Natural Abundance Stable Carbon Isotope Evidence for the Routing and de novo Synthesis of Bone FA and Cholesterol Susan Jima, Stanley H. Ambroseb, and Richard P. Eversheda,* aOrganic Geochemistry Unit, Biogeochemistry Research Centre, School of Chemistry, University of Bristol, Bristol BS8 1TS, United Kingdom, and bDepartment of Anthropology, University of Illinois, Urbana-Champaign, Urbana Illinois 61801 ABSTRACT: This research reported in this paper investigated thought to depend on several factors, including the nutritional the relationship between diet and bone FA and cholesterol in status and digestive physiology of the animal, the turnover rats raised on a variety of isotopically controlled diets compris- rate of the tissue, and its biosynthetic pathway (6). ing 20% C3 or C4 protein (casein) and C3 and/or C4 nonprotein Insights into the relationship between the isotopic composi- or energy (sucrose, starch, and oil) macronutrients. Compound- tion of specific dietary macronutrients and body tissues have δ13 specific stable carbon isotope analysis ( C) was performed on been gleaned from isotopically controlled animal feeding ex- the FA (16:0, 18:0, 18:1, and 18:2) and cholesterol isolated from periments (7–11). Important findings from these studies in- the diet (n = 4) and bone (n = 8) of these animals. The dietary signals reflected by the bone lipids were investigated using lin- clude: (i) an enrichment of 0.8 ± 1.1‰ is observed between the ear regression analysis. δ13C values of bone cholesterol and carbon isotopic composition of whole animals (nematodes, in- stearic (18:0) acid were shown to reflect whole-diet δ13C val- sects, shrimps, snails) and that of their respective diets (7); ues, whereas the δ13C values of bone palmitic (16:0), oleic (ii) the isotopic relationships among the dietary biochemical δ13 δ13 (18:1), and linoleic (18:2) acids reflected dietary FA C values. components of foodstuffs, namely, Ctotal organic matter > Dietary signal differences are a result of the balance between δ13 δ13 δ13 δ13 δ13 Clipid, Ccarbohydrates > Clipid, and Cprotein > Clipid, direct incorporation (or routing) and de novo synthesis of each is inherited by the tissues of animals raised on them (7); of these bone lipids. Estimates of the degree of routing of these (iii) bone collagen δ13C values are biased toward that of the di- bone lipids gleaned from correlations between ∆13C dlipid−wdiet etary protein (10,11); and (iv) bone apatite δ13C values reflect (= δ13C −δ13C ) spacings and ∆13C diet lipid whole diet blipid−wdiet that of the whole diet (10,11). On the molecular level, Hare (= δ13C −δ13C ) fractionations demonstrated bone lipid whole diet δ13 δ15 that the extent of routing, where 18:2 > 16:0 > 18:1 > 18:0 > et al. (9) measured the C and N values of individual col- cholesterol, reflected the relative abundances of these lipids in lagenous amino acids isolated from modern (including labora- the diet. These findings provide the basis for more accurate in- tory-raised) pigs and archaeological bone using preparative sights into diet when the δ13C analysis of bone fatty FA or cho- ion-exchange HPLC, followed by off-line combustion and iso- lesterol is employed. tope ratio mass spectrometry (IRMS). This study showed that Paper no. L9117 in Lipids 38, 179–186 (February 2003). a characteristic pattern existed among the carbon and nitrogen isotope values of the amino acids. Moreover, the comparison of the stable isotope values of essential and nonessential amino Since the 1970s, stable isotope analysis (13C/12C and acids derived from collagen to those present in the diet of the 15N/14N) has provided a direct method with which to explore pigs gave insights into the metabolic pathways that govern trophic interactions in modern and ancient food webs. The ra- these amino acids. tionale behind using stable isotope analysis for dietary recon- The technique of gas chromatography/combustion/isotope struction is based on two well-established observations: ratio mass spectrometry (GC/C/IRMS), originally reported (i) different food groups have characteristically different iso- by Matthews and Hayes (12), allows the separation and mea- tope ratios, and (ii) when these food groups are consumed by surement of the stable isotope ratios of individual compounds an organism, they influence the isotopic composition of its in a sample mixture. GC/C/IRMS requires only nanograms of tissues. Hence, measured isotope values of a consumer’s tis- individual compounds to be introduced to achieve a precision sues serve as a natural tracer for its dietary intake. The dietary of ±0.3‰ for carbon isotope determinations. It is therefore a information or dietary signal obtained from the δ13C analysis much more sensitive and less laborious technique to use than of different consumer tissues reflects different aspects of the preparative HPLC for tracing dietary carbon into consumer diet (1–5). The relationship between dietary macronutrient tissues at the molecular