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Natural Abundance Stable 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 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 . 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. Stable isotopic evi- logical Survey (Finnegan MAT Delta E). dence for the influence of dietary FA on consumer tissues has Chemicals and precautions. All used were of been reported by Rhee et al. (30) and Stott et al. (16). These HPLC grade and purchased from Rathburn Chemicals two studies showed that the δ13C values of non-EFA in (Walkerburn, Scotland). The internal standard n-tetratriacon- human serum and in pig bone are enriched with respect to tane, cholesterol standard, hydroxide pellets, and de- their dietary sources. Stott et al. (16) also demonstrated that rivatizing agent N,O-bis(trimethylsilyl)trifluoroacetamide bone linoleic acid δ13C values, as expected, were highly con- (BSTFA) containing 1% vol/vol trimethylchlorosilane were sistent with dietary values. However, Rhee et al. (30) ob- purchased from the Sigma Chemical Company (Dorset, Eng- served a 3‰ depletion in serum linoleic acid δ13C values with land). All glassware and ceramics employed were washed respect to dietary values. with Decon 90, dried in an oven, and rinsed with chloroform/

TABLE 1 Rat Dietary Compositions and Their Macronutrient Componentsa,b δ13C values (‰) Diet Composition Components and code Protein Energyc Protein 20.0% Sucrose 50.2% Starch 15.5% Oil 5.0% − − − − D2A4 C3 C3 Milk casein ( 24.5) Beet ( 24.2) Rice ( 26.4) Cottonseed ( 27.9) − − − − D3G C3 C4 Milk casein ( 26.3) Cane ( 11.0) Corn ( 10.3) Corn ( 14.9) − − − − D4H C4 C4 Milk casein ( 14.6) Cane ( 11.4) Corn ( 10.6) Corn ( 14.9) − − − − D5I C4 C3 Milk casein ( 14.6) Beet ( 24.2) Rice ( 26.4) Cottonseed ( 27.9) aReference 9; Ambrose, S.H., unpublished data. bOne percent of vitamins, 3.5% of minerals, and 5% cellulose (wood and/or corn) were added to each diet. cEnergy = ∑ (sucrose, starch, and oil components).

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TABLE 2 containing 1% vol/vol trimethylchlorosilane. Twenty micro- Dietary Compositions, Sample Codes, Sex, liters of BSTFA were added to each sample followed by heat- a and Pair of Individual Animals ing at 70°C (Multiblok Lab-Line) for ca. 1 h. Excess BSTFA Dietary composition Sample was removed under a gentle stream of nitrogen. FA were con- Protein Energyb Code Sex Pair verted to their methyl ester derivatives. FA fractions were trans- C C C3 M 2 ferred into fresh screw-capped test tubes and blown down to 3 3 dryness under nitrogen gas. The samples were methylated by C3 C3 C3 F 2 C3 C4 C3P/C4 M 3 adding 100 µL of 14% wt/vol trifluoride/methanol com- C3 C4 C3P/C4 F 3 plex and heating in a water bath at 70°C for 1 h. After cooling, C C C4 F 1 4 4 2 mL of double-distilled water was added and the FAME ex- C C C4 M 2 4 4 × C C C4P/C3 F 2 tracted using diethyl ether (3 2 mL). 4 3 GC. High-temperature GC analyses of the TLE and neu- C4 C3 C4P/C3 M 3 aFirst, second, and third pair animals were sacrificed at 91, 131, and 171 d tral fractions were carried out using a Hewlett-Packard (HP) old, respectively. P = protein; M = male and F = female. 5890 Series II gas chromatograph fitted with a fused-silica b Energy = ∑ (sucrose, starch, and oil components). capillary column (15 m × 0.32 mm i.d.) coated with a di- methyl polysiloxane stationary phase (DB-1, J&W Scientific, methanol (2:1 vol/vol) prior to use. Disposable rubber gloves Folsom, CA; Agilent Technologies, Palo Alto, CA; 0.1 µm were worn throughout the whole experimental procedure, film thickness). The temperature of the oven was held isother- from sample preparation to instrumental analysis, to prevent mally at 50°C (2 min) and then increased to 350°C (20 min) the contamination of samples with finger lipids. Extraction, at a rate of 10°C min−1. FAME GC analyses were performed filtration, and saponification (neutral and acid) blanks were using the chromatograph described above fitted with a fused- used to monitor and