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Interactions Between Smoking, Pulmonary Surfactant Protein B, and Atherosclerosis in the General Population

The Dallas Heart Study
Originally published, Thrombosis, and Vascular Biology. 2011;31:2136–2143



Pulmonary surfactant protein B (SP-B), an alveolar protein normally detectable at only very low concentrations in blood, circulates at higher levels among smokers and those with alveolar injury and inflammation. We hypothesized that SP-B may serve as a marker of the vascular effects of smoking and would thus be associated with subclinical measures of atherosclerosis.

Methods and Results—

Plasma levels of SP-B were measured in 3294 subjects, ages 30 to 65, enrolled in the Dallas Heart Study, a probability-based population sample of Dallas County adults. Coronary artery calcium (CAC) was measured by computed tomography and abdominal aortic plaque (AP) by magnetic resonance imaging. The cohort comprised 29% current and 17% former smokers. The overall prevalence of CAC was 22%, and that of AP was 39%. Median SP-B levels were 5-fold higher among current versus never smokers (P<0.0001) and were significantly correlated with estimated pack-years smoked (Spearman ρ=0.35, P<0.0001). Increasing levels of SP-B also associated with other traditional cardiac risk factors and higher levels of inflammatory biomarkers. In univariable analyses, increasing SP-B quartiles associated with higher prevalence of both CAC and AP (Ptrend<0.0001 for each). In multivariable analyses adjusting for traditional cardiovascular risk factors, SP-B remained associated with AP (OR 1.87 for the 4th versus 1st quartiles, 95% confidence interval 1.39 to 2.51; P<0.0001) but not CAC. An interaction was observed between SP-B, smoking status, and AP (Pinteraction=0.01), such that SP-B associated with AP in current smokers (adjusted OR 2.15 for the 4th versus 1st quartile, 95% confidence interval 1.26 to 3.67; P=0.005) but not in former or never smokers.


Circulating levels of SP-B increase with greater smoking burden and independently associate with abdominal AP among current smokers. Our findings support further investigation of the role of SP-B as a marker of the vascular effects of smoking.


Tobacco smoking promotes atherosclerosis and its complications via direct effects on the vasculature, as well as alterations in local inflammation, platelet function, and antithrombotic, prothrombotic, and fibrinolytic factors.1 Although a causal role of smoking in atherosclerosis has been conclusively established, evidence to support a dose-response effect has been varied. Although several epidemiological studies have shown dose-dependent associations between tobacco smoke exposure and cardiovascular events, such as myocardial infarction, stroke, and cardiovascular death, these effects attenuate quickly after smoking cessation,2 in contrast to the cumulative effects of smoking on other end points, such as cancer. Moreover, studies evaluating dose-dependent effects on subclinical atherosclerosis phenotypes have been conflicting, with some studies suggesting a nonlinear effect,3 and others suggesting a differential effect in peripheral vascular beds versus the coronary circulation.46 Several inflammatory biomarkers are increased in active smokers, and recent studies suggest a possible interaction between smoking, circulating levels of these inflammatory markers, and atherosclerosis phenotypes.7 However, evaluation of a more direct serological measure linking smoking-related toxicity and vascular disease in the general population may provide a means to assess individual risk for smoking-related vascular complications.

See accompanying article on page 1939

Pulmonary surfactant protein B (SP-B) is crucial to lung function.8 It is the oldest member of a family of proteins called saposins, which has been shown to have a significant impact on phospholipid organization.9 The primary role of SP-B is to maintain reduced surface tension, and to enhance the rate, adsorption, and spread of phospholipids during the respiratory cycle, thereby promoting formation and stability of the surfactant monolayer.10 SP-B is normally detectable in the systemic circulation, but only at very low levels.11 However, leakage of surfactant proteins into the circulation has been reported in several diseases that result in damage to the alveolar-capillary membrane, including acute respiratory distress syndrome and chronic heart failure.11,12 Increased levels of SP-B in the circulation have also been reported in tobacco-smoke exposure due to alveolar inflammation and increased lung permeability.13 Given the reported associations of smoking exposure with higher plasma levels of SP-B, we hypothesized that SP-B may serve as a quantitative marker reflecting smoking exposure and its associated vascular risk. To evaluate this hypothesis, we explored the associations and interactions between smoking status, circulating SP-B and several atherosclerotic phenotypes in the Dallas Heart Study, a large, multiethnic, probability-based population cohort.


