The Detection of Biomarkers of Tobacco Smoke Exposure in Wound Fluid

Abstract

Sir: Wound healing complications secondary to smoking are well documented in the clinical literature.1,2 It is unclear, however, whether tobacco constituents work directly on the local wound or primarily by means of systematic mechanisms to impair healing.3 Although tobacco exposure biomarkers have been measured in multiple tissue types, to our knowledge they have not previously been measured in the local wound milieu. We sought to determine the feasibility of measuring these biomarkers in wound fluid and to evaluate their relationship to well-established systemic measures. The proposed study of the effects of smoking on wound healing in head and neck surgery patients includes nonexempt human study research. This study was approved by the University of Minnesota Institutional Review Board (approval no. 0911M74371; approval date, May 21, 2010). All patients gave written informed consent before participation. Eleven current smokers (defined as having smoked within the past month) and three never-smoker controls (<100 lifetime cigarettes) were enrolled in this prospective pilot study (Table 1). All patients received standard preoperative clinical care. Bulb suction surgical drains were placed during surgery as clinically indicated. Wound fluid from surgical drains and urine were collected serially every 8 hours for the first 24 to 48 hours postoperatively. One baseline blood sample was collected during surgery.Table 1: Current Smokers’ Clinical and Tobacco Biomarker DataUrine and whole blood concentrations of total cotinine and total nicotine (free plus cotinine and nicotine N-glucuronides) were determined by gas chromatography–mass spectometry.4 Wound fluid samples were also analyzed by gas chromatography–mass spectometry with additional purification steps: a 400-µl aliquot sample, 500 µl of water, and internal standard were mixed and loaded onto Oasis MCX columns (10 mg sorbent material, 1 cc; Waters Corp., Milford, Mass.) that had been conditioned with 0.5 ml each of methanol, water, and 0.5% formic acid (volume/volume); columns were washed with 0.5 ml each of 0.5% formic acid and methanol, and the cotinine and nicotine were eluted with 1 ml of methylene chloride/isopropanol/ammonium hydroxide (78:20:2). The eluent was further purified by two liquid/liquid extractions as described previously.5 Statistical analysis was completed with SAS 9.2 (SAS Institute, Inc., Cary, N.C.). Levels of cotinine and nicotine in the urine, wound fluid, and blood of controls were at or below the limit of detection as expected. In current smokers, urine cotinine levels were consistent with those reported in the literature. Cotinine was reliably measured in wound fluid (Fig. 1), and wound fluid levels were significantly lower than urine levels at each of the first five 8-hour postoperative intervals (p < 0.001 to 0.004) (Fig. 2). The ratio of urine cotinine to wound fluid cotinine during these intervals ranged from 11:9:1 at interval 1 to 5:7:1 at interval 5. Cotinine levels in both urine and wound fluid declined over time as anticipated, but the decline was less precipitous in wound fluid. Wound cotinine levels also decreased at a rate of 0.14 ng/ml for every minute increase in surgery length. Wound fluid cotinine was correlated with urine cotinine (0.85; p < 0.001) but was not significantly correlated with self-reported cigarettes consumed per day (0.53; p = 0.141). Nicotine levels in wound fluid of current smokers were at or below the limit of detection, whereas nicotine levels in urine and blood were appropriate for cigarette consumption.Fig. 1: Wound fluid cotinine per individual current smoker: Black lines indicate individual subjects; red line indicates mean values for all current smokers.Fig. 2: Mean urine cotinine versus mean wound fluid cotinine in current smokers. Urine cotinine is indicated by the blue line. Wound fluid cotinine is indicated by the red line.These pilot data demonstrate that cotinine is feasibly measured in wound fluid. Although wound cotinine was significantly less than urine cotinine, these levels correlate, showing that wound cotinine reliably quantifies tobacco exposure. With further study, the measurement of biomarkers of tobacco exposure in wound fluid will potentially provide an innovative means by which to evaluate the effect of smoking on wound healing. DISCLOSURE The authors have no financial interest to declare in relation to the content of this article. ACKNOWLEDGMENT Funding for this study was graciously provided by a Lions Club Grant (Minneapolis, Minn.). Amy Anne D. Lassig, M.D. Bevan Yueh, M.D., M.P.H. Department of Otolaryngology–Head and Neck Surgery Sharon E. Murphy, Ph.D. Department of Biochemistry, Molecular Biology, and Biophysics, and Masonic Cancer Center Patricia G. Fernandes, D.D.S. Kathryn M. Banks, B.A. Department of Otolaryngology–Head and Neck Surgery Katherine M. Wickham, B.A. Masonic Cancer Center Anne M. Joseph, M.D., M.P.H. Department of Medicine University of Minnesota Minneapolis, Minn.