locate any contamination that might be silica capillary column (50 m × 0.32 mm i.d.) coated with a introduced during the experimental procedure. polyethylene glycol stationary phase (CP-WAX 52 CB, Var- Preparation of diet and bone samples for lipid extraction. ian; Chrompack, Middleberg, The Netherlands; 0.25 µm film Rat forelimbs were dissected into three different tissue types: thickness). The temperature of the oven was held isother- bone (ulna and radius), skin, and flesh. Bones were manually mally at 40°C (2 min) and then increased to 200°C (10 min) cleaned of adhering flesh, cartilage, and tendons by scraping at a rate of 5°C min−1. was used as the carrier gas, with a scalpel. Half a bone was used for the extraction proce- and FID was used to monitor the column effluent. Data were dure, typically the upper ulna. Rat bone and diet pellets were acquired and analyzed using HP Chemstation software. ground in a pestle and mortar prior to extraction. Liquid ni- GC/MS. GC/MS analyses were performed using a Finni- trogen was employed to aid the bone crushing process. gan 4500 quadrupole mass spectrometer (source temperature, Ranges of rat sample weights are as follows: 0.03 to 0.13 g of 280°C; electron voltage, 35 eV) interfaced to a Carlo Erba powdered bone and 0.83 to 1.41 g of powdered diet pellets. HRGC 5160 Mega series gas chromatograph. The same Extraction of lipids from diet and bone. Samples were columns and temperature programs as described above were transferred into large screw-capped vials and known quanti- used for the TLE, neutral, and FAME fractions. Hydrogen ties of n-tetratriacontane (1 mg mL−1 in chloroform) added as was used as the carrier gas. Data were acquired using an an internal standard. The samples were extracted with chloro- INCOS data system and processed using Interactive Chemi- form/methanol (2:1 vol/vol, 5 to 10 mL) by ultrasonication (3 cal Information Software (ICIS) package. × 1 h, Decon F5200b), where the supernatant was removed GC/C/IRMS. GC/C/IRMS analyses were carried out using and replaced intermittently. The total lipid extract (TLE) was a Varian 3500 gas chromatograph coupled to a Finnigan MAT then concentrated to ca. 5 mL under a gentle stream of nitro- Delta-S isotope ratio mass spectrometer via a Finnigan MAT gen (5 psi) in an evaporation unit (TurboVap LV; Zymarck combustion interface (Pt/CuO) maintained at 850°C. For the Corporation, Hopkinton, MA) with the thermostatic bath set neutral fractions, the GC was fitted with a fused-silica capil- at 40°C. Suspended particulates were removed from the TLE lary column (50 m × 0.32 mm i.d.) coated with a dimethyl by centrifugation (1800 rpm, 20 min; MSE Mistral 1000) and polysiloxane stationary phase (CP-SIL 5 CB, 0.25 µm film filtration through a short pipette column packed with acti- thickness). The temperature of the oven was held at 50°C (2 vated alumina. min) and then increased to 250°C at a rate of 10°C min−1, then Saponification of TLE. Aliquots of TLE were transferred to 300°C (20 min) at 4°C min−1. For the FAME fractions, the into screw-capped test tubes, blown down to dryness under ni- same column and temperature program as described above for trogen gas, and hydrolyzed with 0.5 M methanolic NaOH (2 GC and GC/MS analyses were used. was used as the mL) at 70°C in a water bath for 1 h. After cooling, the mixture carrier gas and the mass spectrometer source pressure was was extracted using hexane (3 × 2 mL), yielding the neutral maintained at 9 × 10−5 Pa. Data were collected and processed cholesterol-containing fraction. The mixture was then acidified using Finnigan MAT Isobase software. Secondary standards × δ13 to pH 3 with 1 M HCl and extracted again using hexane (3 2 (C19 n-alkane or 18:0 FAME) of known C value were mea- mL) to yield the FA fraction. Neutral fractions were converted sured every seventh run to monitor any fluctuations in instru- to their trimethylsilyl (TMS) ether derivatives using BSTFA mental measurements with time. The precision of triplicate

Lipids, Vol. 38, no. 2 (2003) 182 S. JIM ET AL.