Study Population

The Dallas Heart Study is a population-based, probability sample of 6101 Dallas County residents.14 Following an initial in-home visit for collection of survey data, body mass index and measurement of blood pressure, participants between the ages of 30 and 65 were invited to participate in a second visit where they provided in-home fasting blood and urine specimens. Those completing visit 2 were invited to a third visit at the University of Texas Southwestern Medical Center, where imaging studies including cardiac magnetic resonance imaging and electron beam computed tomography were performed. Demographics, blood pressure, and body composition were similar between subjects completing visits 1 and 2, and laboratory data did not significantly differ between those completing visits 2 and 3.14 The study population for the present analyses includes 3294 Dallas Heart Study subjects, ages 30 to 65, who underwent measurement of plasma levels of SP-B at visit 2. A subset of the population underwent electron beam computed tomography to measure coronary artery calcium (CAC; n=2519) and magnetic resonance imaging to measure aortic plaque (AP) prevalence (n=2305).

Definition of Variables

Hypertension was defined as an average (5 repeated measurements) systolic blood pressure ≥140 mm Hg, diastolic blood pressure ≥90 mm Hg, or use of antihypertensive medication. Diabetes was defined as a fasting blood glucose level of ≥126 mg/dL, a nonfasting blood glucose level of ≥200 mg/dL, or prevalent medically treated diabetes. Hypercholesterolemia was defined as a fasting low-density lipoprotein cholesterol ≥160 mg/dL, a total cholesterol ≥240 mg/dL, or use of a statin medication. Hypertriglyceridemia was defined as a fasting triglyceride concentration ≥200 mg/dL. Low high-density lipoprotein cholesterol was defined as high-density lipoprotein cholesterol <40 mg/dL in men and <50 mg/dL in women. Race was self-reported.

Smoking was determined by self-report and categorized in several ways. Current smokers were defined as those who had smoked greater than 100 cigarettes in a lifetime and continued to smoke. Among current smokers, frequency of smoking was subcategorized as daily smoking, defined as smoking every day in the past 30 days, or intermittent smoking, defined as smoking some days in the past 30 days. Former smokers were those who had smoked more than 100 cigarettes in a lifetime but did not currently smoke. Never smokers were those who had smoked fewer than 100 cigarettes in a lifetime. Cumulative smoking exposure was quantified by pack-years among current smokers. Tobacco exposure was also evaluated for smoking cigars and pipes or for chewing tobacco.

Measurement of SP-B and Other Biomarkers

Venous blood was collected in standard blood collection tubes containing citrate EDTA, and samples were maintained at 4°C for ≤4 hours and then centrifuged (1430g for 15 minutes) at 4°C. Plasma was then removed, aliquoted, and frozen at −80°C until assays were performed. SP-B was measured from thawed frozen plasma at Alere, Inc (San Diego, CA) using a proprietary sandwich platform with minimum and maximum detection limits of 1 and 950 ng/mL, respectively. The intraassay coefficient of variation was 10% and interassay coefficient of variation was 11%. Assays were performed by individuals blinded to all clinical data. Samples had been thawed once for aliquoting before biomarker measurement. Tumor necrosis factor-α1 receptor (TNFR1A), myeloperoxidase (MPO), and matrix metalloproteinase-9 were measured on a similar platform: minimum and maximum detection limits for TNFR1A were 0.094 and 100 ng/mL, for MPO they were 3.7 and 250 ng/mL, and for matrix metalloproteinase-9 they were 1.0 and 730 ng/mL. Intraassay and interassay coefficient of variations for TNFR1A were 9% and 10%, for MPO they were 6% and 7%, and for matrix metalloproteinase-9 they were 5% and 7%.

