A potential strategy for diagnosing lung cancer, the leading cause of cancer-related death, is to identify metabolic signatures (biomarkers) of the disease. Although data supports the hypothesis that volatile compounds can be detected in the breath of lung cancer patients by the sense of smell or through bioanalytical techniques, analysis of breath samples is cumbersome and technically challenging, thus limiting its applicability. The hypothesis explored here is that variations in small molecular weight volatile organic compounds (“odorants”) in urine could be used as biomarkers for lung cancer. To demonstrate the presence and chemical structures of volatile biomarkers, we studied mouse olfactory-guided behavior and metabolomics of volatile constituents of urine. Sensor mice could be trained to discriminate between odors of mice with and without experimental tumors demonstrating that volatile odorants are sufficient to identify tumor-bearing mice. Consistent with this result, chemical analyses of urinary volatiles demonstrated that the amounts of several compounds were dramatically different between tumor and control mice. Using principal component analysis and supervised machine-learning, we accurately discriminated between tumor and control groups, a result that was cross validated with novel test groups. Although there were shared differences between experimental and control animals in the two tumor models, we also found chemical differences between these models, demonstrating tumor-based specificity. The success of these studies provides a novel proof-of-principle demonstration of lung tumor diagnosis through urinary volatile odorants. This work should provide an impetus for similar searches for volatile diagnostic biomarkers in the urine of human lung cancer patients.

Individual mice have a unique odor, or odortype, that facilitates individual recognition. Odortypes, like other phenotypes, can be influenced by genetic and environmental variation. The genetic influence derives in part from genes of the major histocompatibility complex (MHC). A major environmental influence is diet, which could obscure the genetic contribution to odortype. Because odortype stability is a prerequisite for individual recognition under normal behavioral conditions, we investigated whether MHC-determined urinary odortypes of inbred mice can be identified in the face of large diet-induced variation. Mice trained to discriminate urines from panels of mice that differed both in diet and MHC type found the diet odor more salient in generalization trials. Nevertheless, when mice were trained to discriminate mice with only MHC differences (but on the same diet), they recognized the MHC difference when tested with urines from mice on a different diet. This indicates that MHC odor profiles remain despite large dietary variation. Chemical analyses of urinary volatile organic compounds (VOCs) extracted by solid phase microextraction (SPME) and analyzed by gas chromatography/mass spectrometry (GC/MS) are consistent with this inference. Although diet influenced VOC variation more than MHC, with algorithmic training (supervised classification) MHC types could be accurately discriminated across different diets. Thus, although there are clear diet effects on urinary volatile profiles, they do not obscure MHC effects.

• Biosensors discriminated between urines collected from donors receiving a rabies vaccine (RV) versus controls • Biosensors discriminated between the urine odors of mice treated with lipopolysaccharide (LPS) from the urine of control mice • LPS-trained biosensors could distinguish between the odors of LPS-treated mouse urine and RV-treated mouse urine.• Discriminations by the biosensors were made on the basis of odor alone.• Volatile metabolites arising from immunization may be products of an innate immune response. a b s t r a c t a r t i c l e i n f o Infections have been shown to alter body odor. Because immune activation accompanies both infection and immunization, we tested the hypothesis that classical immunization might similarly result in the alteration of body odors detectable by trained biosensor mice. Using a Y-maze, we trained biosensor mice to distinguish between urine odors from rabies-vaccinated (RV) and unvaccinated control mice. RV-trained mice generalized this training to mice immunized with the equine West Nile virus (WNV) vaccine compared with urine of corresponding controls. These results suggest that there are similarities between body odors of mice immunized with these two vaccines. This conclusion was reinforced when mice could not be trained to directly discriminate between urine odors of RV-versus WNV-treated mice. Next, we trained biosensor mice to discriminate the urine odors of mice treated with lipopolysaccharide (LPS; a general elicitor of innate immunological responses) from the urine of control mice. These LPS-trained biosensors could distinguish between the odors of LPS-treated mouse urine and RV-treated mouse urine. Finally, biosensor mice trained to distinguish between the odors of RV-treated mouse urine and control mouse urine did not generalize this training to discriminate between the odors of LPS-treated mouse urine and control mouse urine. From these experiments, we conclude that: (1) immunization alters urine odor in similar ways for RV and WNV immunizations; and (2) immune activation with LPS also alters urine odor but in ways different from those of RV and WNV.Published by Elsevier Inc.

Changes in body odor are known to be a consequence of many diseases. Much of the published work on disease-related and body odor changes has involved parasites and certain cancers. Much less studied have been viral diseases, possibly due to an absence of good animal model systems. Here we studied possible alteration of fecal odors in animals infected with avian influenza viruses (AIV). In a behavioral study, inbred C57BL/6 mice were trained in a standard Y-maze to discriminate odors emanating from feces collected from mallard ducks (Anas platyrhynchos) infected with low-pathogenic avian influenza virus compared to fecal odors from non-infected controls. Mice could discriminate odors from non-infected compared to infected individual ducks on the basis of fecal odors when feces from post-infection periods were paired with feces from pre-infection periods. Prompted by this indication of odor change, fecal samples were subjected to dynamic headspace and solvent extraction analyses employing gas chromatography/mass spectrometry to identify chemical markers indicative of AIV infection. Chemical analyses indicated that AIV infection was associated with a marked increase of acetoin (3-hydroxy-2-butanone) in feces. These experiments demonstrate that information regarding viral infection exists via volatile metabolites present in feces. Further, they suggest that odor changes following virus infection could play a role in regulating behavior of conspecifics exposed to infected individuals.

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