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Quantifying dietary macronutrient sources of carbon for bone collagen biosynthesis using natural abundance stable carbon isotope analysis

  • Susan Jim (a1), Vicky Jones (a1), Stanley H. Ambrose (a2) and Richard P. Evershed (a1)


The diets of laboratory rats were isotopically and nutritionally manipulated using purifiedC3 and/or C4 macronutrients to investigate the routing of dietary carbonto bone collagen biosynthesis. Diets were formulated with purified proteins, carbohydrates andlipids of defined composition and natural abundance stable isotope ratios. Bulk protein and constituent amino acid δ13C values determined for whole diet and bone collagen provided the basis for assessing isotopic fractionation and estimating the degree of routing versus synthesis de novo of essential, non-essential and conditionally indispensable amino acids. Essential and conditionally indispensable amino acids were shown to be routed from diet to collagen with little isotopic fractionation whereas non-essential amino acids differed by up to 20‰. Mathematical modelling of the relationships between macronutrient and tissue δ13C values provided qualitative and quantitative insights into the metabolic and energetic controls on bone collagen biosynthesis. Essential amino acids comprise 21·7% of the carbon in collagen, defining the minimum amount of dietary carbon routing. Estimates of 42 and 28% routing were shown for the non-essential amino acids, glycine and aspartate, respectively. In total, the routing of non-essential and conditionally indispensable amino acids was estimated to equal 29·6% of the carbon in collagen. When the contribution of carbon from the essential amino acids is also considered, we arrive at an overall minimum estimate of 51·3% routing of dietary amino acid carbon into bone collagen.