The following analytes were measured previously and the methods have been described: high-sensitivity C-reactive protein,15 N-terminal pro-B-type natriuretic peptide,16 monocyte chemoattractant protein-1,17 interleukin-18,18 osteoprotegerin,19 cystatin C,20 and soluble receptor for advanced glycation end products.21 The homeostasis model assessment of insulin resistance index was calculated as described previously (fasting insulin [μIU/mL]×fasting glucose [mmol/L]/22.5).22

Atherosclerosis Assessment

Electron beam computed tomography scans were performed in duplicate to measure CAC. The scans were scored using the Agatston method, and the results of the 2 scans were averaged. Prevalent CAC was defined as >10 Agatston units, a data-derived threshold selected to maximize the signal-to-noise ratio, as previously described.23 To determine AP prevalence, 6 transverse slices of the infrarenal abdominal aorta were obtained using a 1.5-tesla whole-body magnetic resonance imaging system (Intera, Philips Medical Systems). Investigators blinded to all subject data used the Magnetic Resonance Analytic Software Systems cardiac analysis software package (version 4.2 beta, Medis Medical Imaging Systems Inc) to analyze the images, using a previously published definition of AP as a hyperintense signal volume that protruded ≥1 mm from the endoluminal surface of the aortic wall.24

Death Events

All-cause and cardiovascular mortality were ascertained using National Death Index data through December 31, 2007 (median follow-up time 6.3 [5.9, 6.8] years). Cardiovascular death was defined using ICD10 codes I00 to I99.25 Death status for all participants analyzed was ascertained to the end of 2007 with no loss of follow-up.

Statistical Analysis

Demographic and clinic variables were compared across quartiles of SP-B levels using the χ2 trend test for categorical variables and the Jonckheere-Terpstra test for continuous variables. Correlations between selected biomarkers and SP-B were evaluated by Spearman rank correlation coefficients. Logistic regression was performed to assess associations between SP-B and prevalent CAC and AP in unadjusted models, as well as models adjusting for age, sex, race, diabetes, hypertension, hypertriglyceridemia, hypercholesterolemia, low high-density lipoprotein, body mass index, and smoking exposure. Smoking exposure was entered into models as both a categorical variable (current, former, never) and as a continuous measure of pack-years smoked (never/former=0 pack-years). Additional adjustments were made for cigar, pipe, and snuff tobacco exposure in sensitivity analyses. Because of the highly skewed distribution of SP-B, it was evaluated as both a categorical variable (in quartiles) and as a log-transformed continuous variable. Cox proportional hazards models were used to assess the associations between SP-B and all-cause and cardiovascular death, with adjustment for the same covariates as described above. Proportional hazards assumptions were met for all Cox models. Tests for statistical interactions were performed between smoking (current, former, never), SP-B, and all atherosclerosis phenotypes and death end points. For all interaction tests with P<0.1, stratified analyses were performed in subgroups defined by smoking status. All 2-sided probability values ≤0.05 were considered statistically significant, and no adjustments were made for multiple testing. All analyses were performed using SAS 9.2 (SAS Institute, Cary, NC) and all box-plot figures using GraphPad Prism 5.01 (La Jolla, CA).


Associations of SP-B With Atherosclerosis Risk Factors and Biomarkers

The overall median (interquartile range) level of SP-B was 2.67 ng/mL (1.00, 7.57). Increasing quartiles of SP-B associated with male sex, older age, black race, hypertension, metabolic syndrome, and hypercholesterolemia (Table 1). SP-B did not associate with high-density lipoprotein cholesterol or diabetes. The strongest correlations between SP-B and the continuous variables tested include TNFR1A, MPO, age, monocyte chemoattractant protein-1, cystatin C, osteoprotegerin, interleukin-18, and high-sensitivity C-reactive protein (Table 2). SP-B did not correlate strongly with measures of adiposity (body mass index) or insulin resistance (homeostasis model assessment of insulin resistance index) (Table 1).

