Investigations into the effect of diet on modern human hair isotopic values

Investigations Into the Effect of Diet on ModernHuman Hair Isotopic Values

T.C. O’CONNELL* AND R.E.M. HEDGESResearch Laboratory for Archaeology and the History of Art, University ofOxford, Oxford, OX1 3QJ, United Kingdom

KEY WORDS carbon; nitrogen; isotopic analysis; animal proteinconsumption; diet; humans

ABSTRACT Carbon and nitrogen isotopic analysis of body tissues is oneof the few techniques that can furnish quantitative information about the dietof archaeological humans.

The study of the effects of various diets on modern human isotopic valuescan help to refine palaeodietary theories, and such work also enables thetesting of palaeodietary theories independent of archaeological remains andinterpretations.

This report discusses the use of modern human hair as a sample materialfor isotopic analysis. The biogenic carbon and nitrogen isotopic signal is wellpreserved in hair, and the isotopic values of the keratin can be related to diet.We show that atmospheric and cosmetic contamination of hair keratin doesnot appear to affect the measured isotopic values.

In a small study of Oxford residents, we demonstrate that the magnitude ofthe nitrogen isotopic values of hair keratin reflects the proportion of animalprotein consumed in the diet: omnivores and ovo-lacto-vegetarians havehigher d15N than vegans. There was an observed relationship between thereported amount of animal protein eaten (either meat or secondary animalproducts) and the nitrogen isotopic values within the two groups of omnivoresand ovo-lacto-vegetarians, indicating that an increasing amount of animalprotein in the diet results in an increase in the d15N of hair keratin. Thisprovides the first independent support for a long-held theory that, forindividuals within a single population, a diet high in meat equates to elevatednitrogen isotopic values in the body relative to others eating less animalprotein.

The implications of such results for the magnitude of the trophic level effectare discussed. Results presented here also permit a consideration of theeffects of a change of diet in the short and long term on hair keratin isotopicvalues. Am J Phys Anthropol 108:409–425, 1999. r 1999 Wiley-Liss, Inc.

The ability to make quantitative state-ments about ancient diets has fundamentalimportance for archaeology, since diet re-flects the interaction between demography,economy, environment, and food-productiontechnology. However, direct archaeologicalevidence of diet is often elusive. Since ‘‘youare what you eat,’’ the biochemical analysisof body tissues is an indirect analysis of the

food consumed. The natural distribution ofstable isotopes in biological systems enablesus to quantify aspects of food uptake into thebody, and carbon and nitrogen isotopic analy-

Grant sponsor: SERC/NERC.*Correspondence to: T.C. O’Connell, 6 Keble Road, Oxford,

OX1 3QJ, UK. E-mail: [email protected] 31 October 1997; accepted 16 December 1998.

AMERICAN JOURNAL OF PHYSICAL ANTHROPOLOGY 108:409–425 (1999)

r 1999 WILEY-LISS, INC.

sis of archaeological human bone is often theonly quantitative and objective techniqueavailable for the reconstruction of palaeo-diet (Schwarcz and Schoeninger, 1991).

PALAEODIETARY ANALYSIS

Early dietary interpretations of archaeo-logical human isotopic data were based oncomparison with isotopic values of modernplants and animals (Chisholm et al., 1982,1983; Tauber, 1981; Vogel, 1978; Vogel andvan der Merwe, 1977). The recognition ofsystematic isotopic patterns in natural foodwebs led to laboratory-based animal-feedingstudies in an attempt to quantify the pat-terns observed (Ambrose and Norr, 1993;DeNiro and Epstein, 1978, 1981; Katzen-berg and Krouse, 1989; Nakagawa et al.,1985; Tieszen and Boutton, 1988; Tieszen etal., 1983; Tieszen and Fagre, 1993). Theseexperiments supplied primary informationabout the fate of macronutrients in thebody’s metabolic processes and have pro-vided the basic tenets that underlie thecurrent theories of palaeodietary reconstruc-tion.