GC/C/IRMS analyses carried out on each sample was shown C4 caseins during processing, prior to the formulation of the δ13 to be ±0.3‰. FA and cholesterol C values were corrected diets. The C4 casein retained a significant proportion of its for the addition of derivatizing carbon. lipids and this resulted in the presence of C30 to C44 TAG, and 10:0 and 12:0 FA in the TLE and FAME, respectively, of diets D4H (Figs. 1g and 1i) and D5I (Figs. 1j and 1l). Bone RESULTS AND DISCUSSION TLE (Fig. 1m) were characterized by the presence of Diet and bone lipid compositions. Figure 1 presents partial FFA, cholesterol, and C48 to C54 TAG. gas chromatograms of the TLE, neutral, and FAME fractions Trends in the δ13C values of diet and bone components. of the four diets and of bone sample C4P/C3 (C4 protein with Figure 2a compares whole diet and dietary lipid with bone δ13 C3 energy; see Table 2 for sample codes), female, third pair. lipid, collagen, and apatite C values for the C3 and C4 an- Dietary lipid compositions reflect that of the oil (cottonseed imals. All bone lipid δ13C values are depleted with respect to or corn oil) and casein constituting the diets. The cottonseed collagen or apatite values, and this finding is consistent with and corn oil exhibited very similar lipid distributions charac- the relative depletion in 13C that occurs during lipid biosyn- terized by the presence of C32 and C36 DAG and C48 to C54 thesis with respect to other biochemical pathways (32,33). δ13 TAG. Milk fat displays a TAG distribution in the range of C28 The majority of bone lipid C values are highly consistent to C54 but is characterized by a greater abundance of the C36 with dietary lipid values. Small, positive diet-to-bone frac- ∆13 δ13 −δ13 to C44 TAG (31). Differences in dietary lipid distributions can tionations ( Cblipid−dlipid = Cbone lipid Cdiet lipid) be explained by the differential lipid extraction of the C3 and are observed for 16:0, 18:1, 18:2, and cholesterol, where ∆13 mean Cblipid−dlipid fractionations are +0.9, +0.7, +1.5, and − ∆13 0.5‰, respectively. For 18:0, a larger Cblipid−dlipid frac- tionation of +3.6‰ toward whole-diet δ13C values is ob- served, which suggests that a greater degree of de novo syn- thesis may have occurred with 18:0 when compared to the other non-EFA or cholesterol. However, a true assessment of

FIG. 1. Partial gas chromatograms of the total lipid extracts (a, d, g, j, and m), saponified trimethylsilylated neutral (b, e, h, k, and n), and FAME fractions (c, f, i, l, and o) of diets D2A4 (a, b, and c), D3G (d, e, and f), D4H (g, h, and i), D5I (j, k, and l) and bone bC4P/C3 (m, n, and o). 1 = FFA; 2 = sugar; 3 = cholesterol; 4 = β-sitosterol; 5 = internal stan- FIG. 2. Whole diet, diet and bone lipid, collagen, and apatite δ13C val- dard; 6 = C32 and C36 DAG; 7a = C30 to C44 TAG; 7b = C46 to C54 TAG; ues for: (a) C3 and C4 animals, and (b) C3P/C4 and C4P/C3 animals. 8 = C10:0 FAME; 9 = C12:0 FAME; 10 = C14:0 FAME; 11 = C16:0 FAME; Each bone data point represents the mean value for the analyses of two 12 = C16:1 FAME; 13 = C18:0 FAME; 14 = C18:1 FAME; and 15 = C18:2 animals. WDIET = whole diet; CHOL = cholesterol; COLL = collagen; FAME. APAT = apatite; d = diet and b = bone.

