Nitric oxide diffusing capacity versus spirometry in the early diagnosis of emphysema in smokers

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1 Respiratory Medicine (2009) 103, 1892e1897 available at journal homepage: Nitric oxide diffusing capacity versus spirometry in the early diagnosis of emphysema in smokers I. van der Lee a, *, H.A. Gietema b, P. Zanen c,d, R.J. van Klaveren e, M. Prokop b, J.-W.J. Lammers d, J.M.M. van den Bosch c a Spaarne Hospital, Department of Pulmonary Diseases, Hoofddorp, The Netherlands b University Medical Center, Department of Radiology, Utrecht, The Netherlands c St Antonius Hospital, Department of Pulmonary Diseases, Nieuwegein, The Netherlands d University Medical Center, Department of Pulmonary Diseases, Utrecht, the Netherlands e Department of Pulmonology, Erasmus Medical Center, Rotterdam, The Netherlands Received 5 August 2008; accepted 8 June 2009 Available online 7 July 2009 KEYWORDS COPD; Emphysema; Diffusion capacity; Spirometry; Nitric oxide Summary The diffusion capacity for nitric oxide (DLNO) is independent of pulmonary capillary blood volume and equals the membrane diffusing capacity. Therefore the DLNO could be more sensitive in detecting alveolar destruction than the DLCO. We measured flow-volumes curves, DLNO, DLCO, the transfer coefficients KNO (DLNO/VA) and KCO (DLCO/VA) and performed computed tomography (CT) scans in 263 randomly selected heavy smokers. Subjects with areas 1% of the total lung volume showing an attenuation < 950 Hounsfield Units were considered to have emphysema. In 36 subjects emphysema was diagnosed with CT, a low KNO was present in 94 subjects, and in 95 subjects a FEV1/FVC ratio <70% was seen. The area under the ROC curve for detection CT-based emphysema was for the KNO, for the KCO and for FEV1/FVC, meaning that the KNO has a slightly higher sensitivity to detect emphysema than the KCO and FEV1/FVC. The positive predictive value of KNO however was low (34.7%), while the negative predictive value of KNO was very high (98.2%), indicating an emphysema exclusion test. The DLNO/DLCO ratio is significantly higher in the study group compared to normal subjects. ª 2009 Elsevier Ltd. All rights reserved. Introduction * Correspondence to: I. van der Lee, Spaarne Ziekenhuis, Department of Pulmonary Diseases, PO Box 770, 2130 AT Hoofddorp, The Netherlands, Tel.: þ ; fax: þ address: vdlee@tiscali.nl (I. van der Lee). The pathological findings in chronic obstructive pulmonary disease (COPD) are chronic bronchitis, small airways disease and emphysema. 1 Chronic bronchitis is clinically characterized by cough and increased sputum production; airway obstruction can be measured by spirometry /$ - see front matter ª 2009 Elsevier Ltd. All rights reserved. doi: /j.rmed

2 Early diagnosis of emphysema in smokers 1893 However, a diagnosis of emphysema, especially early in the disease, is often more difficult to make. Emphysema can be quantified by measuring the percentage of low attenuation areas on high resolution computed tomography (HRCT) scans, which has good correlation with the amount of emphysema present in lung specimens. 2 The HRCT emphysema indices are stronger correlated with the carbon monoxide (CO) diffusion capacity (DLCO) and coefficient (KCO) than with the FEV 1 and FEV 1 /FVC ratio. 3e5 Nitric oxide (NO) has a much stronger affinity for haemoglobin than CO, therefore the NO diffusion capacity (DLNO) is not limited by the pulmonary capillary blood volume (Vcap) and equals the true membrane diffusing capacity (Dm). 6e8 Because emphysema is characterized by loss of functional alveoli, 9 and thus a decrease in Dm, we hypothesized that the DLNO is more sensitive in diagnosing emphysema than the DLCO. We are aware of one other study measuring the DLNO in subjects with COPD, 10 in which a very short breathholding time of 3 s was used, which makes the results difficult to compare with studies in which a breathholding time of 10 s was used. A simultaneous single breath DLNO and DLCO measurement gives additional information about the cause of the diffusion deficit. 