Archaeological human diet has been ana-lysed by extrapolating the results of thesesystematic investigations into the isotopicrelationship between diet and body tissuesin animals. Although animal studies provideimportant information about relationshipsbetween diet and body tissues, it is of ques-tionable validity to use these results as abasis for the interpretation of specific hu-man dietary situations. Although the basicphysiology of humans and animals is simi-lar, the question of size, growth, and effi-ciency of assimilation of the diet may callinto question the possibility of using ani-mals as direct comparisons for humans. Inaddition, the effect of growth (when theanimal is in negative nitrogen balance) onisotopic values has not been quantified, andthe majority of animal studies are per-formed on animals that are either still grow-ing or that have not been maintained in asteady state for a long period of time. Thestudy of the effects of various diets on mod-ern human isotopic values should providedirect information on the detailed isotopiceffects of particular dietary variations andcan be used to refine and extend palaeodi-

etary theories. Systematic studies of dietaryeffects on modern humans also permit theindependent testing of palaeodietary theo-ries developed via animal studies.

Isotopic analysis of modernhuman tissues

The effect of dietary variation on the isoto-pic values of modern (living) human bodytissues has rarely been directly studied.Part of the problem has been one of samplematerial. Archaeological human isotopicanalyses are usually of bone collagen, sincebones are often the only part of the bodyrecovered after significant burial periods.Most previous animal isotopic studies havealso concentrated on bone collagen and therelationship between bone collagen isotopicvalues and diet. An advantage of using bonecollagen for palaeodietary analysis is that ithas a long turnover period and as suchreflects the diet consumed over a period ofabout 10 years (Stenhouse and Baxter, 1979),although this figure is debated and may besomewhere in the region of 2–20 years,depending on the age and health of theindividual and the type of bone considered(Ambrose, 1993). However, bone is not areadily available sample material from mostmodern living humans. In most investiga-tions, hair has been used as a representativesample material of all body tissues(Minagawa, 1992; Nakamura et al., 1982;Webb et al., 1980; Yoshinaga et al., 1996),while some have used hair, urine, and bloodplasma as indicators of body isotopic values(Katzenberg and Krouse, 1989; Schoeller etal., 1986).

Hair keratin has been used as an alterna-tive modern human body tissue because it iseasy to sample and the limited animal dataavailable suggest that, like collagen, keratinisotopic values closely reflect diet. The car-bon isotopic values of the hair protein kera-tin correlate well with diet, being enrichedby 11–2‰ relative to dietary protein (De-Niro and Epstein, 1978; Jones et al., 1981;Katzenberg and Krouse, 1989; Tieszen andFagre, 1993). The nitrogen isotopic values ofmost body proteins including collagen, kera-tin, and muscle protein are very similar, andthese correlate well with diet, generallyenriched by 2–3‰ (Ambrose, 1993; DeNiro

410 T.C. O’CONNELL AND R.E.M. HEDGES

and Epstein, 1981; Hare et al., 1991; Nak-agawa et al., 1985; Sealy et al., 1987). Hairappears to be representative of the isotopiccomposition of human tissues, although ithas a much faster turnover time than colla-gen, which may affect the measured isotopicvalues. This is discussed in more detail later.

Some of the previous modern hair studieshave examined how the variations in anindividual’s hair keratin isotopic values canbe related to the average isotopic values ofthe typical diet of their geographical loca-tion (Katzenberg and Krouse, 1989;Minagawa, 1992; Nakamura et al., 1982;Wada et al., 1991). Other studies have esti-mated the isotopic enrichment between dietand hair keratin in modern humans (Schoel-ler et al., 1986; Yoshinaga et al., 1996). Inagreement with the available animal isoto-pic data, these studies of modern humanhair suggest that hair isotopic values doreflect the isotopic composition of the diet.However, such work has not quantitativelyinvestigated the effect of dietary variationon the isotopic values in the human body.

Studying modern human diet using iso-topic analysis of hair. These previousstudies have shown that hair can be used asisotopically representative of human bodytissues and suggest that hair from modernhumans can be used to study the effects ofdifferent dietary composition on the isotopicvalues of the human body. We have used thisapproach to investigate one possible effect:that differing levels of meat intake (strictlydefined as animal protein) have a measur-able influence on human body tissue isotopicvalues. A decision to eat or not to eat meatand animal products is a common moderndietary choice. Therefore, the isotopic analy-sis of modern human hair from individualswith varying diets can be used to study theeffect of levels of animal protein on theisotopic values of human body tissues.

Levels of animal protein in human diet

Estimation of the amount of meat or ani-mal protein intake in the diet of archaeologi-cal populations is important in establishingthe type of palaeoeconomy (e.g., an agricul-tural subsistence as opposed to a pastoral orhunting society). Methods of assessing diet

and specifically the importance of animalprotein consumption include analysis of fau-nal remains, tooth microwear, and palaeopa-thology; however, these techniques are quali-tative. Isotopic analysis has been suggestedas a possible quantitative method of estab-lishing animal protein consumption in ar-chaeological humans.