level. Adopting a molecular approach components and consumer tissue types is complex and is not only increases the specificity of dietary investigations but also can circumvent many of the problems associated with the *To whom correspondence should be addressed at Organic Geochemistry Unit, Biogeochemistry Research Centre, University of Bristol, School of effects of contamination encountered in bulk isotope determi- Chemistry, Cantock’s Close, Bristol BS8 1TS, United Kingdom. nations. GC/C/IRMS has allowed dietary insights to be E-mail: [email protected] gleaned using individual amino acids (13,14), FA (15–17), Abbreviations: BSTFA, bis(trimethylsilyl)trifluoroacetamide; GC/C/IRMS, gas chromatography/combustion/isotope ratio mass spectrometry; TLE, total and cholesterol (16,18–20; Jim, S., Evershed, R.P., and Am- lipid extract; TMS, trimethylsilyl. brose, S.H., unpublished data) as indicators of diet. Copyright © 2003 by AOCS Press 179 Lipids, Vol. 38, no. 2 (2003) 180 S. JIM ET AL. The aim of this paper was to use GC/C/IRMS to assess the MATERIALS AND METHODS relative importance of routing and synthesis de novo for each bone lipid to gain a better understanding of the dietary signal Sample description. Holtzman albino rats were raised on a va- reflected in bone FA and cholesterol δ13C values. To this end, riety of purified and pelletized diets comprising 20.0% pro- it is necessary to consider the different metabolic pathways tein, 50.2% sucrose, 15.5% starch, 5.0% oil, 5% fiber, 3.5% that affect their occurrence in bone. Linoleic acid (18:2) is an minerals, and 1% vitamins. One day after insemination, EFA that cannot be synthesized de novo in higher mammals sperm-positive, 90-d-old female rats were placed on diets that (21,22). It must therefore be directly incorporated or routed their offspring would consume. Birth occurred 21 d after in- from the diet, and thus bone linoleic acid δ13C values are ex- semination, and weaning occurred 21 to 23 d later. The sexes pected to reflect dietary values. Non-EFA (16:0, 18:0, and were separated prior to sexual maturity. Normal room tem- 18:1) and cholesterol can be both absorbed directly from the perature (20°C) was maintained, and food and water were diet and synthesized de novo in the body from acetyl-CoA. provided ad libitum. Male and female pairs were sacrificed at Acetyl-CoA is the common metabolite formed from the ca- 91, 131, and 171 d after birth. Eight rat forelimbs from four tabolism of dietary lipids, carbohydrates, and proteins (or distinct diets were sampled for lipid analysis, in addition to from tissue glycogen and fat stores). All the carbon atoms of the whole diets and dietary oils. The dietary compositions and the common FA, two-thirds of the carbon in carbohydrates, the δ13C values of individual dietary components comprising and approximately half of the carbon skeleton of amino acids the diets are shown in Table 1. Table 2 lists all the animals contribute to the acetyl-CoA pool (23). Hence, δ13C values of that were studied. 1 synthesized FA and cholesterol are expected to reflect whole- Bulk δ 3C analysis of whole diet, dietary macronutrients, diet δ13C values with a bias toward dietary lipid and carbohy- bone collagen, and bone apatite. Bone collagen and apatite drate values. However, the overall dietary signal of bone non- extraction procedures and δ13C measurements summarized EFA and cholesterol is dependent on the relative importance here are described in detail in Ambrose and Norr (10). Lipids of the processes of routing vs. de novo synthesis of these were extracted from clean ground bone using petroleum ether. lipids. In humans, it has been estimated that the amount of Collagen was extracted by demineralization with 0.1 M HCl, cholesterol synthesized per day (typically 1.0 to 1.5 g) is at treated with 0.125 M NaOH, solubilized at 95°C, and freeze- least twice that of the daily dietary intake for an average dried. Whole diet, dietary macronutrients, and bone collagen Western diet (24). Approximately half of the dietary choles- were combusted at >800°C with Cu, CuO, and Ag foil in evac- terol will be absorbed by the intestine and the other half ex- uated sealed quartz tubes. Bone apatite carbonate was pre- creted; thus, dietary cholesterol can be estimated to contribute pared by deproteinization with NaOCl, treated with 1 M acetic ca. 20% of total body cholesterol. Dietary FA compositions acid to remove adsorbed carbonate, freeze-dried, and reacted have been shown to greatly influence the FA compositions in under vacuum with 100% H3PO4 at 25°C. CO2 was cryogeni- rat bone marrow (25), human serum and plasma lipids cally distilled off-line and analyzed on dual inlet isotope ratio (26,27), and human bone and blood phospholipids (28,29), mass spectrometers at the Anthropology Department, Univer- providing semiquantitative evidence for the direct incorpora- sity of Illinois (Nuclide 6-60 RMS), or the Illinois State Geo- tion of dietary FA into consumer tissues.