Various biomolecular components preserved in domesticated animal bones recovered from the Nubian site of Qasr Ibrim are used for dietary reconstruction of their foddering and foraging behaviours. Utilising models of the biochemical correlations with the dietary components and their turnover rates, the bulk stable isotope values of bone collagen and apatite combined with compound-specific stable isotope values of the collagenous amino acids (essential and non-essential) provided long-term indicators of the diet of cattle and sheep/goats from the site. Cattle appear to have predominantly consumed C4 plants, such as sorghum (Sorghum bicolor bicolor Moench.) and millet (Panicum miliaceum L.), during the later periods at the site, suggesting that cattle were subjected to differing feeding strategies over the period of occupation of the site. Furthermore, the δ13C values of the individual fatty acids (n-hexadecanoic and -octadecanoic acids) preserved in the bones provide short-term indicators of the animals' diet. The application of a new model based on δ13C values of the bone apatite and fatty acids indicates differences in the long- and short-term diets of sheep/goat, which are less obvious in cattle.

Pigs were raised on six isotopically controlled diets to examine the dietary macronutrients used in the synthesis of bulk bone isochemical components (apatite, collagen and lipids) and individual compounds (bone fatty acids, cholesterol and amino acids from collagen). delta C-13 values of apatite and bulk bone lipids reflected those of the whole diet, with Delta C-13(apatite-whole diet)=10.2 +/-1.3% and Delta C-13(bone lipids-whole diet)=-2.4 +/-0.7%. A wide variation observed in the Delta C-13(collagen-whole diet) values (0.5 to 6.1%) was hypothesized to reflect the relative importance of (i) the direct incorporation of essential amino acids, and (ii) the balance between direct incorporation and de novo synthesis of non-essential amino acids. Linear regression (n=6) was used to assess the relationship between the delta C-13 values of whole diet and bulk bone components and individual compounds. Whole diet delta C-13 values showed a strong correlation with those of bone cholesterol (R-2=0.85) and glutamate (R-2=0.96) in collagen. The essential amino acids leucine (Delta (13)C(collagen leu diet leu)0.5 +/-1.2%) and phenylalanine (Delta C-13(collagen phe-diet phe)=-0.6 +/-0.6%) showed little isotopic fractionation between diet and bone collagen.

Rats were raised on a variety of isotopically controlled diets comprising 20% C3, C4 or marine protein and C3 and/or C4 non-protein or energy (i.e. sucrose, starch and oil) macronutrients. Compound specific stable carbon isotope (δ13C) analysis was performed on the cholesterol isolated from the diet (n=7 ) and bone (n=15 ) of these animals and the values compared with bulk δ13C measurements of bone collagen and apatite. The dietary signals reflected by these three bone biochemical components were investigated using linear regression analysis. δ13C values of bone cholesterol were shown to reflect whole diet δ13C values, collagen to reflect mainly dietary protein values and apatite to reflect whole diet values. Further correlations between dietary protein-to-energy spacings (Δ13Cprot-engy = δ13Cprotein – δ13Cenergy) and whole diet-to-bone component fractionations (Δ13Cbcomp-wdiet = δ13Cbone component – δ13Cwhole diet) indicates that for hypothetical diets where protein δ13C values are equal to energy values, fractionations between whole diet and bone biochemical fractions are –3.3‰ for cholesterol, +5.4‰ for collagen and +9.5‰ for apatite. Moreover, the narrow range of variation observed in apatite-to-cholesterol spacings (Δ13Capat-bchol) suggests that cholesterol δ13C values can potentially also be used as an independent test for the isotopic integrity of apatite δ13C values. These insights into bone cholesterol, collagen and apatite dietary signals, diet-to-bone fractionations and bone component-to-bone component spacings provide the basis for more accurate interpretations of the dietary behaviour of archaeological populations and food webs when the δ13C analysis of bone is employed.