It is known that body tissues such as bonecollagen and hair keratin are more positivein d15N by 13‰ relative to diet (DeNiro andEpstein, 1981; Hare et al., 1991). This re-sults in increasing d15N values as the foodchain is ascended: carnivores have a higherd15N than the herbivores on which they feed,while herbivores have a higher d15N thanplants (Schoeninger and DeNiro, 1984). Theincrease in d15N up the food chain is termedthe trophic level effect; it is a consequence ofand is equivalent in magnitude to the enrich-ment in 15N levels between an individual’sdiet and its body. From the trophic leveleffect, it has been argued that a diet low inmeat (a low mean dietary d15N value) pro-duces low d15N values in the body proteins ofthe consumer and one high in meat (a highmean dietary d15N value) produces highd15N values in the consumer.

Although this argument has been appliedto archaeological humans in an attempt toestimate the animal protein content of theirdiet, the postulated theory has not yet beendemonstrated. It is necessary, in order toavoid circular arguments, to prove a directrelationship between increasing levels ofanimal protein consumption and human ni-trogen isotopic values independent of thearchaeological evidence.

Modern human carbon isotopic varia-tion and animal protein consumption.Work by Webb et al. (1980) attempted toassess the specific effects of animal proteinintake on hair isotopic values from individu-als within the same population; however,they measured only the carbon isotopic val-ues. After the d13C of hair keratin fromomnivores and ovo-lacto-vegetarians (thoseindividuals eating no animal flesh but con-suming secondary animal protein such asmilk and eggs) in Australia and New Zea-land was measured, there was found to belittle effect on d13C from the consumption of

411EFFECT OF DIET ON HUMAN HAIR ISOTOPIC VALUES

secondary animal protein as opposed to meat.In retrospect, the results are not surprising.As Webb et al. (1980) discuss, isotopic valuesof food in the literature show that all animal-derived protein from the same individual—achicken and its eggs, a cow’s meat and itsmilk—are isotopically equivalent in bothcarbon and nitrogen and can all be classedas animal protein for the sake of isotopicdietary reconstruction (Katzenberg andKrouse, 1989; Minagawa, 1992; Schoeller etal., 1986). Consequently, there is no funda-mental carbon isotopic difference betweenthe dietary components of omnivores andovo-lacto-vegetarians and therefore no ex-pected difference between their carbon isoto-pic values.

Nitrogen isotopic variation. To be ableto comment on the dietary composition ofarchaeological humans from nitrogen isoto-pic values, we must understand which fac-tors affect these values. As stated previously,the primary source of human nitrogen isoto-pic variation is diet. Secondary influencessuch as climate, rainfall, and animal physiol-ogy may also play a part (e.g. water stress,pregnancy, and lactation) but have not yetbeen fully quantified (Ambrose and DeNiro,1986; Grocke et al., 1997; Koch, 1997; Sealyet al., 1987).

When the effect of diet on nitrogen isotopicvalues is considered, there are two sorts ofvariation: diet composition (marine vs. ter-restrial, amount of animal protein, etc.) andalso baseline variation in the food chaind15N. Plant nitrogen isotopic variation isknown to vary widely between ecosystems,owing to climate, environmental conditions,and the nitrogen content and isotopic valuesof soils (Handley et al., 1994; Handley andRaven, 1992; Heaton, 1987). This makescomparisons between archaeological popula-tions difficult unless there are referenceplant and animal food source materials avail-able.

Therefore, for nitrogen isotopic values ofarchaeological human samples to be used toinvestigate meat or animal protein consump-tion in archaeological humans, there are twoareas that must be investigated. Interecosys-tem nitrogen isotopic variation must be as-sessed to investigate the range of variation

in nitrogen isotopic values and to determinewhether nitrogen isotopic values can becompared between populations. In addition,we need to show whether, within a singlepopulation, variations in diet compositionresult in observable variations in d15N. Inthis study we investigate the latter point byexamining whether the level of dietary ani-mal protein determines the d15N of bodytissues. This has not been previously demon-strated.