11,12 In case of decreased Vcap, the DLCO will lower but the DLNO not, therefore the DLNO/DLCO ratio will increase. With a decreased alveolocapillary membrane surface, both the DLNO and DLCO will be hampered, and the DLNO/DLCO ratio will be unchanged. A lowering of the ratio is also possible, as the DLNO is influenced by alveolocapillary membrane surface only. For example, subjects with pulmonary hypertension have a higher DLNO/DLCO ratio than normal subjects. 12,13 The DLNO/DLCO ratio is independent of exercise intensity, 14 although others found a slight decrease from rest to exercise using a rebreathing technique. 15 Recent research in healthy subjects showed an inverse relationship between the DLNO/DLCO ratio and the thickness of alveolar membrane and capillary sheet. 16 The main question of this study was whether the DLNO is more sensitive in diagnosing emphysema in a large group of heavy smokers than the DLCO. The secondary question was whether the DLNO/DLCO ratio differs in subjects with emphysema compared to healthy individuals. Methods Subjects All subjects were participating in the NELSON-project, a Dutch-Belgian multi-centre lung cancer screening trial. 17 The subjects were all male, 50e75 years of age with a smoking history of at least 20 pack years. The NELSONproject was approved by the Dutch ministry of health and by the local ethics committee; informed consent was obtained from all participants. A random selection of the screened subjects performed pulmonary function testing. Pulmonary function testing Spirometry and flow-volume curves were obtained via pneumotachography, according to ERS guidelines. 18 No reversibility testing was done. A FEV1/FVC ratio <70% was labelled as abnormal, according to the GOLD classification. 19 The DLNO and DLCO were measured in one single breath maneuver, with test gas containing a mixture of CO 0.25%, He 9.17%, and NO 8 ppm with balance air. A small amount NO from a separate tank containing 750 ppm NO in nitrogen (Hoekloos Medical; Schiedam, the Netherlands) was added to the test gas directly before each measurement to dilute to 8 ppm. The procedure was performed with an effective breathholding period of 10 s (Jones and Meade method 20 ), discard volume of 750 ml, and a sample volume 750 ml. Standard DLCO measurement was performed on a MasterLab Pro (Erich Jaeger GmbH, Germany). NO-concentration was measured in inspiratory and expiratory gas mixture with the aid of a chemoluminescence analyzer (CLD 77 AM; Eco Physics; Zurich, Switzerland (lower detection limit 0.02e 0.05 parts per billion (ppb); upper detection limit 10 ppm; reaction time 0.1 s)). The chemoluminescence analyzer was calibrated with 5 ppm NO in nitrogen and NO-free air on a weekly basis. All connections between the NO analyzer and the inspiratory and expiratory bags were made of polytetrafluoroethylene (Teflon; DuPont; Wilmington, DE) or stainless steel, which do not interact with NO. The alveolar volume (VA) and DLCO were calculated according to European Respiratory Society recommendations, 21 the DLNO was calculated according to the method described by Borland et al. 6 Endogenous NO levels and CO backpressure were ignored, and smoking was allowed until 24 hours before testing. A correction for haemoglobin (Hb) levels was not made, because Stam et al. 22 showed that in healthy volunteers Hb correction only has a very limited effect. The DLCO and DLNO were corrected to BTPS conditions, and a minimum of two measurements was performed, in which a variability of 10% or less for the VA and DLCO was acceptable. All pulmonary function values are given as a mean with standard deviation (SD), and as percentage of predicted values (%pred) 18,21,23. For the DLNO we used references equations that we published previously. 24 Values below 1.64*SD were considered abnormal. The DLNO/DLCO and FEV 1 /FVC ratios are expressed as absolute values. CT Scanning and emphysema quantification The CT scan was performed on a 16 detector-row scanner (Mx8000 IDT or Brilliance 16P, Philips Medical Systems, Cleveland, OH), scan time was 12 s from apex to base, in spiral mode with mm collimation, 1.0 mm reconstruction thickness, without contrast-injection. After automatic lung segmentation connecting all areas below 500 Hounsfield Units (HU) starting in the trachea and excluding the main bronchi, quantification of emphysema was done with a density mask method, 25 with a threshold of 950 HU. Emphysema scores (ES) were calculated as the volume with an attenuation < 950 HU indexed to total lung volume. Subjects with an ES 1% were considered to suffer from emphysema. 3,26,27 Statistics Values are given as means with standard deviations (SD) and as percentage of predicted values. Spearman correlation