Corresponding author

*Corresponding author: Richard Evershed, fax 44 (0)117 9251295, email


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Ambrose, SHIsotopic analysis of paleodiets: methodological and interpretive considerations. In Investigation of Ancient Human Tissue, Chemical Analyses in Anthropology, Sandford, MKLanghorne, PAGordon and Breach Scientific 1993 59130
Ambrose, SH, Butler, BM, Hanson, DB, Hunter-Anderson, RL & Krueger, HWStable isotopic analysis of human diet in the Marianas Archipelago, Western Pacific. Am J Phys Anthropol 1997 104 343361
Ambrose, SH & Norr, LExperimental evidence for the relationship of the carbon isotope ratios of whole diet and dietary protein to those of bone collagen and carbonate. In Prehistoric Human Bone – Archaeology at the Molecular Level, Lambert, JB and Grupe, GBerlinSpringer-Verlag 1993 137
Atasoglu, C, Guliye, AY & Wallace, RJUse of stable isotopes to measure de novo synthesis and turnover of amino acid-C and -N in mixed micro-organisms from the sheep rumen in vitro. Br J Nutr 2004 91 253261
Ayliffe, LK, Cerling, TE, Robinson, T, West, AG, Sponheimer, M, Passey, BH, Hammer, J, Roeder, B, Dearing, MD & Ehleringer, JRTurnover of carbon isotopes in tail hair and breath CO2 of horses fed an isotopically varied diet. Oecologia 2004 139 1122
Ball, RO, Atkinson, JL & Bayley, HSProline as an essential amino acid for the young pig. Br J Nutr 1986 55 659668
Bocherens, H & Drucker, DTrophic level isotopic enrichment of carbon and nitrogen in bone collagen: case studies from recent and ancient terrestrial ecosystems. Int J Osteoarchaeol 2003 13 4653
Brenna, JTNatural 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 12521262
Chisholm, BS Reconstruction of prehistoric diet in British Columbia using stable carbon isotopic analysis. PhD Thesis, Simon Fraser University. 1986
Chung, TK & Baker, DHA note on the dispensability of proline for weanling pigs. Anim Prod 1993 56 407408
Craig, HIsotopic standards for carbon and oxygen and correction factors for mass-spectrometric analysis of carbondioxide. Geochim Cosmochim Acta 1957 12 133149
DeNiro, MJ & Epstein, SInfluence of diet on the distribution of carbon isotopes in animals. Geochim Cosmochim Acta 1978 42 495506
Docherty, G, Jones, V & Evershed, RP2001) Practical and theoretical considerations in the gas chromatography/combustion/isotope ratio mass spectrometry δ13C analysis of small polyfunctional compounds. Rapid Commun Mass Spectrom 2001 15 730738
Francois, CJ & Glimcher, MJIsolation and amino acid composition of alpha chains of chicken bone collagen. Biochim Biophys Acta 1967 133 91
Fuller, MF & Garlick, PJHuman amino acid requirements: can the controversy be resolved? Annu Rev Nutr 1994 14 217241
Gannes, LZ, Martínez de Rio, C & Koch, PNatural abundance variations in stable isotopes and their potential uses in animal physiological ecology. Comp Biochem Physiol 1998 119A 725737
Guillet, C, Boirie, Y & Walrand, SAn integrative approach to in-vivo protein synthesis measurements: from whole tissues to specific proteins. Curr Opin Clin Nutr Metab Care 2004 7 531538
Hare, PE, Fogel, ML, Stafford, TW Jr, Mitchell, AD & Hoering, TCThe isotopic composition of carbon and nitrogen in individual amino acids isolated from modern and fossil proteins. J Archaeol Sci 1991 18 277292
Howland, MR, Corr, T, Young, SMM, Jones, V, Jim, S, van derMerwe, NJ, Mitchell, AD & Evershed, RPExpression of the dietary isotope signal in the compound-specific δ13C values of pig bone lipids and amino acids. Int J Osteoarchaeol 2003 13 5465
Jim, S The development of bone cholesterol δ13C values as a new source of palaeodietary information: models of its use in conjunction with bone collagen and apatite δ13C values. PhD Thesis University of Bristol 2000
Jim, S, Ambrose, SH & Evershed, RPStable carbon isotopic evidence for the routing and de novo synthesis of bone fatty acids and cholesterol. Lipids 2003 38 179186
Jim, S, Ambrose, SH & Evershed, RPStable carbon isotopic evidence for differences in the biosynthetic origin of bone cholesterol, collagen and apatite: implications for their use in palaeodietary reconstruction. Geochim Cosmochim Acta 2004 68 6172
Jim, S, Jones, V, Copley, MS, Ambrose, SH & Evershed, RPEffect of hydrolysis on the δ13C values of amino acids derived from polypeptides and proteins. Rapid Commun Mass Spectrom 2003 17 22832289
Krueger, HW & Sullivan, CHModels for carbon isotope fractionation between diet and bone. In Stable Isotopes in Human Nutrition Turnlund, JS & Johnson,, PEWashington, DCAmerican Chemical Society 1984 205220
Lee-Thorp, JA, Sealy, JC & van der Merwe, NJStable carbon isotope ratio differences between bone collagen and bone apatite, and their relationship to diet. J Archaeol Sci 1989 16 585599
Lee-Thorp, JA, Sponheimer, M & van der Merwe, NJWhat do stable isotopes tell us about hominid dietary and ecological niches in the Pliocene? Int J Osteoarchaeol 2003 13 104113
Macko, SA, Uhle, ME, Engel, MH & Andrusevich, VStable nitrogen isotope analysis of amino acid enantiomers by gas chromatography/combustion/isotope ratio mass spectrometry. Anal Chem 1997 69 926929
Morrison, DJ, Dodson, B, Slater, C & Preston, T13C natural abundance in the British diet: implications for 13C breath tests. Rapid Commun Mass Spectrom 2000 144 13211324
O'Brien, DM, Fogel, ML & Boggs, CLRenewable and nonrenewable resources: amino acid turnover and allocation to reproduction in lepidoptera. Proc Natl Acad Sci USA 2002 99 44134418
Reeds, PDispensable and indispensable amino acids for humans. J Nutr 2000 130 S1835S1840
Savidge, WB & Blair, NESeasonal and within-plant gradients in the intramolecular carbon isotopic composition of amino acids of Spartina alterniflora. J Exp Mar Biol Ecol 2004 308 151167
Schwarcz, HP2000) Some biochemical aspects of carbon isotopic paleodiet studies. In Biogeochemical Approaches to Paleodietary Analysis, Ambrose, SH and Katzenberg, MANew YorkKluwer/Plenum 2000 189209
Tieszen, LL & Boutton, TWStable carbon isotopes in terrestrial ecosystem research. In Stable Isotopes in Ecological Research, Rundel, PW, Ehleringer, JR and Nagy, KALondonSpringer 1988 167195
Tieszen, LL & Fagre, TEffect of diet quality and composition on the isotopic composition of respiratory CO2, bone collagen, bioapatite, and soft tissues. In Prehistoric Human Bone – Archaeology at the Molecular Level, Lambert, JB and Grupe, GBerlinSpringer-Verlag 1993 121155
Tuross, N, Fogel, ML & Hare, PEVariability in the preservation of the isotopic composition of collagen from fossil bone. Geochim Cosmochim Acta 1988 52 929935
van Klinken, GJNote on the isolation of single amino acids from fossil bone. Appl Geochem 1989 4 271
Voet, D & Voet, JGAmino acid biosynthesis. In Biochemistry, New YorkJohn Wiley and Sons 1995 764776
Wu, CC, MacCoss, MJ, Howell, KE, Matthews, DE & Yates, JR IIIMetabolic labelling of mammalian organisms with stable isotopes for quantitative proteomic analysis. Anal Chem 2004 76 49514959
Young, VR & El-Khoury, AEThe notion of the nutritional essentiality of amino acids, revisited, with a note on the indispensable amino acid requirements in adults. InAmino Acid Metabolism and Therapy in Health and Nutritional Disease, Cynober, LABoca Raton, FLCRC Press 1995 191232



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