Lipids, Vol. 38, no. 2 (2003) ROUTING AND DE NOVO SYNTHESIS OF BONE LIPIDS 183 the extent of routing or de novo synthesis can only be gleaned tions (21,22) where isotopic fractionation may be introduced. when the δ13C values from the C3P/C4 and C4P/C3 animals Correlations between diet and bone lipid δ13C values. The are also taken into consideration. The small but significant relationships between diet (whole diet, protein, energy, FA, ∆13 Cblipid−dlipid fractionation of +1.5‰ shown for the EFA and cholesterol) and bone (collagen, apatite, FA, and choles- 18:2 must be caused by isotopic fractionation occurring dur- terol) δ13C values were investigated using linear regression. ing assimilation, transport, or catabolism. Table 3 summarizes the R2 values observed between each diet Figure 2b presents the δ13C values for the C3P/C4 and and bone component, and the most significant correlations are C4P/C3 animals. Here, bone lipid δ13C values are depleted highlighted in bold. Collagen and apatite δ13C values were with respect to collagen or apatite values for the C4P/C3 ani- shown to correlate best with dietary protein (R 2 = 0.77, P ≤ mals only; for the C3P/C4 animals, bone collagen displayed 0.01) and whole diet (R 2 = 0.99, P = 0.001) values, respec- a comparable δ13C value to the bone FA. The reasons for this tively [as was also demonstrated for the data presented in Am- will become apparent when we consider the diet-to-bone lipid brose and Norr (10)], and these findings are consistent with isotopic relationships in more detail. For both diets and in ac- those from the Tieszen and Fagre (11) study. The most signifi- cordance with the C3 and C4 animals, small although not nec- cant correlations observed for the bone lipids are consistent ∆13 ∆13 essarily positive Cblipid−dlipid fractionations are seen for with the interpretations of the Cblipid−dlipid fractionations 16:0, 18:1, 18:2 where absolute mean values are 0.1, 1.2, and above and are shown in Figure 3. As expected, bone linoleic 1.9‰, respectively. In contrast to the C3 and C4 animals, a δ13C values correlated extremely well with dietary values (R2 ∆13 ≤ small absolute mean Cblipid−dlipid fractionation of 1.1‰ is > 0.99, P 0.001), providing further evidence for its direct in- ∆13 observed for 18:0, and a large absolute mean Cblipid−dlipid corporation from the diet. It was postulated that the majority fractionation of 7.9‰ is observed for cholesterol. When these of bone 16:0 and 18:1 FA were also routed directly from the findings are considered alongside those shown for the C3 and diet, and indeed, the highest R2 values of 0.97 (P ≤ 0.001) and C4 animals, a clearer assessment of the relative importance 0.95 (P = 0.001), respectively, were shown with respect to of routing and de novo synthesis for each of the bone lipids their corresponding dietary FA. Conversely, the majority of ∆13 can be gained. Comparable Cblipid−dlipid fractionations are bone 18:0 FA and cholesterol was postulated to result from shown for 18:2 in all four diets, demonstrating that bone 18:2 de novo synthesis, and this hypothesis is corroborated by a δ13C values are independent of macronutrient isotopic differ- higher degree of correlation with whole diet δ13C values rather ences. This finding is expected for linoleic acid, which must than with their corresponding dietary FA values. Certainly, the ∆13 2 be derived solely from the diet. Comparable