Stable carbon isotope diagnostics of mammalian metabolism, a high-resolution isotomics approach using amino acid carboxyl groups

Autoři: Brian Fry aff001;  James F. Carter aff002
Působiště autorů: Australian Rivers Institute, Griffith University, Nathan, Queensland, Australia aff001;  Queensland Health Forensic and Scientific Services, Coopers Plains, Queensland, Australia aff002
Vyšlo v časopise: PLoS ONE 14(10)
Kategorie: Research Article
doi: 10.1371/journal.pone.0224297


The carbon isotopic compositions of amino acids are increasingly measured to characterize diets and metabolic response to diets. We report a new high-resolution system to measure the stable carbon isotopic composition of carboxyl atoms within amino acids. The automated system used HPLC to separate amino acids followed by addition of ninhydrin for decarboxylation and transfer of the evolved CO2 to a stable isotope ratio mass spectrometer for δ13CCARBOXYL measurement. The ninhydrin reaction was conducted at acidic pH (1.5) and elevated temperature (160 oC) giving yields close to 100% for most common amino acids. Eight mammalian keratin samples from herbivores (kudu and caribou), omnivores (humans) and carnivores (bowhead and humpback zooplanktivorous whales) were analysed with this new system. The data provide an initial calibration of reference materials to be used in studies of this type and is the first report of carboxyl carbon isotope distributions in mammals. Results showed widespread 13C enrichments in both essential and non-essential amino acid carboxyl groups, likely linked to decarboxylation of amino acids during normal metabolism. Analyses of non-essential amino acid isotope profiles showed (1) consistent and general taxon-level metabolic differences between the herbivore, human and whale samples, (2) marked differences among individual humans, ruminants and whales (3) evidence for gluconeogenesis in the wildlife samples, and (4) extensive 13C enrichment likely associated with fasting in the humpback whale sample. Future mammalian research related to the metabolism of growth, reproduction, aging and disease may benefit from using this technique. Values obtained for internationally available samples USGS42 and USGS43 (Tibetan and Indian human hair) provide a first characterization of reference materials for δ13CCARBOXYL profiles.

Klíčová slova:

Amino acid analysis – Amino acid metabolism – Deer – High performance liquid chromatography – Humpback whales – Isotopes – Keratins – Whales


1. Mosora F, Marechal R, Manil P, Duchesne J. Sur le rapport isotopique 13C/12C dans une tumeur ajligne chez le Rat. C R Acad Sci Hebd Seances Acad Sci D. 1972;275(4):599–601. 4342849

2. Mosora F, Lacroix M, Duchesne J. Radicaux libres et rapport isotopique 13C/12C dans des tumeurs vegetales. C R Acad Sci Hebd Seances Acad Sci D. 1972;275(15):1701–2. 4629943

3. Park R, Epstein S. Metabolic fractionation of 13C and 12C in plants. Plant Physiol. 1961;36(2):133–8. doi: 10.1104/pp.36.2.133 16655481

4. Larsen T, Ventura M, O'Brien DM, Magid J, Lomstein BA, Larsen J. Contrasting effects of nitrogen limitation and amino acid imbalance on carbon and nitrogen turnover in three species of Collembola. Soil Biol Biochem. 2011;43(4):749–59. doi: 10.1016/j.soilbio.2010.12.008

5. Abelson PH, Hoering TC. Carbon isotope fractionation in formation of amino acids by photosynthetic organisms. Proc Natl Acad Sci U S A. 1961;47:623–32. doi: 10.1073/pnas.47.5.623 13681011

6. Savidge WB, Blair NE. Seasonal and within-plant gradients in the intramolecular carbon isotopic composition of amino acids of Spartina alterniflora. J Exp Mar Bio Ecol. 2004;308(2):151–67. doi: 10.1016/j.jembe.2004.02.010

7. Meier-Augenstein W. Stable isotope forensics: methods and forensic applications of stable isotope analysis. 2nd ed. Hoboken, NJ: John Wiley & Sons,; 2018.