Breast-feeding and weaning studies.Weaning studies have shown an observabletrophic level effect in humans, with breast-feeding infants enriched by about 3‰ ind15N relative to their mother (Fogel et al.,1989; Katzenberg and Pfeiffer, 1995;O’Connell and Hedges, unpublished data).Since infants have a high animal proteinintake, it might be suggested that this sig-nal demonstrates that a high animal proteinintake results in enriched nitrogen isotopicvalues. But a trophic level signal in humansowing to breast-feeding cannot be comparedto variation in adult human d15N owing todifferences in diet selection and composi-tion.

The d15N signal in breast-feeding infantsindicates an enrichment in nitrogen isotopicvalues between diet and body tissues. How-ever, it does not indicate that, given a choiceof foods, the amount of animal protein con-sumed affects the d15N of an individual’sbody tissues. To demonstrate this effect, wemust compare the nitrogen isotopic values ofbody tissues of individuals within a singlepopulation who have varying levels of ani-mal protein intake, and, for such a compari-son to be valid, the available animal andvegetable protein that may be consumed byeach individual must be assumed to havesimilar d15N values. This is not the case withbreast-fed infant and mother, since the nitro-gen isotopic values of the diets of the infantand mother are not comparable (breast-fedinfants are one step directly above theirmothers in the food chain). In addition,infants are in a rapid state of growth, andthis may affect their isotopic values to somedegree. The weaning signal therefore cannotconfirm that varying d15N values can be seenin adult humans (in a metabolic steady


state) from varying animal protein intakealone.

This study. We propose to test the hypoth-esis that the level of animal protein in anadult individual’s diet produces a detectableand quantifiable signal in the d15N of theirbody tissues. We have compared the isotopicvalues of human hair from adults in thesame population who have varying meatand animal protein consumption.

CONSIDERATIONS IN USINGHAIR KERATIN

Before using hair keratin to study theeffects of a change in diet composition on theisotopic values of human body tissues, wemust consider both the effects of the rate ofhair growth and metabolic turnover on hairisotopic values. Unlike most body proteinswhich are remodelled throughout an ani-mal’s lifetime, hair is unusual in that theprotein is not reabsorbed. The carbon andnitrogen isotopic values of proteins with aslow turnover rate, such as bone collagen,reflect an average of the body’s dietary pro-tein intake over a long period of time (Sten-house and Baxter, 1979), but the carbon andnitrogen isotopic values of hair along thehair shaft length approximate to a linearrecord of the most recent diet. With a growthrate of about 1cm each month (Saitoh et al.,1969), a fairly short length of 6 cm covers thediet of the last 6 months. This unique prop-erty may used to monitor such parametersas seasonal dietary change. White (1993;White and Schwarcz, 1994) has used d13Cand d15N in hair to determine seasonalchange in diet. The change of 2–4‰ that shefound in the d13C values of Nubian mummyhair can be attributed to a shift in diet fromC3 to C4 plants. However, it is not knownhow great a dietary shift is required beforethe signal shows in the isotopic values ofhair or how long the time period is before thechange in diet is registered in the isotopicvalues of hair. If a seasonal diet change iscyclical and we do not know the time periodrequired for humans to reach isotopic equi-librium after a change in diet, then wecannot use such isotopic analyses to quan-tify by how much the diet has changed.

Physical considerations

Nakamura et al. (1982) showed that anisotopic shift resulting from dietary changecaused by a change of geographical locationtakes at least 6–12 days to show in the d13Cof adult male beard hair, since it takes aminimum of 6 days for the growing hair toemerge from the skin (Saitoh et al., 1969;Valkovic, 1977). In addition, when the timelag for stable isotope ratios to be reflected inhair is considered, it must be rememberedthat at any one time individual hair folliclesall over the scalp are at different stages inthe growth cycle. The hair growth cycle hasthree time phases: the long anagen, or grow-ing, phase, the short transitional catagenphase, and the intermediate telogen, or rest-ing, phase. In humans, each follicle’s growthpattern is independent of the ones around it,so hairs that are adjacent on the scalp andassumed to be contemporaneous can be outof step with each other. Human scalp hairfollicles spend on average 3 or more years inthe anagen phase, 1–2 weeks in the catagenphase, and 3–4 months in the telogen phase(Valkovic, 1977). Thus, any sample takenwill have an average of 88% of hairs growingand 11% static, and therefore approximately10% of the isotopic signal will be between0–3 months behind in reflecting the diet.