Cblipid−dlipid low R of 0.21 (P > 0.05) observed between diet and bone fractionations are also shown for 16:0 and 18:1, suggesting cholesterol indicates that the direct incorporation of choles- that the majority of these nonessential bone lipids are also di- terol from the diet is not the dominant pathway. ∆13 ∆13 rectly incorporated from the diet. Conversely, Cblipid−dlipid Correlations between Cblipid−wdiet fractionations and ∆13 fractionations for 18:0 and cholesterol have been shown Cdlipid−wdiet spacings. Greater insight into the extent of to be influenced by macronutrient isotopic differences, sug- routing of dietary lipids into bone lipids can be gleaned from ∆13 gesting that a major proportion of these lipids are derived plotting Cblipid−wdiet fractionations against their correspond- ∆13 δ13 −δ13 from de novo synthesis. For all four diets, absolute mean ing Cdlipid−wdiet spacings (= Cdiet lipid Cwhole diet). We ∆13 2 Cblipid−dlipid fractionations for 16:0, 18:0, 18:1, 18:2, and focus here on the R values and gradients (m) of the regression cholesterol are 0.5, 2.3, 0.9, 1.7, and 4.2‰, respectively. If equations in Figure 4 to interpret diet-to-bone lipid relation- the magnitudes of these spacings are considered to be a mea- ships. R2 values ranged from 0.67 to 0.96 (P ≤ 0.05), showing sure of the extent of routing of the non-EFA and cholesterol, that these relationships correlated well/very well with each then this can be estimated to be of the order 16:0 > 18:1 > other. Significant differences in the gradients of these regres- ∆13 18:0 > cholesterol. The Cblipid−dlipid fractionation ob- sion lines are observed, demonstrating the varying degrees to served for linoleic acid is significant, demonstrating that bone 18:0 δ13C values are consistently more enriched by 1.7‰ with respect to dietary values. This fractionation obviously TABLE 3 R2 Values from Linear Correlations of Diet and Bone δ13C Valuesa cannot be taken as a measure of the extent of routing and must 2 arise due to isotopic fractionation occurring during metabolic R values processes other than de novo synthesis. Linoleic acid is the Diet component most abundant FA in these diets but is one of the minor FA Bone component Whole diet Protein Energy Lipidb observed in bone (Figs. 1c, 1f, 1i, 1l, 1o). If we postulate that 16:0 0.92 0.07 0.80 0.97 the diet provides more than enough linoleic acid for these an- 18:0 0.95 0.04 0.87 0.84 imals, then the enrichment observed in bone linoleic δ13C 18:1 0.93 0.01 0.90 0.95 acid values is consistent with a kinetic isotope effect occur- 18:2 0.91 0.02 0.99 0.996 ing during its catabolism or conversion to other metabolites, Cholesterol 0.91 0.18 0.73 0.21 Collagen 0.38 0.77 0.15 NA e.g., prostaglandins and eicosanoids. Certainly, the formation Apatite 0.99 0.02 0.94 NA of prostaglandins and eicosanoids from linoleic acid involves aBoldfaced values indicate the most significant correlations. enzyme-catalyzed chain elongation and desaturation reac- bLipid refers to corresponding lipid in the diet. NA, not applicable.

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FIG. 3. Linear correlations of: (a) diet and bone 16:0 FA, (b) whole diet and bone 18:0 FA, (c) diet and bone 18:1 FA, (d) diet and bone 18:2 FA, and (e) whole diet and bone cholesterol δ13C values. WDIET = whole diet; CHOL = cholesterol; d = diet; b = bone; and dotted line is where x = y.