Table 1. Clinical and Biological Variables by Quartile of Pulmonary Surfactant Protein B

VariableQ1 (n=873)Q2 (n=773)Q3 (n=825)Q4 (n=823)P Trend
SP-B, ng/mL<1.01.0 to 2.62.7 to 7.67.6 to 829.7
Age, y39 (33, 47)43 (36, 51)45 (37, 54)47 (40, 54)<0.0001
Men341 (39%)331 (43%)402 (49%)381 (46%)0.000
Black345 (40%)362 (47%)464 (56%)525 (64%)<0.0001
White294 (34%)237 (31%)251 (30%)192 (23%)<0.0001
Hispanic209 (24%)148 (19%)99 (12%)98 (12%)<0.0001
Hypertension212 (25%)212 (28%)323 (40%)355 (44%)<0.0001
Diabetes81 (9%)100 (13%)104 (13%)101 (12%)0.072
Metabolic syndrome225 (29%)255 (33%)293 (36%)300 (36%)0.001
Hypercholesterolemia80 (9%)95 (12%)125 (15%)131 (16%)<0.0001
Low high-density lipoprotein356 (41%)316 (41%)336 (41%)359 (44%)0.27
Current smokers94 (11%)117 (15%)264 (32%)486 (59%)<0.0001
Former smokers137 (16%)153 (20%)172 (21%)94 (12%)0.056
Never smokers641 (73%)502 (65%)389 (47%)240 (29%)<0.0001
Pack-years (current smokers)2.9 (0.7, 8.0)6.9 (1.7, 19.8)13.1 (5.3, 24.0)17.5 (8.0, 29.0)<0.0001

Levels of continuous variables are reported as median and interquartile range (IQR) and categorical variables as percentages. SP-B indicates pulmonary surfactant protein B.

Table 2. Spearman Rank Correlations of Biomarkers With Pulmonary Surfactant Protein B in Smokers and Nonsmokers

CoefficientP ValueCoefficientP Value
Cystatin C0.130.00010.21<0.0001

TNFR1A indicates tumor necrosis factor receptor 1α; MPO, myeloperoxidase; MCP-1, monocyte chemoattractant protein-1; hsCRP, high-sensitivity C-reactive protein; IL-18, interleukin-18; NT-proBNP, N-terminal pro-B-type natriuretic peptide; BMI, body mass index; eGFR, estimated glomerular filtration rate; HOMA-IR, homeostasis model assessment of insulin resistance index; LDL-C, low-density lipoprotein cholesterol; HDL-C, high-density lipoprotein cholesterol; MMP-9, matrix metalloproteinase-9; sRAGE, soluble receptor for advanced glycation end products; MCP-1, monocyte chemoattractant protein-1.

Associations of SP-B and Tobacco Exposure

Median SP-B levels were markedly higher among current versus never smokers (7.83 ng/mL versus 1.64 ng/mL; P<0.0001) and significantly correlated with estimated pack-years smoked (Spearman ρ=0.35, P<0.0001). SP-B levels were highest among current smokers, intermediate among former smokers, and lowest among never smokers (Ptrend <0.001; Figure 1a); among current smokers, SP-B exhibited a graded association with increasing tobacco exposure as measured by pack-years smoked (Ptrend<0.0001; Figure 1b). Moreover, among current smokers, higher levels of SP-B were found in subjects who smoked every day in the past 30 days, as compared with those who only smoked intermittently over the same time period (P<0.0001; Figure 1c). Higher levels of SP-B were found in subjects who smoked cigars or pipes than those who did not smoke tobacco products (Table 3). In contrast, no difference in SP-B levels was found between subjects who chewed tobacco versus those who did not (Table 3).

Figure 1.

Figure 1. a, Pulmonary surfactant protein B (SP-B) in current, former, and never smokers. b, Pulmonary surfactant protein B in current smokers by pack-years. c, Pulmonary surfactant protein B in current smokers by smoking frequency.

