Gas Signatures From Cultured Human Leukocytes

Abstract

Rationale. The presence of leukocytes in airways indicates substantial inflammation. We wondered whether volatile organic compounds produced by these cells could be detected and ultimately used as noninvasive biomarkers. We recently demonstrated that distinct gases were produced by transformed promyelocitic cells (J. transl Med, 2009 7:31). We hypothesized that neutrophils and peripheral blood mononuclear cells (PBMCs) could generate cell-type specific gases which could be detected even in the parts-per-billion and trillion (ppbv, pptv) range using appropriate technologies.Methods. This study was approved by the UCI IRB. Neutrophils and PBMCs were isolated from the peripheral blood of healthy donors (n=10, 18-65 yrs old). The cells were resuspended in RPMI 1640 supplemented with 10% FCS. The vials containing cell suspensions (30 x 10 cells/10mL) were placed inside bioreactors (that we specially designed for this purpose) in an incubator at 37°C for 24 h. The headspace 6 gas was then collected and analyzed using a GC-MS system built originally to detect trace volatile organic compounds in the atmosphere. We performed 10 separate experiments and compared target gases between media alone and cell culture conditions using paired t-tests.Results. In the cultured neutrophils, acetaldehyde in the headspace was significantly elevated (220±140 (SD) ppbv) compared with media (90±20 ppbv, p=0.024). In contrast, the level of acetaldehyde from PBMCs was 30±20 ppbv, significantly below media alone (p<0.001) In . cultured PBMCs, we found 2 unidentified gases (A and B, 260±110 pptv and 80±70 pptv) greater than media alone (p<0.002). For both neutrophils and PBMCs hexanaldehyde (1±1 ppbv, 0.6±0.2 ppbv, respectively) was below medium alone (4±2 ppbv).Conclusions. Human primary immune cells produce volatile gases that can be measured in trace amounts. This study demonstrates in vitro for the first time the ability to detect basal levels of acetaldehyde produced by unperturbed cultured neutrophils. Moreover, the data suggest that different leukocyte subtypes have different gas profiles (e.g., no acetaldehyde was found in PBMCs). Intriguingly, the data suggest that some gases such as acetaldehyde in PBMCs and hexanaldehyde in both neutrophils and PBMCs appear to be metabolized by the cultured cells. Finally, we measured several as yet unidentified gases produced by leukocytes. Detection of key gases in human breath may eventually reveal the extent and type of pulmonary leukocyte infiltration. This abstract is funded by: National Institutes of Health (R01-HL-080947 to D.M.C); and the Physical Sciences Dean's Innovation fund (D.R. B.) Am J Respir Crit Care Med

Non-invasive phenotyping using exhaled volatile organic compounds in asthma

“…Exhaled VOCs may also be able to discriminate breath samples by the dominant inflammatory cell type. Cultured white cells both release and use VOCs,9 10 and it is likely that the predominant airway inflammatory cell type will be associated with specific VOC profiles. Non-invasive assessment of metabolites in the breath therefore offers the potential for the discovery of novel biomarkers and for providing insights into inflammatory pathways.…”

Methodology validation, intra-subject reproducibility and stability of exhaled volatile organic compounds

“…How then can we explain the lower levels of aldehydes in the breath of our COPD patients? It may be that the aldehydes in the inhaled air are in fact used as a substrate in the inflammatory process, and indeed neutrophils (the predominant inflammatory cell type in COPD) have been shown to use hexanal in vitro [12]. Basanta et al had also shown similar trend of reproducibility in aldehydes, alkanes and aromatic hydrocarbons [3].…”