Comprehensive characterisation of pulmonary and serum surfactant protein D in COPD

Abstract

Background: Pulmonary surfactant protein D (SP-D) is considered as a candidate biomarker for the functional integrity of the lung and for disease progression, which can be detected in serum. The origin of SP-D in serum and how serum concentrations are related to pulmonary concentrations under inflammatory conditions is still unclear.

Methods: In a cross-sectional study comprising non-smokers (n=10), young--(n=10), elderly smokers (n=20), and smokers with COPD (n=20) we simultaneously analysed pulmonary and serum SP-D levels with regard to pulmonary function, exercise, repeatability and its quaternary structure by native gel electrophoresis. Statistical comparisons were conducted by ANOVA and post-hoc testing for multiple comparisons; repeatability was assessed by Bland-Altman analysis.

Results: In COPD, median (IQR) pulmonary SP-D levels were lower (129(68) ng/ml) compared to smokers (young: 299(190), elderly: 296(158) ng/ml; p<0.01) and non-smokers (967(708) ng/ml; p<0.001). The opposite was observed in serum, with higher concentrations in COPD (140(89) ng/ml) as compared to non-smokers (76(47) ng/ml; p<0.01). SP-D levels were reproducible and correlated with the degree of airway obstruction in all smokers. In addition, smoking lead to disruption of the quaternary structure.

Conclusions: Pulmonary and serum SP-D levels are stable markers influenced by smoking and related to airflow obstruction and disease state. Smaller subunits of pulmonary SP-D and the rapid increase of serum SP-D levels in COPD due to exercise support the translocation hypothesis and its use as a COPD biomarker.

Trial registration: no interventional trial.

Figure 1

SP-D levels in BAL and Serum. SP-D levels in BAL (A) serum (B) and the ratio of BAL/serum SP-D levels (C) of healthy, non-smoking subjects (H, open squares n = 10), young smokers (S1, black squares, n = 10), elderly smokers (S2, open triangles, n = 20) and smokers with COPD (C, black triangles n = 20). Log-transformed individual data points are provided together with the respective mean of the log transformed data. Values are displayed on a logarithmic scale. *p < 0.05, **p < 0.01, ***p < 0.001 compared to H, ^#p < 0.05, ^##p < 0.01 compared to C.

Figure 2

Repeatability of SP-D measurements. Bland-Altman plot to assess the repeatability of SP-D levels in BAL (A) and serum (B) of elderly smokers (S2) and smokers with COPD between two samplings with a time delay of about 34 ± 10 days. The coefficient of reliability (derived from one-way ANOVA as the ratio of variance among subjects to total variance) is given as intraclass correlation coefficient (ICC).

Figure 3

Correlation of lung function measurements and SP-D levels. Correlation of SP-D levels in BAL (A), serum (B) or the ratio of SP-D in BAL and serum (C) with FEV₁/FVC in smokers (n = 50, young and elderly smokers and smokers with COPD). FEV₁: forced expiratory volume in 1 s, FVC: forced vital capacity. Correlations were significant with p < 0.001, r = 0.62 (A); p = 0.013, r = 0.-0.35 (B) and p < 0.001, r = 0.60 (C).

Figure 4

Kinetics of SP-D levels in serum during constant load exercise. Kinetics of SP-D levels in serum during constant load exercise taken at 4 different time points: at rest (rest), after 5 min of steady state exercise (5 min), at the end of loaded exercise (end), and 20 min after exercise (recovery). Change in SP-D serum concentrations over time in elderly smokers (S2, open triangles, dashed line, n = 15) and smokers with COPD (C, black triangles, n = 15) are shown. Data are given on the vertical axis on a logarithmic scale as mean ± SEM, * p < 0.05 compared to time point "rest".

Figure 5

Quaternary structure of pulmonary SP-D. The quaternary structure of SP-D in BAL: A) equal amounts of SP-D were loaded as indicated by equal band intensities of the SP-D monomer (43 kDa) in SDS page under reduced conditions and immunoblotting (upper panel A and B). The quaternary structure of SP-D in bronchoalveolar lavage from 4 healthy subjects (H) and smokers (S1) is demonstrated by blue native electrophoresis and immunoblotting (lower panel A) as well as for 3 smokers with COPD (C) (lower panel B). Due to very low SP-D levels in BAL of COPD subjects the displayed intensity of SP-D bands is not comparable between A and B.

Figure 6

Oxidative induced disruption of the quaternary structure of pulmonary SP-D. Blue native electrophoresis of surfactant protein-D after incubation with an oxygen radical donor (+). Disruption of the multimeric structure (*) can be chemically induced by exposing rr-SP-D or BAL from healthy subjects to 74 mM of the oxidizing agent 2,2-Azo-bis-(2-amidinopropane)-dihydrochloride (+).