Table 3. Pulmonary Surfactant Protein B and Other Tobacco Use

Current UseNo Current UseP Value
Cigars (n=118 of 3208)5.95 (2.07, 13.29)2.57 (1, 7.31)<0.0001
Pipes (n=13 of 3208)8.15 (4.86, 10.42)2.62 (1, 7.51)0.02
Snuff (n=55 of 3208)2.38 (1.00, 6.40)2.65 (1, 7.55)0.39

Levels of pulmonary surfactant protein B are reported as median (25th, 75th percentile) by current tobacco use status.

Associations of SP-B With Atherosclerosis

The overall prevalence of CAC was 22% and AP 39%. CAC prevalence (Ptrend<0.0001) and AP prevalence (Ptrend<0.0001) increased across higher quartiles of SP-B. Similarly, when SP-B was analyzed as a log transformed continuous variable, univariable associations were observed with CAC and AP (P<0.0001 for each; Table 4). In multivariable models adjusting for traditional atherosclerosis risk factors, former and current smoking, and pack-years of tobacco exposure, a graded association remained between higher SP-B quartiles and AP (OR 1.87 for the 4th versus 1st quartile, 95% confidence interval (CI) 1.39 to 2.51; P<0.0001; Table 4). Similar results were observed when log SP-B was entered into the model as a continuous variable (P<0.0001; Table 4). These findings were not changed by further adjustment for cigar and pipe smoking. In contrast, after multivariable adjustment, associations of SP-B with CAC were attenuated and no longer significant (Table 4).

Table 4. Overall Association of Pulmonary Surfactant Protein B With Atherosclerotic Phenotypes and Mortality

OR (95% CI)P ValueOR (95% CI)P Value
CAC >10n=2485
    Log SP-B*1.54 (1.41 to 1.69)<0.00011.10 (0.97 to 1.25)0.15
    Q11.0 (referent)1.0 (referent)
    Q21.62 (1.18 to 2.24)0.0031.13 (0.78 to 1.65)0.52
    Q32.57 (1.90 to 3.46)<0.00011.13 (0.78 to 1.62)0.52
    Q43.75 (2.80 to 5.03)<0.00011.33 (0.91 to 1.94)0.14
    Former smoking1.27 (0.94 to 1.73)0.13
    Current smoking2.63 (1.81 to 3.83)<0.0001
    Pack-years1.04 (0.93 to 1.16)0.54
    Log SP-B*1.60 (1.47 to 1.75)<0.00011.24 (1.11 to 1.38)<0.0001
    Q11.0 (referent)1.0 (referent)
    Q21.70 (1.31 to 2.19)<0.00011.32 (1.00 to 1.74)0.05
    Q32.39 (1.87 to 3.06)<0.00011.44 (1.09 to 1.89)0.01
    Q43.84 (3.01 to 4.92)<0.00011.87 (1.39 to 2.51)<0.0001
    Former smoking1.28 (0.99 to 1.66)0.06
    Current smoking1.36 (1.00 to 1.84)0.05
    Pack-years1.21 (1.08 to 1.37)0.002
HR (95% CI)P ValueHR (95% CI)P Value
Overall deathn=2788
    Log SP-B*1.57 (1.35 to 1.83)<0.00011.20 (1.00 to 1.46)0.05
    Q11.0 (referent)1.0 (referent)
    Q21.30 (0.62 to 2.73)0.490.94 (0.45 to 1.99)0.87
    Q33.57 (1.92 to 6.63)<0.00011.70 (0.90 to 3.25)0.10
    Q44.60 (2.51 to 8.44)<0.00011.85 (0.95 to 3.59)0.07
    Former smoking1.84 (1.12 to 3.03)0.02
    Current smoking2.38 (1.37 to 4.14)0.002
    Pack-years0.96 (0.82 to 1.12)0.60
Cardiovascular deathn=2788
    Log SP-B*1.46 (1.15 to 1.85)0.0021.16 (0.86 to 1.56)0.32
    Q11.0 (referent)1.0 (referent)
    Q21.44 (0.54 to 3.87)0.471.08 (0.40 to 2.94)0.87
    Q32.61 (1.08 to 6.28)0.031.32 (0.53 to 3.31)0.56
    Q42.97 (1.25 to 7.07)0.011.30 (0.50 to 3.38)0.59
    Former smoking1.17 (0.55 to 2.49)0.69
    Current smoking1.49 (0.63 to 3.54)0.36
    Pack-years0.98 (0.74 to 1.30)0.88