3 1894 I. van der Lee et al. coefficients were used to assess significant relationships; the area under the curve (AUC) of the receiver operator curves (ROC) were used to assess the capability of the pulmonary function parameters to signal the absence or the presence of emphysema. One-way ANOVA was used to compare differences between groups. All statistics were calculated with SPSS for Windows release 17 (SPSS Inc, Chicago, USA). Results A total of 263 male subjects were included in the study. Characteristics of the study population are displayed in Table 1. Emphysema (ES 1) was present in 36 subjects (13.6%). According to spirometric criteria, 168 subjects had normal spirometry or were in GOLD 0, 68 subjects were in GOLD 1, 26 subjects were in GOLD 2, and 1 subject was in GOLD class Significant negative correlations were observed between all diffusion capacity parameters, the FEV1/FVC ratio and the ES (Table 2). The mean DLNO/DLCO in the entire sample ratio was 4.9, which is significantly higher (p < 0.001) than the value of 4.4 we measured earlier in healthy subjects, 24 and higher than the value of 4.3 measured by Borland. 6 The AUC of the ROC curves (Fig. 1) showed highest values for KNO (%pred) to detect emphysema; KCO (%pred) and FEV 1 /FVC had slightly lower values (Table 3). The sensitivities and specificities of all parameters to detect emphysema are given in Table 4, as well as the positive (PPV) and negative (NPV) predictive values. The NPV of KNO is very high indicating that a normal KNO virtually excludes emphysema. The low PPV values indicate that an abnormal KCO, KNO or FEV 1 /FVC are often present in the absence of emphysema. This is graphically shown in Fig. 2. Subjects with low KNO values, subjects with emphysema on CT and subjects with a FEV1/FVC <70% form partially overlapping groups, which can be illustrated with a non-proportional Venn diagram (Fig. 3). The mean DLNO/ DLCO ratio was 5.0 (SD 0.49) in all subjects with abnormal test results (i.e. low KNO, FEV1/FVC <70% or ES > 1%, thus all subjects included in the Venn diagram), versus 4.8 (SD 0.37) in subjects with normal test results, which is a statistical significant difference (p < 0.001). Table 2 Correlation coefficients of the emphysema score versus pulmonary function data (*p < 0.01). Emphysema score FEV 1 /FVC DLCO, %predicted KCO, %predicted DLNO, %predicted KNO, %predicted Discussion 0.43* 0.26* 0.38* 0.29* 0.50* The main finding of this study is the fact that the KNO has (slightly) greater power for the detection of CT-based emphysema than the KCO. Another important finding of this study is the fact that the DLNO/DLCO ratio in this group of heavy (ex) smokers is significantly higher than in healthy subjects. The values we use for the DLNO/DLCO ratio in healthy subjects is similar to those found by other researchers: Aguilaniu et al. found a DLNO/DLCO ratio of and Zavorsky et al. published a ratio of 4.52 in his earlier study. 14 The DLNO/DLCO ratio in the study group reaches values found in subjects with pulmonary arterial hypertension. 11e13 The DLNO/DLCO ratio is even more increased in the group of subjects with abnormal results for FEV1/FVC, KNO or emphysema score. Unfortunately, the study group is too small to measure a dose response relationship between pack years smoking and the DLNO/DLCO ratio or other pulmonary function tests. Because of the variable individual susceptibility for the toxic effects of cigarette smoke, very large cohorts are needed to find a significant relationship between pack years smoking and Table 1 Characteristics of the study population, n Z 263. Mean Range SD Age, years e Height, m e VC, %pred e FEV 1 /FVC, %pred e FEV 1, %pred e FEV 1 /FVC e DLCO, %pred e KCO, %pred e DLNO, %pred e KNO, %pred e DLNO/DLCO ratio e ES (emphysema score) e Figure 1 ROC curves of pulmonary function parameters as a diagnostic for CT-based emphysema.