∆13 which Cblipid−wdiet fractionations depend on the difference from the diet. Significantly, the extent of routing of the non- between dietary lipid and whole-diet δ13C values. The largest EFA and cholesterol reflected their relative abundances in the gradient is observed for linoleic acid where m = 0.97, and this diet. Although tissue lipid compositions have been shown to can be interpreted as indicating that 97% of the carbon atoms be greatly influenced by dietary lipids (24–28), this is the first in bone 18:2 are derived from the diet, reflecting the direct in- stable isotopic experimental evidence at natural abundance to corporation of this EFA from the diet. Thus, from Figure 4, the show that FA are absorbed proportionately from the diet. The degree of routing of diet-to-bone lipids can be estimated to be potential of using bone FA and cholesterol δ13C values as in- of the order 18:2 (97%) > 16:0 (80%) > 18:1 (64%) > 18:0 dicators of diet has therefore been shown in this study. How- (48%) > cholesterol (21%). Again, these estimates are entirely ever, the interpretation of lipid δ13C values from ecological consistent with previous interpretations of routing as discussed and archaeological studies, where there are likely to have above and, moreover, reflect the relative abundances of these been temporal changes in diet, requires knowledge of their lipids in the diet. The estimated percentage of routing for cho- turnover rates. Bone FA and cholesterol turnover rates have lesterol corresponds to the predicted 20% discussed above and been estimated from the study of rats that were subjected to compares well with an estimate of 16% (34) where a further diet-switch experiments (Jim, S., Evershed, R.P., and Am- set of animals raised on marine protein were also considered. brose, S.H., unpublished data). In summary, the δ13C values of the major lipids found in bone were shown to reflect different dietary signals. δ13C val- ACKNOWLEDGMENTS ues of bone cholesterol and stearic (18:0) acid reflected We thank the Wellcome Trust for providing the Bioarchaeology Stu- whole-diet δ13C values, whereas the δ13C values of bone dentship (047442/Z/96/Z) and Fellowship (057166/Z/99/Z) for this palmitic (16:0), oleic (18:1), and linoleic (18:2) acids re- research. NERC is thanked for financial support for mass spectrom- flected dietary FA δ13C values. Differences in their dietary etry facilities (GR 3/2951, GR 3/3758, and FG 6/36/01). We also thank Jim Carter and Andrew Gledhill for technical assistance with signals were attributed to the different extents to which di- GC/MS and GC/C/IRMS. Controlled diet experiments were sup- etary lipids were routed to bone. Linoleic acid is an EFA, and ported by the National Science Foundation, USA (BNS 9010937 and the routing estimate of 97% confirms its direct incorporation SBR 9212466), and the University of Illinois Research Board. Con-

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∆ ∆13 FIG. 4. Double correlations of Cdlipid−wdiet ∆13 spacings vs. corresponding Cblipid−wdiet frac- tionations: (a) 16:0 FA, (b) 18:0 FA, (c) 18:1 FA, (d) 18:2 FA, and (e) cholesterol. WDIET = whole diet; CHOL = cholesterol; d = diet; and b = bone. trolled diet experiment protocols were approved by the Office of 8. DeNiro, M.J., and Epstein, S. (1981) Influence of Diet on the Laboratory Animal Care (OLAC), University of Illinois, Urbana- Distribution of Nitrogen in Animals, Geochim. Cos- Champaign. mochim. Acta 45, 341–351. 