This study investigates the effects of hydrolysis on the delta(13)C values of individual amino acids (IAAs) derived from polypeptide standards, and modern and ancient bone collagen. All IAAs were derivatised to their trifluoroacetyl/isopropyl (TFA/IP) esters for delta(13)C determination using gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). Firstly, authentic single poly amino acid standards (SPAAs; n = 5) were hydrolysed for 4,10, 24 and 48 h. As expected, IAA yields increased as a function of hydrolysis time. Significantly, it was only after 24 h of hydrolysis that IAA delta(13)C values were statistically identical to bulk SPAA values for all five standards. The accuracy of IAA delta(13)C values was thus shown to be a function of yield; however, poly phenylalanine demonstrated accurate IAA delta(13)C values with yields of only 1.4 and 4.3%, after 24 and 48 h of hydrolysis time, respectively. Authentic mixed poly amino acid standards (MPAAs; n = 5) comprising two different amino acids were then hydrolysed for 24 h. Percentage recoveries ranged from 36-95%. Estimates of bulk MPAA delta(13)C values calculated from measured IAA delta(13)C values agreed within experimental error with measured bulk MPAA values for three out of the five standards. Finally, the experimental procedure was applied to modem rat (MBCs; n = 20) and ancient ovicaprine and bovine (ABCs; n=27) bone collagen samples where the delta(13)C values of 12 out of its 18 constituent amino acids were determined. Estimated bulk MBC and ABC delta(13)C values were calculated from constituent amino acid delta(13)C values using mass balance. With the exclusion of three ABC samples, calculated bulk bone collagen delta(13)C values (delta(13)C(BCcal)) were shown to correlate extremely well with measured bone collagen values (delta(13)CB(Cmes)) for both modern and ancient samples, 2 where R-2 = 0.91 and 0.84, respectively. Significantly, the variation between calculated and measured bone collagen values (Delta(13)C(BCcal-BCmes)) exhibited similar ranges for both MBC (from -2.6 to +1.2parts per thousand) and ABC (from -2.7 to +2.2parts per thousand) samples, providing evidence for the preservation of intact collagen in the ancient samples. These results demonstrate that the experimental procedures employed in the acid hydrolytic cleavage of peptides or proteins to their constituent amino acids does not involve significant isotopic fractionation. Copyright (C) 2003 John Wiley Sons, Ltd.

Rats were raised on a variety of isotopically controlled diets comprising 20% C-3, C-4 or marine protein and 70% C-3 and/or C-4 energy (sucrose, starch and lipid) components. Isotope ratio monitoring-gas chromatography/mass spectrometry (irm-GC/MS) was used to investigate the effect on the stable carbon isotopic composition of their flesh cholesterol as a result of varying the isotopic composition of the protein and energy components in their diets. The delta(13)C values of cholesterol obtained for the pure C-3 and C-4 fed rats compared to within 1.5parts per thousand to the results of an analogous study performed on pigs raised on monotonous C-3 and C-4 diets. Distinct delta(13)C values of cholesterol were found for all rats raised on different diets. The faster turnover rate of flesh cholesterol with respect to bone collagen and apatite was demonstrated from the analysis of an animal which was fed a pure C-3 diet at birth and then switched to a pure C-4 diet at 131 days old and sacrificed 40 days later. Comparisons of the cholesterol delta(13)C values obtained from studies of this type with those measured on cholesterol recovered from ancient human bone may provide the basis for more accurate interpretations of the dietary behaviour of past populations when cholesterol is used as a new corroboratory source of palaeodietary information.

Cholesterol and its diagenetic congeners have been detected in a wide range of human and animal bones dating to the Mesolithic period. Using isotope ratio monitoring-gas chromatography/mass spectrometry (irm-GC/MS), it is possible to determine accurately compound specific δ13C values of preserved cholesterol with high precision (± 0·4 per mil). The δ13C value of cholesterol provides information complementary to that derived from collagen and apatite stable isotopes for use in palaeodietary studies. Potential advantages of using cholesterol are that: (1) the δ13C value will be biased towards the original isotopic composition of the carbohydrates and fats in the diet, (2) its δ13C value will retain isotopic integrity, i.e., will not be diagenetically altered, provided its carbon skeleton is characterized, and (3) that its relatively rapid rate of turnover provides a means of investigating carbon cycling on a shorter time scale than collagen. Laboratory animal feeding experiments have shown that cholesterol faithfully derives its isotopic signature from the diet and has a faster turnover rate than collagen. This paper reviews aspects of the methodologies employed in recording δ13C values from cholesterol preserved in skeletal materials and presents new results obtained from modern and archaeological humans, and studies of experimental animals.