Nitrogen and Carbon Stable Isotope Composition Informs Metabolic Routing in Blarina brevicauda Tissues Altai Perry1, Ryan Stephens2, MS, and Erik Hobbie3, PhD University of New Hampshire AP (1) Department of Chemical Engineering, RS (2) Department of Natural Resources, EH (3) Earth Systems Research Center Abstract Isotopic routing is a manifestation of metabolic process and the kinetic isotope effect. Using mass spectrometry for 13C and 15N, muscle lipid, muscle protein, hair, and cecum of Blarina brevicauda was analyzed. Hair had the highest 15N signal followed by muscle protein and finally by muscle lipid; muscle protein had the highest 13C signal followed by hair and finally by muscle lipids. These are consistent with the expected results from kinetic isotope effects in metabolism such as The higher content of non-essential amino acids in muscle protein relative to keratin should increase muscle 13C:12C ratios. Formation of lipids from acetyl-coA should lead to 13C-depleted lipids relative to body protein and bulk food sources. Gastrointestinal contents were collected and analyzed to obtain an estimate of bulk diet isotopic composition Goals Determine the extent to which isotopic routing affects 13C and 15N in the tissues of wild Blarina brevicauda. Gather preliminary data and results on a larger diet reconstruction project involving several more species of small mammals. Develop methods of predicting heavy isotope fractionation by bridging empirical data and theoretical biochemical concepts. Moving Forward Background 15N is depleted by processes that excrete ammonia within animals (denitrification, deamination)1. 15N is enriched based upon trophic level1 Northern short-tailed shrews are primarily carnivores2. 13C is discriminated against during fatty acid synthesis during the creation of Acetyl-CoA3. 13C tends to be enriched through integration of essential amino acids. Collagen has a higher amount of essential amino acid integration than muscle protein4. The kinetic isotope effect results in a decrease in reaction rate and relative abundance in heavy isotopes5. Species and Compound Figure 2: A schematic of the movement of 13C and 15N through animals shows that relative isotopic abundance in tissues is governed by rate-determining reactions and abundance of essential amino acid integration. Scat (cecum is used as surrogate) heavy isotope integration is indicative of bulk diet (shown as “Food” in the schematic). While deamination provides an mechanism for 15N depletion, it serves mostly as a possible explanation for the relative enrichment of essential amino acids compared to nonessential amino acids. Essential amino acids tend to also be enriched in 13C due to a similar mechanism. While they tend to have low nitrogen content, lipids also tend to have high rates of 15N discrimination based upon the results of this study. Methods Results Twenty northern short-tailed shrews (Blarina brevicauda) were captured at Bartlett Experimental Forest in New Hampshire. Muscle, hair, and gastrointestinal contents were collected. The muscle lipids were extracted using a Bligh and Dwyer extraction 6 and then ground into a fine powder using liquid nitrogen. The hair was cleaned using chloroform and methanol (2:1 vol.) to wash off contaminants. All samples were freeze-dried and analyzed for %N, %C, d15N, and d13C with an isotope ratio mass spectrometer linked to an elemental analyzer (EA-IRMS). From figure 1, both d13C and d15N followed similar trends across the tissue types, even though d15N experienced larger variation overall. However, the variance differs greatly where the highest variance in d15N was in lipid and the highest variance in d13C was in hair. From figure 3 it can be seen that the means roughly fall on a line. By running a multilinear regression on the data, individual animals as a factor had a larger effect on d15N than the type of tissue examined. This accounts for the large variance in d15N in figures 1 and 3. Figure 4: Lipid and protein samples from all five species investigated as part of the larger study are plotted in d15N-d13C space (while MIPE is plotted, there are not enough samples to construct an ellipse). From figure 4, it is seen that all protein samples are relatively enriched in both 13C and 15N. The change in diet simply shifts the total 15N integration while no horizontal shifts in d13C seem to occur, hinting that once a line in d15N-d13C space is created, a corresponding bulk 15N can be found, indicative of bulk diet. Moving forward, stomach, cecum, and hair from all species will be integrated into the model in the hopes of developing accurate diet reconstruction techniques robust enough to describe wildlife behavior and its changes Conclusions Acknowledgements We thank Corina Danielson, Amanda Adams, and Nimesh Bhattarai for laboratory assistance. The U.S. Forest service collected the specimens used in this study. This work was supported by the U.S. National Science Foundation, grant ARC1108074 and an NSF Research Experience for Undergraduates grant.  While the average stable isotope composition of the types of tissues are substantially different, the size of the variation and the high dependence on individual show that some of the highest factors in heavy isotope composition (especially that of 15N) is highly dependent on an individual’s diet. Blarina brevicauda has been known to consume many different kinds of insects2 whose chitin makeup vary considerably. Chitin is heavily deleted in 15N4 and can vary bulk 15N consumption. Lipid samples are heavily depleted in both 15N and 13C, hinting that nitrogen integration into lipids, while small and limited to specific types, is heavily depleted. References Adams, T. S.; Sterner, R. W. Limnol. Oceanogr. 2000, 45 (3), 601–607. No, M. S.; Sarah, B.; Choate, J. R.; Genoways, H. H. Mamm. Species 1986, 261 (261), 1–9. Cunnane, S. C. Fed. Eur. Biochem. Soc. 1992, 306 (2), 273–275. Hobbie, E. A. Rapid Commun. Mass Spectrom. 2017, 31 (7), 639–648. Northrop, D. B. Biochemistry 1975, 14 (12), 2644–2651. Bligh, E.; Dyer, W. Can. J. Biochem. 1959, 37 (8). Figure 3: Heavy-isotopic ratio signals of muscle lipid (L), cecum (C), muscle protein (P), and hair (H), each are represented by their first letter. The boundary of the ellipses represent the 95% confidence interval of each population. The means of the population are the center of the ellipses. In general, hair varies the most out of all of the tissues. Figure 1: Boxplots of the d13C and d15N values are shown. In general, d15N has a tighter spread and shows more dependence on the type of tissue.