Adjusted models include age, sex, race/ethnicity, hypertension, diabetes, hypercholesterolemia, low HDL-C, hypertriglyceridemia, BMI, smoking status (current, former, never smoker), and pack-years smoked. Odds ratios for CAC and AP are derived from logistic regression models. Hazard ratios for all-cause death (n=125) and cardiovascular death (n=52) are derived from Cox proportional hazards models. CI indicates confidence interval; CAC, coronary artery calcium; SP-B, pulmonary surfactant protein B; Q, quartile; AP, aortic plaque; HR, hazard ratio.

*OR per 1 SD increment in SP-B;

OR per 10 pack-year increment.

Current smoking was significantly associated with both CAC and AP in unadjusted and adjusted analyses (Table 4). Number of pack-years smoked was significantly associated with AP but not CAC in analyses adjusted for smoking status, SP-B levels, and traditional risk factors (Table 4).

Interactions Between SP-B, Smoking, and AP

A significant interaction was observed between SP-B quartile, smoking status, and AP (Pinteraction=0.01). In subgroups defined as current, former, or never smokers, SP-B was associated with AP after adjustment for traditional risk factors and pack-years in current smokers (adjusted OR 2.15 in the 4th quartile, 95% CI 1.26 to 3.67; P=0.005) (Figure 2). In contrast, no significant association was seen between SP-B and AP in former or never smokers (Figure 2). Among current smokers, levels of SP-B were significantly higher among those with compared to without prevalent AP (10.9 ng/mL [4.5, 19.1] versus 6.2 ng/mL [2.2, 12.9]; P<0.0001), a difference that persisted after adjustment for self-reported pack-year exposure (P<0.0001). Adjustment for inflammatory markers TNFR1A, high-sensitivity C-reactive protein, cystatin C, and interleukin-18 separately did not significantly attenuate the interaction between SP-B, smoking status, and AP (Pinteraction≤0.03 for SP-B quartile×smoking with additional inflammatory markers).

Figure 2.

Figure 2. Association of pulmonary surfactant protein B (SP-B) with prevalent aortic plaque (AP), stratified by smoking status.

Associations of SP-B With All-Cause and Cardiovascular Disease Mortality

In univariable analysis, SP-B was significantly associated with overall mortality and cardiovascular disease mortality (Table 4). This association was attenuated for both overall and cardiovascular disease mortality when adjusted for traditional risk factors (Table 4). There was an interaction between log SP-B and smoking for overall mortality (Pinteraction=0.05), but stratified analyses by smoking status did not reveal any significant associations of SP-B with mortality among smokers in adjusted models (data not shown). Although current smoking and former smoking were significantly associated with overall mortality adjusted for traditional risk factors, there was no association between pack-years smoked and overall mortality in unadjusted or adjusted analyses (Table 4).


In a large, multiethnic, probability-based population study, we report novel associations between circulating levels of a pulmonary surfactant protein, SP-B, and cardiovascular risk factors, inflammatory biomarkers, and atherosclerotic phenotypes. We found that increasing levels of SP-B correlated remarkably with multiple measures of self-reported smoking exposure, including current smoking status, cumulative smoking burden, and frequency of smoking. Moreover, higher levels of SP-B were found in those who smoked cigars or pipes but not in those who chewed tobacco, further supporting the relationship between tobacco inhalation and higher circulating levels of SP-B.