4 Early diagnosis of emphysema in smokers 1895 Table 3 Area under the curve (AUC) with 95% confidence intervals (95% CI) of the receiver operator curves (ROC) for detecting of emphysema (ES 1). AUC p 95%CI FEV 1, %pred < e0.761 FEV 1 /FVC, %pred < e0.880 DLCO, %pred < e0.833 KCO, %pred < e0.887 DLNO, %pred < e0.815 KNO, %pred < e0.938 pulmonary function tests. The increased DLNO/DLCO ratio is observed in the complete study sample, where most subjects had no CT-detectable emphysema. The Roughton and Forster equation 29 assumes that DmCO and Vcap are independent components by stating that the total DLCO resistance is a sum of two resistances. However, capillary blood flow is a prerequisite for a DmCO to exist and so DmCO must be dependent on capillary blood flow. Furthermore, research has pointed out that the DmCO is dependent on haemoglobin concentration, and indeed a relationship between DmCO and Vcap exists. 30 If the DmCO and the Vcap components are dependent variables, the separation of DLCO in these two components is a pure mathematical concept, and simplifies the involved pathophysiology. Another important argument against the use of DmCO and Vcap is the fact that the correct calculation is still open for debate: disagreement exists on the correct Q- value needed for the Roughton and Forster equation. Using DLNO to obtain an estimate for DmCO is also under debate: some researchers use a Z and others use a Z in the DmCO Z DLNO/a equation. Therefore, in calculating DmCO and Vcap one uses parameters that are based on an assumption of independency, that have non-assessed clinical value 31 and the involved physiological principles have been simplified. Furthermore, an ongoing but still unresolved debate which way DmCO should be calculated 7,32,33 is present. By using the DLNO/DLCO ratio, we can avoid these unresolved methodological problems; we expect that the clinical utility of the DLNO/DLCO ratio will be greater than the use of the components DmCO and Vcap. As DLNO is not governed by capillary blood volume and DLCO is, a strong relationship between DLNO/DLCO and Dm/Vcap is logical, because the same factors influence both ratios. 28 These are the main reasons that we choose NOT to use calculated DmCO and Vcap values, but only DLNO/DLCO ratios instead. Figure 2 Scatterplot of pulmonary function parameters versus CT-based emphysema. The population in this study is slightly older than the population we used for reference equations, 24 Zavorsky et al. 33 showed that age dependent effects for the DLNO as well as the DLCO are present. Whether the DLNO/DLCO ratio is age dependent is not clear. Although further research on this item is necessary, we are convinced that the small age difference cannot explain the difference in the DLNO/DLCO ratio in healthy subjects and heavy smokers. The cause of alveolar destruction in emphysema is subject of research, but until now it is not clear whether apoptosis of alveolar epithelial cells or apoptosis of endothelial cells are the origin of the pathological destruction. 34 In experimental animal models, blockage of vascular endothelial growth factor (VEGF) resulted in endothelial cell apoptosis and subsequent development of emphysema. 35 It is conceivable that microvascular malfunctioning is present in an early stage of the disease, later followed by the development of tissue loss or measurable emphysema. Another argument can be found in the development of pulmonary hypertension secondary to COPD, which can be seen in a subgroup of patients with severe COPD. The classic explanation is that tissue hypoxia leads to vasoconstriction, which in the end becomes irreversible due to remodeling of the vascular wall. More recent insights points at a completely different cause: microvascular changes are the key factor in the development of secondary pulmonary Table 4 Sensitivity, specificity, positive predictive values (PPV) and negative predictive values (NPV) of the measured parameters to detect emphysema. Sensitivity Specificity PPV NPV DLCO 58.3% 81.5% 33.3% 92.5% DLNO 50.0% 81.9% 30.5% 91.2% KCO 88.9% 57.3% 24.8% 97.0% KNO 91.7% 72.7% 34.7% 98.2% FEV 1 /FVC 77.8% 70.1% 29.5% 95.2% Figure 3 Non-proportional Venn diagram: the presence of emphysema on CT (n Z 36), a decreased KNO (n Z 94) and FEV1/FVC < 70% (n Z 95) are partially overlapping entities.

