Measurement of C-reactive Protein, Procalcitonin and Neutrophil Elastase in Saliva of COPD Patients and Healthy Controls

Correlation to Self-Reported Wellbeing Parameters

Neil Patel; John Belcher; Gary Thorpe; Nicholas R Forsyth; Monica A Spiteri

Disclosures

Respiratory Research. 2015;16(62) 

In This Article

Abstract and Introduction

Abstract

Background: Saliva is increasingly promoted as an alternative diagnostic bio-sample to blood; however its role in respiratory disease requires elucidation. Our aim was to investigate whether C-reactive protein (CRP), procalcitonin (PCT) and neutrophil elastase (NE) could be measured in unstimulated whole saliva, and to explore differences between COPD patients and controls with normal lung function. We also determined the relationship between these salivary biomarkers and self-reported COPD-relevant metrics.

Methods: Salivary CRP, PCT and NE levels were measured at each of 3 visits over a 14-day period alongside spirometry and a daily self-assessment dairy in 143 subjects: 20 never-smokers and 25 smokers with normal spirometry; 98 COPD patients [GOLD Stage I, 16; Stage II, 32; Stage III, 39; Stage IV, 11]. Twenty-two randomly selected subjects provided simultaneous blood samples.

Results: Levels of each salivary biomarker could distinguish between the above cohorts. Significant differences remained for salivary CRP and NE (p < 0.05) following adjustment for age, gender, sampling time, gum disease and total co-morbidities; but not for BMI except for salivary NE, which remained higher in smokers compared to non-smokers and stable COPD subjects (p < 0.001). Patients with acute COPD exacerbations had a median increase in all 3 salivary biomarkers (p < 0.001); CRP: median 5.74 ng/ml, [interquartile range (IQR) 2.86–12.25], PCT 0.38 ng/ml, [IQR 0.22–0.94], and NE 539 ng/ml, [IQR 112.25–1264]. In COPD patients, only salivary CRP and PCT levels correlated with breathing scores (r = 0.14, p < 0.02; r = 0.13, p < 0.03 respectively) and sputum features but not with activities of daily living. Salivary CRP and PCT concentrations strongly correlated with serum counterparts [r = 0.82, (95 % CI: 0.72–0.87), p < 0.001 by Spearman's; and r = 0.53, (95 % CI: 0.33–0.69), p < 0.006 respectively]; salivary NE did not.

Conclusions: CRP, PCT and NE were reliably and reproducibly measured in saliva, providing clinically-relevant information on health status in COPD; additionally NE distinguished smoking status. All 3 salivary biomarkers increased during COPD exacerbations, with CRP and PCT correlating well with patient-derived clinical metrics. These results provide the conceptual basis for further development of saliva as a viable bio-sample in COPD monitoring and exacerbation management.

Introduction

Saliva is increasingly used as a non-invasive easily accessible bio-sample for point-of-care diagnostics instead of blood[1,2] to inform on infection,[3–7] drugs[8] and disease states,[9–18] including airways inflammation. Salivary eosinophil cationic protein can differentiate between asthmatic and healthy subjects.[19] Increased salivary CRP and haptoglobin levels are demonstrated in childhood allergic asthma;[20] raised salivary leukotriene levels differentiate aspirin-intolerant asthmatics from tolerant counterparts.[21]

Biomarkers in various body fluids have been associated with Chronic Obstructive Pulmonary Disease (COPD) pathogenesis and clinical outcome.[22,23] Serum and sputum CRP are elevated in COPD patients and healthy smokers,[24,25] with moderate inverse correlation of serum CRP to Forced Expiratory Volume in 1 s (FEV1).[26] Serum CRP increases during exacerbations;[27–32] with high levels at 14 days post-exacerbation predicting re-exacerbation within 50 days and poor outcome.[33] Serum Procalcitonin (PCT) shows strong correlation to bacterial exacerbations,[34] guiding antibiotic prescriptions.[29,35] Neutrophil elastase (NE), mediator of airway pathogenesis,[36,37] is known to be elevated in smokers[38] and COPD patients,[39] has a negative correlation with FEV1 in patients with expiratory volumes below 40 % predicted[40] and increases further during exacerbations.[41]

The importance of CRP, PCT and NE in COPD has been clearly demonstrated. Yet despite the merits that saliva could offer to practical monitoring of COPD and its exacerbations, only two studies have explored its potential clinical role.[42,43] The aim of our study was therefore to investigate levels of CRP, PCT and NE in unstimulated whole saliva using commercially-validated and modified enzyme-linked immunoassays (ELISA) and to determine differences between patients with COPD and controls with normal lung function. Target biomarkers were measured at 3 time points within a 14-day period. As smoking can influence steady-state biomarker levels,[44] control groups included life-long never-smokers and current smokers. COPD data were analysed relative to the Global Initiative for Chronic Obstructive Lung Disease (GOLD) stage (percentage predicted FEV1),[45] which alongside MRC scores and self-assessment scores provided information for correlations between target salivary biomarkers and COPD-relevant clinical metrics. For further validation, randomly chosen participants also provided simultaneous blood samples.

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