Circulating plasma levels of SP-B associated with both prevalent coronary and peripheral atherosclerosis phenotypes, but after adjustment for smoking exposure and other risk factors, an independent association was observed only for abdominal aortic atherosclerosis. Moreover, this association was modified by smoking status, such that SP-B was independently associated with abdominal AP among current smokers but not among former or never smokers. This observation is concordant with the clinical observation of a stronger association of smoking with peripheral arterial disease than coronary artery disease.4 We found that compared with smokers without AP, those with AP had higher SP-B levels even after adjusting for self-reported pack-year exposure, supporting the concept that biomarkers of alveolar toxicity may help to quantify smoking dose-exposure and help to explain variability in the adverse vascular effects of smoking.

The associations of smoking dose-exposure with atherosclerosis phenotypes and mortality were mirrored consistently by the associations of SP-B levels with these end points. For example, current smoking independently associated with both coronary and aortic atherosclerosis in our study sample; however, among current smokers, a dose-response association with pack-years smoked was seen only with aortic atherosclerosis, consistent with prior reports.46,2628 In a parallel fashion, SP-B was independently associated with aortic but not coronary atherosclerosis. In contrast, although current smoking independently associated with all-cause mortality, pack-years smoked did not, mirroring the absence of an association of SP-B with all-cause mortality.

It is interesting to note that considerable variability in SP-B levels was observed among individuals reporting similar tobacco exposures. This variability may have resulted from inaccurate self-reporting, differences in the type of cigarettes smoked, the mode of smoking, degree of inhalation, and “downstream” factors, such as the ability of the lungs to clear toxins and defend against injury to the alveolar-capillary membrane. Importantly, renal clearance did not appear to play a role in circulating SP-B levels, consistent with prior reports.29

Biology of SP-B

Mature SP-B is a hydrophobic 79-amino-acid peptide of low molecular mass (≈18 kDa) that associates as a thiol-dependent homodimer and so far has been detected only in the lumen of the alveolus, in alveolar type II cells, and in alveolar macrophages.10 SP-B is normally associated with complexes of surfactant phospholipids, likely making it too large to breach the alveolar-capillary membrane.30 However, labeling studies of alveolar type II cells have shown that some of the protein is secreted into the alveolus as a hydrophilic, monomeric proprotein and processing intermediate with molecular weights of ≈45 and ≈25 kDa, respectively.31 Because these forms are considerably smaller, they can more readily breach the alveolar-capillary membrane.

The route by which proteins leak through the alveolar-capillary membrane into the circulation is not completely understood. Doyle et al measured levels of surfactant proteins in patients with acute respiratory failure and postulated that the alveolar-capillary membrane acts as a sieve.11 With respiratory disorders that cause inflammation and damage to the membrane, a dynamic alteration occurs to the radius of the pores, allowing proteins that are normally restricted by their size to cross the membrane and enter the circulation.11 A large concentration gradient of >1500:1 of SP-B between the alveoli and plasma supports the notion that SP-B may flow naturally down its concentration gradient into the circulation during states that alter the permeability of the alveolar-capillary membrane.29

Is SP-B a Biomarker of the Cumulative Vascular Effects of Tobacco Smoke?

Although evidence supports a dose-response effect of smoking on cardiovascular events, demonstration of a linear and cumulative effect on subclinical atherosclerosis has not been consistent. The dose-response effect seen with tobacco exposure differs among vascular beds, with weaker dose-response effects seen in the coronary arteries than the aorta and peripheral arteries.5,6,27,28 The toxic effects of smoking may vary among individuals with similar apparent tobacco exposures because of multiple factors, including interindividual variability in delivery of toxins to lung parenchyma, pulmonary defense mechanisms to clear inhaled toxins, nicotine pharmacokinetics, environmental factors, and tissue-specific responses to the vascular changes induced by inhaled tobacco smoke.