9. Hare P.E., Fogel, M.L., Stafford, T.W., Jr., Mitchell, A.D., and Hoering, T.C. (1991) The Isotopic Composition of Carbon REFERENCES and Nitrogen in Individual Amino Acids Isolated from Modern 1. van der Merwe, N.J. (1982) Carbon Isotopes, Photosynthesis, and Fossil Proteins, J. Archaeol. Sci. 18, 277–292. and Archaeology, Am. Sci. 70, 596–606. 10. Ambrose, S.H., and Norr, L. (1993) Experimental Evidence for 2. Chisholm, B.S. (1989) Variation in Diet Reconstructions Based the Relationship of the Carbon Isotope Ratios of Whole Diet and on Stable Carbon Isotopic Evidence, in The Chemistry of Pre- Dietary Protein to Those of Bone Collagen and Carbonate, in historic Human Bone (Price, T.D., ed.), pp. 10–37, Cambridge Prehistoric Human Bone—Archaeology at the Molecular Level University Press, Cambridge. (Lambert, J.B., and Grupe, G., eds.), pp. 1–37, Springer-Verlag, 3. Lee-Thorp, J.A., Sealy, J.C., and van der Merwe, N.J. (1989) Berlin. Stable Carbon Isotope Ratio Differences Between Bone Colla- 11. Tieszen, L.L., and Fagre, T. (1993) Effect of Diet Quality and gen and Bone Apatite, and Their Relationship to Diet, J. Ar- Composition on the Isotopic Composition of Respiratory CO2, chaeol. Sci. 16, 585–599. Bone Collagen, Bioapatite and Soft Tissues, in Prehistoric 4. Schwarcz, H.P. (1991) Some Theoretical Aspects of Isotope Pa- Human Bone—Archaeology at the Molecular Level (Lambert, leodiet Studies, J. Archaeol. Sci. 18, 261–275. J.B., and Grupe, G., eds.), pp. 121–155, Springer-Verlag, Berlin. 5. Schwarcz, H.P. (2000) Some Biochemical Aspects of Carbon 12. Matthews, D.E., and Hayes, J.M. (1978) Isotope-Ratio-Moni- Isotopic Paleodiet Studies, in Biogeochemical Approaches to toring Gas Chromatography–Mass Spectrometry, Anal. Chem. Paleodietary Analysis (Ambrose, S.H., and Katzenberg, M.A., 50, 1465–1473. eds.), pp. 189–209, Kluwer Academic/Plenum, New York. 13. Fantle, M.S., Dittel, A.I., Schwalm, S.M., Epifanio, C.E., and 6. Koch, P.L., Fogel, M.L., and Tuross, N. (1994) Tracing the Fogel, M.L. (1999) A Food Web Analysis of the Juvenile Crab, Diets of Fossil Animals Using Stable Isotopes, in Methods in Callinectes sapidus, Using Stable Isotopes in Whole Animals Ecology, Stable Isotopes in Ecology and Environmental Science and Individual Amino Acids, Oecologia 120, 416–426. (Lajtha, K., and Michener, R.H., eds.), pp. 63–92, Blackwell 14. O’Brien, D.M., Fogel, M.L., and Boggs, C.L. (2002) Renewable Scientific Publications, Oxford. and Nonrenewable Resources: Amino Acid Turnover and Allo- 7. DeNiro, M.J., and Epstein, S. (1978) Influence of Diet on the cation to Reproduction in Lepidoptera, Proc. Nat. Acad. Sci. Distribution of Carbon Isotopes in Animals, Geochim. Cos- USA 99, 4413–4418. mochim. Acta 42, 495–506. 15. Pond, C.M., and Gilmour, I. (1997) Stable Isotopes in Adipose

Lipids, Vol. 38, no. 2 (2003) 186 S. JIM ET AL.

Tissue Fatty Acids as Indicators of Diet in Arctic Foxes (Alopex Pressure, Serum Lipids, and Fatty Acids in Populations on a lagopus), Proc. Nutr. Soc. 56, 1067–1081. Lake-Fish Diet or on a Vegetarian Diet in Tanzania, Lipids 31, 16. Stott, A.W., Davies, E., Tuross, N., and Evershed, R.P. (1997) S309–S312. Monitoring the Routing of Dietary and Biosynthesized Lipids 27. Vidgren, H.M., Ågren, J.J., Schwab, U., Rissanen, T., Hänni- through Compound-Specific Stable Isotope (δ13C) Measure- nen, O., and Uusitupa, M.I.J. (1997) Incorporation of n-3 Fatty ments at Natural Abundance, Naturwissenschaften 84, 82–86. Acids into Plasma Lipid Fractions, and Erythrocyte Membranes 17. Hammer, B.T., Fogel, M.L., and Hoering, T.C. (1998) Stable and Platelets During Dietary Supplementation with Fish, Fish Carbon Isotope Ratios of Fatty Acids in Seagrass and Redhead Oil, and Docosahexaenoic Acid-Rich Oil Among Healthy Ducks, Chem. Geol. 152, 29–41. Young Men, Lipids 32, 697–705. 