Changes in volatile compounds of mouse urine as it ages: Their interactions with water and urinary proteins Jae Kwak a,b, ⁎, Claude C. Grigsby b , George Preti a,c , Mateen M. Rizki d , Kunio Yamazaki a,1 , Gary K. Beauchamp a a Monell Chemical Senses Center, 3500 Market Street, Philadelphia, PA 19104, USA b Human Signatures Branch, Forecasting Division, Human Effectiveness Directorate, Air Force Research Laboratory, Wright-Patterson AFB, Dayton, OH 45433, USA c Department of Dermatology, School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA d Department of Computer Science and Engineering, Wright State University, 3640 Colonel Glenn Highway, Dayton, OH 45435, USA HIGHLIGHTS • As mouse urine ages, substantial changes occur in the release of urinary VOCs. • Large amounts of some VOCs are lost as urine ages, whereas other VOCs increase. • The VOCs previously dissolved in water exhibit an increased release as urine dries. • Some VOCs decrease slightly due to their binding with mouse urinary proteins (MUPs). • MUP ligands whose headspace concentrations increase as urine ages are identified. abstract article info Article history: Received 4 October 2012 Received in revised form 6 April 2013 Accepted 7 August 2013 Available online 16 August 2013 Keywords: Age of mouse urine Volatile organic compounds (VOCs) Major urinary proteins (MUPs) Hydration status Gas chromatography–mass spectrometry (GC–MS) Metabolite Differentiation and Discovery Lab (MeDDL) Mice release a variety of chemical signals, particularly through urine, which mediate social interactions and endocrine function. Studies have been conducted to investigate the stability of urinary chemosignals in mice. Neuroendocrine and behavioral responses of mice to urine samples of male and female conspecifics which have aged for different amounts of time have been examined, demonstrating that the quality and intensity of sig- naling molecules in urine change over time. In this study, we monitored changes in volatile organic compounds (VOCs) released from male and female mouse urine following aging the urine samples. Substantial amounts of some VOCs were lost during the aging process of urine, whereas other VOCs increased. Considerable portions of the VOCs which exhibited the increased release were shown to have previously been dissolved in water and subsequently released as the urine dried. We also demonstrated that some VOCs decreased slightly due to their binding with the major urinary proteins (MUPs) and identified MUP ligands whose headspace concentra- tions increased as the urine aged. Our results underscore the important role of MUPs and the hydration status in the release of VOCs in urine, which may largely account for the changes in the quality and intensity of urinary signals over time. © 2013 Elsevier Inc. All rights reserved. 1. Introduction Mice release a variety of chemical signals, particularly through urine, which mediate social interactions and endocrine function. For example, substances present in male urine advance the onset of female puberty, and induce and synchronize estrus [1–3]. Pregnant or lactating female urine stimulates female puberty [4], whereas urinary chemosignals de- rived from grouped female mice delay estrous cycles [5,6]. Pregnancy is terminated when pregnant females are exposed to the urine derived from an unfamiliar male [7]. When male mice are exposed to female mouse urine, they produce ultrasonic vocalizations that attract females [8]. Mice also recognize individual odor differences in urine of conspe- cifics caused by genetic differences. For example, male mice recognize the olfactory signature of urine marks derived from other males and countermark presumably to advertise their presence and ownership [9]. Mice can discriminate the odor differences between their own urine and the urine derived from others carrying different genetic back- ground as well as from those having a small genetic difference in major histocompatibility complex (MHC) genes (reviewed in [10]). As a result, mice prefer to mate with conspecifics whose odor signatures are differ- ent from their own (reviewed in [11]). Physiology & Behavior 120 (2013) 211–219 ⁎ Corresponding author at: Human Signatures Branch, Forecasting Division, Human Effectiveness Directorate, Air Force Research Laboratory, Wright-Patterson AFB, Dayton, OH 45433, USA. Tel.: +1 937 938 3790; fax: +1 937 656 6898. E-mail addresses: jaekwak@hotmail.com (J. Kwak), Claude.Grigsby@wpafb.af.mil (C.C. Grigsby), preti@pobox.upenn.edu (G. Preti), mateen.rizki@wright.edu (M.M. Rizki), beauchamp@monell.org (G.K. Beauchamp). 1 Deceased. 0031-9384/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.physbeh.2013.08.011 Contents lists available at ScienceDirect Physiology & Behavior journal homepage: www.elsevier.com/locate/phb