8. Rashaid AHB, Harrington PB, Jackson GP. Amino acid composition of human scalp hair as a biometric classifier and investigative lead. Anal Methods. 2015;7(5):1707–18. doi: 10.1039/C4AY02588A

9. Fry B, Carter JF, Yamada K, Yoshida N, Juchelka D. Position-specific 13C/12C analysis of amino acid carboxyl groups—automated flow-injection analysis based on reaction with ninhydrin. Rapid Commun Mass Spectrom. 2018;32(12):992–1000. doi: 10.1002/rcm.8126 29575301

10. Choy K, Smith CI, Fuller BT, Richards MP. Investigation of amino acid δ13C signatures in bone collagen to reconstruct human palaeodiets using liquid chromatography-isotope ratio mass spectrometry. Geochim Cosmochim Acta. 2010;74(21):6093–111. doi: 10.1016/j.gca.2010.07.025

11. Kaleta C, de Figueiredo LF, Werner S, Guthke R, Ristow M, Schuster S. In silico evidence for gluconeogenesis from fatty acids in humans. PLoS Comput Biol. 2011;7(7):e1002116. doi: 10.1371/journal.pcbi.1002116 21814506

12. Godin J-P, Ross AB, Rezzi S, Poussin C, Martin F-P, Fuerholz A, et al. Isotopomics: A top-down systems biology approach for understanding dynamic metabolism in rats using [1,2-13C2] acetate. Anal Chem. 2010;82(2):646–53. doi: 10.1021/ac902086g 20028023

13. Adeniyi-Jones SK, Karnovsky ML. Oxidative decarboxylation of free and peptide-linked amino acids in phagocytizing guinea pig granulocytes. J Clin Invest. 1981;68(2):365–73. doi: 10.1172/JCI110264 6267101

14. Berg JM, Tymoczko JL, Stryer L, Gatto GJ. Biochemistry. 7th ed. New York: W.H. Freeman and Co.; 2012.

15. McCullagh J, Gaye‐Siessegger J, Focken U. Determination of underivatized amino acid δ13C by liquid chromatography/isotope ratio mass spectrometry for nutritional studies: the effect of dietary non‐essential amino acid profile on the isotopic signature of individual amino acids in fish. Rapid Commun Mass Spectrom. 2008;22(12):1817–22. doi: 10.1002/rcm.3554 18473333

16. Newsome SD, Fogel ML, Kelly L, del Rio CM. Contributions of direct incorporation from diet and microbial amino acids to protein synthesis in Nile tilapia. Funct Ecol. 2011;25(5):1051–62. doi: 10.1111/j.1365-2435.2011.01866.x

17. Newsome SD, Wolf N, Peters J, Fogel ML. Amino acid δ13C analysis shows flexibility in the routing of dietary protein and lipids to the tissue of an omnivore. Integr Comp Biol. 2014;54(5):890–902. doi: 10.1093/icb/icu106 25104856

18. DeNiro MJ, Epstein S. Mechanism of carbon isotope fractionation associated with lipid synthesis. Science. 1977;197:261–263. doi: 10.1126/science.327543 327543

19. Melzer E, and Schmidt HL. Carbon isotope effects on the pyruvate dehydrogenase reaction and their importance for relative carbon-13 depletion in lipids. J Biol Chem. 1987;17:8159–8164.

20. Exley RA, Mattey DP, Clague DA, Pillinger CT. Carbon isotope systematics of a mantle "hotspot": a comparison of Loihi Seamount and MORB glasses. Earth Planet Sci Lett. 1986;78(2–3):189–99. doi: 10.1016/0012-821X(86)90060-9

21. Brenna JT. Natural intramolecular isotope measurements in physiology: elements of the case for an effort toward high-precision position-specific isotope analysis. Rapid Commun Mass Spectrom. 2001;15(15):1252–62. doi: 10.1002/rcm.325 11466780

22. Tcherkez G, Ghashghaie J, Griffiths H. Methods for improving the visualization and deconvolution of isotopic signals. Plant, Cell Environ. 2007;30(8):887–91. doi: 10.1111/j.1365-3040.2007.01687.x 17617817

23. Coplen TB, Qi H. USGS42 and USGS43: Human-hair stable hydrogen and oxygen isotopic reference materials and analytical methods for forensic science and implications for published measurement results. Forensic Sci Int. 2012;214(1):135–41. doi: 10.1016/j.forsciint.2011.07.035 21852055

24. Wassenaar LI, Hobson KA. Two keratin standards (δ2H, δ18O) for daily use in wildlife and forensic isotope studies. ISOECOL VII; Aug 9–13; Fairbanks, Alaska2010.

25. Wassenaar L, Hobson K. Comparative equilibration and online technique for determination of non-exchangeable hydrogen of keratins for use in animal migration studies. Isot Environ Health Stud. 2003;39(3):211–7. doi: 10.1080/1025601031000096781 14521282

26. Eisenmann P. Interpreting the feeding ecology of Southern hemisphere humpback whales through biochemical assessment. PhD Thesis: Griffith University; 2016.