5 1896 I. van der Lee et al. hypertension, irrespective of pulmonary arterial oxygen tension. 36 This point at the possibility that vascular changes are key factors in the cause and the consequence of the progression of COPD. The focus of pulmonologists on airway obstruction is logical: it is the most distinguishing feature of COPD, and strongly correlated with performance, quality of life and survival. On the other hand, smoking induces a lot of systemic effects, e.g. arteriosclerosis and delayed wound healing due to endothelial dysfunction. These effects are based on haematological spreading of toxic components of cigarette smoke, why should the lungs be spared of these effects? The focus of pulmonologists has always been more on local air borne effects of smoking, instead of systemic effects. It is remarkable to see (Fig. 3) that of the 133 subjects with one or more abnormal test results (e.g. low KNO, low FEV1/ FVC, etc), only 95 are diagnosed as COPD according to the GOLD approach: using only spirometry 38 diseased subjects are missed. There are 30 subjects with decreased KNO but normal spirometry and no emphysema on CT. This could point at interstitial lung disease, however after carefully reviewing the scans this proved not to be the case. Obviously, other processes than emphysema are responsible for this phenomenon; microvascular changes are a good candidate for the explanation of this phenomenon. Due to the design of this study this hypothesis cannot be tested, further research will be necessary to resolve this item. The study results indicate that the combination of spirometry and diffusion capacity detects almost all cases of HRCT-diagnosed emphysema. On the other hand, abnormal results for spirometry and diffusion capacity are not specific for the presence of emphysema. A strong correlation between the emphysema score and the DLNO cannot be found, as the non-emphysematous lung parts do not influence the CT-based emphysema score. 37 Emphysematous tissue ventilates poorly and is less accessible for test gas, and therefore cannot contribute to the uptake of NO or CO. Emphysema will betray itself by lowered diffusion capacity, but any diffusing capacity measure is also influenced by the status of the better ventilating lung zones. In other words, the diffusing capacity represents the functional status of non-emphysematous lung tissue. 37 Emphysema could be considered as the last stage of an ongoing disease process of the lung parenchyma, influencing pulmonary microstructures first, which cannot be visualized by CT scanning. The microscopical damage leads to functional impairment and our results are pointing to hypothesis that microvascular pathology is an important early phenomenon. Based on our observations, early detection of emphysema should also be performed with measurement of the diffusion capacity instead of spirometry only. The GOLD criteria 19 are based on spirometry only. By widening the clinical spectrum of COPD than just airway obstruction solely, the measurement of the diffusing capacity leads to better insight in the pathophysiology of COPD. An ongoing discussion in pulmonary literature is present concerning the focus on spirometry only in GOLD criteria. It seems that the reason for this choice is that spirometry is widely available in many countries, as well with general practioners as in hospitals; the measurement of the diffusion capacity is limited to pulmonary function labs. Based on our results, the combination of spirometry and the diffusion capacity detects almost all cases of CT-based emphysema. No great difference has been observed between CO and NO diffusion capacity. Using this approach, CT scanning for the quantification of emphysema is not necessary, except in clinical trials in which quantification of emphysema is an end-point. Spirometry, gas transfer and CT-based emphysema are complementary and partially overlapping entities (Fig. 3). In this population-based study in heavy (ex) smokers the correlation between pulmonary function tests and CTbased assessment of emphysema is weaker than reported earlier. 38 This is not unexpected, because our subjects were not selected on the presence of COPD by pulmonary function testing. In studies in which such work-up bias was present, one might expect stronger correlations, due to the presence of more severe disease. As a consequence, the correlation between for example the FEV 1 /FVC ratio and the emphysema score is low: 8 out of 36 subjects with emphysema on CT had a FEV 1 /FVC ratio above 70%. In conclusion, spirometry is very important for the diagnosis of emphysema, but it only measures airway obstruction. The KNO is a very sensitive measure for the detection of CT-based emphysema. The DLNO/DLCO ratio is increased in (ex)smokers, which could be explained by the hypothesis that pulmonary endothelial dysfunction precedes extensive parenchymal loss ultimately leading to airway obstruction. Further studies are needed to explore this concept, in which the DLNO/DLCO ratio could be used as a sensitive tool to detect microvascular malfunctioning. Conflict of interest None of the authors have a conflict of interest to declare in relation to this work. References 1. Hogg JC. Pathophysiology of airflow limitation in chronic obstructive pulmonary disease. 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