18. Stott, A.W., Evershed, R.P., Jim, S., Jones, V., Rogers, J.M., 28. Alam, S.Q., Kokkinos, P.P., and Alam, B.S. (1993) Fatty Acid Tuross, N., and Ambrose, S.H. (1999) Cholesterol as a New Composition and Arachidonic Acid in Alveolar Source of Palaeodietary Information: Experimental Approaches Bone of Rats Fed Diets with Different Lipids, Calcified Tissue and Archaeological Applications, J. Archaeol. Sci. 26, 705–716. Int. 53, 330–332. 19. Jim, S. (2000) The Development of Bone Cholesterol δ13C Val- 29. Rambjør, G.S., Wålen, A.I., Winsor, S.L., and Harris, W.S. ues as a New Source of Palaeodietary Information: Models of (1996) Eicosapentaenoic Acid Is Primarily Responsible for Hy- Its Use in Conjunction with Bone Collagen and Apatite δ13C potriglyceridemic Effect of Fish Oil in Humans, Lipids 31, Values, Ph.D. Thesis, University of Bristol, England, pp. S45–S49. 117–123. 30. Rhee, S.K., Reed, R.G., and Brenna, J.T. (1997) Fatty Acid Car- 20. Jim, S., Stott, A.W., Evershed, R.P., Rogers, J.M., and Ambrose, bon Isotope Ratios in Humans on Controlled Diets, Lipids 32, S.H. (2001) Animal Feeding Experiments in the Development 1257–1263. of Cholesterol as a Palaeodietary Indicator, in Archaeological 31. Mottram, H.R., and Evershed, R.P. (2001) Elucidation of the Sciences ’97 (Millard, A., ed.), pp. 68–77, BAR International Composition of Bovine Milk Fat Triacylglycerols Using High- Series 939, Oxford. Performance Liquid Chromatography–Atmospheric Pressure 21. Voet, D., and Voet, J.G. (1995) Biochemistry, 2nd edn., pp. Chemical Ionisation Mass Spectrometry, J. Chromatogr. A 926, 687–688, John Wiley & Sons, New York. 239–253. 22. McGarry, J.D. (1997) Lipid Metabolism I: Utilization and Stor- 32. DeNiro, M.J., and Epstein, S. (1977) Mechanisms of Carbon age of Energy in Lipid Form, in Textbook of Biochemistry with Isotope Fractionation Associated with Lipid Synthesis, Science Clinical Correlations (Devlin, T.M., ed.), pp. 361–393, Wiley- 197, 261–263. Liss, New York. 33. Hayes, J.M. (1993) Factors Controlling 13C Contents of Sedi- 23. Krebs, H.A., and Lowenstein, J.M. (1960) The Tricarboxylic mentary Organic Compounds: Principles and Evidence, Mar. Acid Cycle, in Metabolic Pathways (Greenberg, D.M., ed.), Vol. Geol. 113, 111–125. 1, pp. 129–203, Academic Press, New York. 34. Jim, S., Ambrose, S.H., and Evershed, R.P. (in press) Stable 24. Sabine, J.R. (1977) Cholesterol, p. 81, Marcel Dekker, New Carbon Isotopic Evidence for Differences in the Biosynthetic York. Origin of Bone Cholesterol, Collagen and Apatite: Implications 25. Das, S.K., Scott, M.T., and Adhikary, P.K. (1975) Effect of the for Their Use in Palaeodietary Reconstruction, Geochim. Cos- Nature and Amount of Dietary Energy on Lipid Composition of mochim. Acta. Rat Bone Marrow, Lipids 10, 584–590. 26. Pauletto, P., Puato, M., Angeli, M.T., Pessina, A.C., [Received July 12, 2002, and in revised form February 24, 2003; Munhambo, A., Bittolo-Bon, G., and Galli, C. (1996) Blood accepted February 27, 2003]

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