27. Eisenmann P, Fry B, Holyoake C, Coughran D, Nicol S, Nash SB. Isotopic evidence of a wide spectrum of feeding strategies in southern hemisphere humpback whale baleen records. PLoS One. 2016;11(5):e0156698/1-e/20. doi: 10.1371/journal.pone.0156698 27244081

28. Matthews CJD, Ferguson SH. Seasonal foraging behaviour of Eastern Canada-West Greenland bowhead whales: an assessment of isotopic cycles along baleen. Mar Ecol: Prog Ser. 2015;522:269–86. doi: 10.3354/meps11145

29. Moore S, Stein WH. Chromatographic determination of amino acids by the use of automatic recording equipment. Methods Enzymol. 1963;6:819–31. doi: 10.1016/0076-6879(63)06257-1

30. Metges CC, Daenzer M. 13C gas chromatography-combustion isotope ratio mass spectrometry analysis of N-pivaloyl amino acid esters of tissue and plasma samples. Anal Biochem. 2000;278(2):156–64. doi: 10.1006/abio.1999.4426 10660457

31. Jim S, Jones V, Copley MS, Ambrose SH, Evershed RP. Effects of hydrolysis on the δ13C values of individual amino acids derived from polypeptides and proteins. Rapid Commun Mass Spectrom. 2003;17(20):2283–9. doi: 10.1002/rcm.1177 14558127

32. Krummen M, Hilkert AW, Juchelka D, Pesch R. A new concept for isotope ratio monitoring liquid chromatography/mass spectrometry. Rapid Commun Mass Spectrom. 2004;18(19): 2260–2266. doi: 10.1002/rcm.1620 15384146

33. Smith CI, Fuller BT, Choy K, Richards MP. A three-phase liquid chromatographic method for δ13C analysis of amino acids from biological protein hydrolysates using liquid chromatography-isotope ratio mass spectrometry. Anal Biochem. 2009;390(2):165–72. doi: 10.1016/j.ab.2009.04.014 19379706

34. Graham CE, Waitkoff HK, Hier SW. Amino acid content of some scleroproteins. J Biol Chem. 1949;177:529–32. 18110430

35. Carter JF, Yates HSA, Tinggi U. Isotopic and Elemental Composition of roasted coffee as a guide to authenticity and origin. J Agric Food Chem. 2015;63(24):5771–9. doi: 10.1021/acs.jafc.5b01526 26001050

36. Van Slyke DD, Dillon RT, MacFadyen DA, Hamilton P. Gasometric determination of carboxyl groups in free amino acids. J Biol Chem. 1941;141:627–69.

37. Hamilton PB, Van Slyke DD, Lemish S. The gasometric determination of free amino acids in blood filtrates by the ninhydrin carbon dioxide method. J Biol Chem. 1943;150:231–50.

38. Block RJ, Bolling D, Brand FC, Schein A. The composition of keratins. The amino acid composition of hair, wool, horn, and other euketatins. J Biol Chem. 1939;128:181–6.

39. Van Sande M. Hair amino acids: normal values and results in metabolic errors. Arch Dis Child. 1970;45(243):678–81. doi: 10.1136/adc.45.243.678 5477681

40. Rustad JR. Ab initio calculation of the carbon isotope signatures of amino acids. Org Geochem. 2009;40(6):720–3. doi: 10.1016/j.orggeochem.2009.03.003

41. Hayes JM. Fractionation of carbon and hydrogen isotopes in biosynthetic processes. Rev Mineral Geochem. 2001;43:225–77. doi: 10.2138/gsrmg.43.1.225

42. Bada JL, Schoeninger MJ, Schimmelmann A. Isotopic fractionation during peptide bond hydrolysis. Geochim Cosmochim Acta. 1989;53(12):3337–41. doi: 10.1016/0016-7037(89)90114-2

43. Silfer JA, Engel MH, Macko SA. Kinetic fractionation of stable carbon and nitrogen isotopes during peptide bond hydrolysis: Experimental evidence and geochemical implications. Chem Geol. 1992;101(3–4):211–21. doi: 10.1016/0009-2541(92)90003-N

44. Aguilar A, Gimenez J, Gomez-Campos E, Cardona L, Borrell A. δ15N value does not reflect fasting in mysticetes. PLoS One. 2014;9(3):e92288. doi: 10.1371/journal.pone.0092288 24651388

45. Choy K, Nash SH, Kristal AR, Hopkins S, Boyer BB, O'Brien DM. The carbon isotope ratio of alanine in red blood cells is a new candidate biomarker of sugar-sweetened beverage intake. J Nutr. 2013;143(6):878–84. doi: 10.3945/jn.112.172999 23616504

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2019 Číslo 10