Multiple studies have investigated the role of biomarkers in assessing the variable toxicity resulting from inhaled tobacco smoke.32 Blood and urine levels of cotinine, a nicotine metabolite, have been shown to correlate with both active and passive smoking exposure and are associated with cardiovascular disease mortality and atherosclerosis.3337 In addition, expired air carbon monoxide levels have also been shown to correlate with smoking exposure and cardiovascular disease.37 However, the relationship between levels of these markers and self-reported pack-years is not linear.38 Furthermore, there is marked variation in levels of cotinine and carbon monoxide at similar levels of pack-year exposure, indicating variable pharmacokinetics and supporting the notion that smoking may have variable downstream effects.39,40 Though nicotine metabolites may be associated with smoking toxicity, they are not a direct measure of smoking-related tissue toxicity, as SP-B is. Studies of nonspecific systemic inflammatory markers, such as C-reactive protein, fibrinogen, and leukocyte count, have also shown increased levels in smokers,7 and several studies have suggested interactions between smoking, biomarker levels, and cardiovascular disease.41

Biomarkers of direct lung toxicity have been limited mostly to assessment of bronchoalveolar lavage fluid or expired air. The pulmonary surfactant proteins (SP-A, -B, -D) are detectable at low levels in the circulation and, except for SP-D, are specific to lung tissue, making them attractive blood-based biomarkers for lung-related smoking toxicity.29 Circulating levels of SP-B have been shown to correlate with tobacco exposure,13 as well as with increased pulmonary microvascular pressures in conditions such as acute respiratory failure and heart failure.12 Our novel findings of significant correlations between SP-B and several inflammatory biomarkers that have previously been associated with the development and progression of atherosclerosis, including TNFR1A,42,43 MPO,44 osteoprotegerin,19 monocyte chemoattractant protein-1,17 and cystatin C,45,46 support a link between alveolar injury from smoking and inflammation and atherosclerosis. In contrast, no association was seen between SP-B and C-reactive protein, matrix metalloproteinase-9, or monocyte chemoattractant protein-1, suggesting that SP-B may reflect specific inflammatory pathways related to the vascular effects of smoking.

Study Limitations

Several study limitations must be noted. First, our cross-sectional associations should not be interpreted to suggest a causal relationship of SP-B, smoking, and aortic atherosclerosis. Indeed, we believe it is more likely that SP-B is a marker than a mediator of tobacco-smoke related vascular risk. Cumulative pack-year exposure was not available for former smokers. Circulating SP-B reflects several proprotein and processing intermediates of varying molecular weights,29 and the high-throughput methodology for determining plasma levels used in this study did not allow characterization of these different moieties or direct biological activity of the measured analyte. Other reported markers of smoking exposure and smoking-related toxicity were not measured, precluding direct comparison of biomarkers of smoking toxicity. Nonfatal cardiovascular events were not available at the time of analysis.


Circulating levels of SP-B are increased in proportion to the burden of inhaled tobacco exposure in the general population. Higher levels of SP-B independently associate with aortic atherosclerosis and correlate with a dose-response effect in smokers, suggesting that SP-B may be a useful marker of the dose-dependent vascular effects of smoking. These findings require confirmation in other study samples and should prompt further investigation into the role this protein may play as a marker of the interaction between inhaled toxins and the development and progression of atherosclerosis.

Sources of Funding

Grant support for the Dallas Heart Study was provided by the Donald W. Reynolds Foundation at the University of Texas Southwestern Medical Center, Dallas, TX, and by US Public Health Service General Clinical Research Center Grant M01-RR00633 from National Institutes of Health, National Center for Research Resources - Clinical Research. This publication was supported in part by the North and Central Texas Clinical and Translational Science Initiative (National Institutes of Health Grant UL1 RR024982). Assay measurements for SP-B were provided by Alere, Inc (San Diego, CA).


Dr de Lemos has received grant support from Roche Diagnostics and Alere, Inc (formerly Biosite) and consulting income from Johnson and Johnson and Tethys Biomedical. Dr McGuire has received grant support from Alere and consulting income from Tethys Biomedical.


Drs Nguyen and Rohatgi contributed equally to this work.

Correspondence to James A. de Lemos, MD,
University of Texas Southwestern Medical Center, 5909 Harry Hines Blvd, HA 9.133, Dallas, TX 75390-9047
. E-mail


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