. Obviously haemolysis would irrepairably damage the endothelial lining .In the mid 2000s great interest was aroused by cell-free haem based blood substitutes, the” holy grail” of trauma medicine. A bovine haem based product became available which was licenced for human use in South Africa which has a major problem of HIV blood contamination .It underwent US Navy trials but was withdrawn because of safety concerns. However it retained its US canine veterinary licence and that gave Hsia and colleagues at the Houston Group the opportunity to investigated its effect on DL,NO and DL,CO in three dogs and the Cambridge group to do a parallel in vitro study using horse blood in their oxygenator model. Almost identical increases with cell free haem were noted proving that the resistance of the blood to NO transfer was significant (Borland et al, 2010). This resistance could lie in plasma the red cell membrane or interior. The Cambridge group have attempted to alter each barrier in turn (Borland et al, 2014). They added a high molecular weight polysaccharide to the extracellular fluid to alter viscosity and they hoped diffusivity. They compared horse to (much larger) human red cells and altered the intracellular diffusion barrier by progressively converting oxyhaemoglobin to methaemoglobin by adding nitrite. Only altering the interior altered D,NO. Two bioengineering groups have pointed out that colloids do not obey the Stokes Einstein Law (Miller, 1924). In other words diffusivity is independent of viscosity. The case for or against a plasma barrier therefore needs further work. In 2010 the Pennsylvania group measured the rate of uptake of NO by human red cells in a Continuous Flow rapid reaction apparatus at pO2 75-350 torr and a
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1:40 suspension of human red cells and obtained a value for θNO of 2.8 ml ml-1 min-1 torr-1 (Botros et al unpublished observations.) US groups particularly studying DL,NO on exercise (Coffman et al 2016,Hsia, 2002, Tamhane et al , 2001) have consistently adopted the view that θNO is infinite and that α, the ratio of DMNO/DMCO exceeds 2. The basis for this is that their approach results in values for DM that are believable and increase on exercise. One intriguing explanation for α exceeding 2 might be a membrane carrier; Aquaporin-1 has been claimed as an NO carrier at physiological concentrations (Herrera et al, 2006).However if there were a carrier saturation should occur.This would give lower transfer rate constants at higher concentrations. In a single individual DL,NO at 0.7 ppm was 142 ml /min/mmHg whereas at 40ppm it was 158 ml/min/mmHg (C Borland unpublished observations.) This finding does not support a membrane carrier hypothesis. The Bordeaux group have recently published a value for θCO which uniquely yields believable (and similar) values for DM and Vc using both the multiple oxygen DLCO approach and via DL,NO (Guenard et al, 2016). Hughes and van der Lee have advocated the DL,NO / DL,CO ratio avoiding speculation about θNO and indeed about θCO about which there seems almost as much disagreement as to whether the 1987 or 1957 values should be used. If the 1987 values for θCO are used negative values for DM are often calculated. Forster himself (Forster, 1987) underplays DM regarding it as what gets left after calculating Vc. His interpretation of DM includes the entire diffusion path from alveolus across the alveolar capillary membrane through plasma and red cell membrane to the haemoglobin layer immediately adjacent to the membrane. Hughes and van der Lee suggest that the DL,NO / DL,CO ratio (Hughes and van der Lee, 2013) which will reflect DM/Vc is a better interpretation than calculating suspect numbers. However it is important to remember that anaemia and free haemoglobin from haemolysis will also increase the DL,NO / DL,CO ratio.
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One of the major strengths of DL,CO has been the development of prediction equations derived from measurements on normal individuals (Crapo and Morris 1981,Gulsvik et al 1992,Knudson et al 1987,Miller et al 1983,Neas et al 1996, Paoletti et al 1985, Pesola et al 2004,Robers et al 1991). However, in the past 10 years, several other groups have developed prediction equations for DL,NO; in children, for example (Thomas et al., 2014), and in Caucasian adults (Aguilianu 2008,van der Lee 2007, Zavorsky et al., 2008). The data in adults from those studies have recently been amalgamated in the first ERS Task Force Document on DL,NO so that a more representative prediction equation for DL,NO was made (Zavorsky et al., 2017). Over thirty years on from the original measurements of DL,NO on volunteers the test remains enigmatic and not in widespread use. By contrast the single breath DL,CO was in use in all respiratory function laboratories twenty five years after it was described.NO is a more unstable gas and because of controversial patent laws on its human use more expensive. Sensitive analysers are more expensive. Cheaper analysers demand a shorter breathhold time. The ERS task force has now achieved consensus on the optimal inspired concentration, breathhold time and sample collection together with reference equations. We are therefore confident that the test will gain widespread international acceptance and use.
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