Menssana Research (United States)

There is widespread interest in the development of tools to estimate radiation exposures. Exhaled breath provides a novel matrix for assessing biomarkers that could be correlated with exposures. The use of exhaled breath for estimating radiation exposure is warranted, as studies have shown that external exposure to ionizing radiation causes oxidative stress that accelerates lipid peroxidation of polyunsaturated fatty acids, liberating alkanes and alkane metabolites that are excreted in the breath as volatile organic compounds (VOCs). As a proof of principle study, small groups (n = 4) of Göttingen minipigs were whole-body irradiated with gamma rays delivered by a 60Co source at absorbed doses of 0, 0.25, 0.5, 0.75, 1, 1.25, 2, and 4 Gy. Additional groups (n = 4) were treated with lipopolysaccharide (LPS) or granulocyte colony stimulating factor (G-CSF), with and without concurrent 60Co exposure, at an absorbed dose of 1 Gy. Breath and background air VOC samples were collected on days -3, -2, -1, 0 pre-irradiation, then at 0.25, 24, 48, 72, and 168 h post-irradiation. VOCs were analyzed by automated thermal desorption with two-dimensional gas chromatography and time-of-flight mass spectrometry (ATD GCxGC TOF MS). The results show significant changes in 58 breath VOCs post-irradiation, mainly consisting of methylated and other derivatives of alkanes, alkenes, and benzene. Using a multivariate combination of these VOCs, a radiation response function was constructed, which was significantly elevated at 15 min post irradiation and remained elevated throughout the study (to 168 h post irradiation). As a binary test of radiation absorbed doses ≥ 0.25 Gy, the radiation response function distinguished irradiated animals from shams (0 Gy) with 83-84% accuracy. A randomly derived radiation response function was robust: When half of the biomarkers were removed, accuracy was 75%. An optimally derived function with two biomarkers was 82% accurate. As a binary test of radiation absorbed doses ≥ 0.5 Gy, the radiation response function identified irradiated animals with an accuracy of 87% at 15 min post irradiation and 75.5% at 168 h post irradiation. Treatment with LPS and G-CSF did not affect the radiation response function. This proof-of-principle study supports the hypothesis that breath VOCs may be used for estimating radiation exposures. Further studies will be required to validate the sensitivity and specificity of these potential biomarkers.

Viral infections cause increased oxidative stress, so a breath test for oxidative stress biomarkers (alkanes and alkane derivatives) might provide a new tool for early diagnosis. We studied 33 normal healthy human subjects receiving scheduled treatment with live attenuated influenza vaccine (LAIV). Each subject was his or her own control, since they were studied on day 0 prior to vaccination, and then on days 2, 7 and 14 following vaccination. Breath volatile organic compounds (VOCs) were collected with a breath collection apparatus, then analyzed by automated thermal desorption with gas chromatography and mass spectroscopy. A Monte Carlo simulation technique identified non-random VOC biomarkers of infection based on their C-statistic values (area under curve of receiver operating characteristic). Treatment with LAIV was followed by non-random changes in the abundance of breath VOCs. 2, 8-Dimethyl-undecane and other alkane derivatives were observed on all days. Conservative multivariate models identified vaccinated subjects on day 2 (C-statistic = 0.82, sensitivity = 63.6% and specificity = 88.5%); day 7 (C-statistic = 0.94, sensitivity = 88.5% and specificity = 92.3%); and day 14 (C-statistic = 0.95, sensitivity = 92.3% and specificity = 92.3%). The altered breath VOCs were not detected in live attenuated influenza vaccine, excluding artifactual contamination. LAIV vaccination in healthy humans elicited a prompt and sustained increase in breath biomarkers of oxidative stress. A breath test for these VOCs could potentially identify humans who are acutely infected with influenza, but who have not yet developed clinical symptoms or signs of disease.

Ethane and pentane in breath are markers of oxidative stress, produced by ROS-mediated lipid peroxidation of n-3 and n-6 polyunsaturated fatty acids (PUFAs), but little is known about other n-alkanes in normal human breath. We investigated the spectrum of alkanes in normal human alveolar breath, and their variation with age. Fifty normal humans were studied (age range 23–75, median 35). Volatile organic compounds (VOCs) in alveolar breath were captured on sorbent traps and assayed by gas chromatography and mass spectroscopy. Alveolar gradients (concentration in breath minus concentration in ambient room air) of alkanes were determined. C4–C20 alkanes were observed in breath and room air. Their mean alveolar gradients were negative from C4 to C12 and positive from C13 to C20. The mean alveolar gradients of four alkanes (C5–C8) were significantly less negative in the older subjects (p < 0.05). There were no significant differences between males and females. Normal human breath contained a spectrum of alkanes which may include new markers of oxidative stress. The mean rate of clearance (via cytochrome p450) exceeded the mean rate of synthesis (by ROS-mediated oxidative stress) for C4–C12 alkanes, while synthesis was greater than clearance for C13–C20 alkanes. The elevated alkane profile in older subjects was consistent with an age-related increase in oxidative stress, though an age-related decline in alkane clearance rate may have contributed.