Metabolic considerations

The initial effects of any isotopic variationwithin the body caused by a change in dietcan be seen within days (Nakamura et al.,1982). However, the transition to an isotopicsteady state reflecting the new diet will takea longer period of time. Most proteins in thebody are constantly being resorbed and re-modelled (excluding hair, fingernails, andskin). Protein synthesis rates in the humanbody are such that every day the bodytypically produces three to five times asmuch protein as the average daily proteinintake (Davidson and Passmore, 1979). Thus,approximately one-quarter of the amino ac-ids required for protein regeneration withinthe body is supplied by the dietary intake,and the rest is taken from the breakdownproducts (amino acids) of other body pro-teins, or from the body protein pool. Thisprotein pool acts as an isotopic buffer and


reduces the effect of short-term isotopic fluc-tuation in the diet. Similarly, after a changein diet isotopic composition, the body pro-teins formed prior to the dietary change area reservoir of amino acids that prevent theisotopic values of newly synthesised bodytissues from immediately shifting to theisotopic composition of the new diet. There-fore, after a change in diet, the isotopicvalues of all body proteins change graduallyas the protein pool is slowly modified to theisotopic value of the new diet.

It is not known how long the body proteinpool in humans takes to isotopically equili-brate after a change in diet. In a study onyoung steers, Jones et al. (1981) showed thatcattle hair took at least 74 days to equili-brate after a shift from a C4 to C3 diet. Itwould be expected that cattle would take alonger time to reach equilibrium than hu-mans, since cattle have a larger proteinreservoir within the body which should takelonger to equilibrate to the new isotopiccomposition. However, no previous studyhas examined the isotopic equilibration timefor humans.

MATERIALS AND METHODS

In this study, hair samples were collectedand isotopically analysed from 28 individu-als with varying diets, and the results corre-lated with the amount of animal proteinthey consumed. The effects on hair isotopicvalues of a change in diet in the short andlong term were studied to consider the ef-fects of metabolic turnover on the isotopicsignal measured. Prior to these experi-ments, we considered how best to measurethe isotopic signal in the hair sample andwhat effects atmospheric and cosmetic con-tamination might have on hair isotopic val-ues. In the experimental section, we detailconsiderations of isotopic analysis and alsoexperiments performed to investigate theeffects of contamination using both animal(previously untreated) and human hair as atest material.

Dietary variation and the effect on hairisotopic values

The isotopic values of hair keratin fromindividuals with differing diets in the samepopulation were measured. Hair samples

were collected from local adult Oxford resi-dents. The samples were taken from eachsubject in the same way. A small hair sample(15–20 hairs) was cut from the crown of thescalp, the scalp end of the sample wrappedin micropore tape to anchor the strands, andthe hair stored in a plastic bag until it wasanalysed. Analysis was as described in thefollowing methodological section. All sub-jects gave details of their diet over the yearprior to sampling and of their hair careprocedure, such as the frequency of washingand any special treatments or dyes used.Diet details taken included listing the typesof protein, carbohydrate, fats, and sugarsconsumed as well as the frequency of con-sumption.

Twenty-eight individuals gave hairsamples, and the subjects were placed intoone of three groups: omnivores (numbering14), ovo-lacto-vegetarians (numbering six)and vegans (numbering eight). Vegans werethose consuming no animal produce at all.Others who did not eat meat or marine foodsbut who did eat secondary animal productssuch as eggs and dairy produce were placedin the ovo-lacto-vegetarian group. There wasa wide range in the reported frequency ofconsumption of secondary animal productsin this group, ranging from daily to rarely.The third group, omnivores, were those thatate meat, secondary animal products, andmarine foods. Again there was a wide rangeof reported frequency of animal protein con-sumption, from daily to once or twice weekly.None of the subjects had a significant directconsumption of C4 plants such as maize.None of the omnivores reported more thanan infrequent consumption of marine foods.All 28 had not significantly changed theireating habits in the 3 years prior to sam-pling.

Effect of a change in dietary composi-tion. It was possible to investigate theeffects of a change in diet on the isotopicvalues of hair in both the short term and inthe long term. Short term effects were ob-served by analysing hair from one indi-vidual (not included in the main study) whohad changed from an omnivorous to a vegandiet 15 months prior to giving a hair sample.The length of hair taken (23 cm) covers the


time period before and after the change indiet. Hair was also sampled from the sub-ject’s husband, who had been a vegan forover 5 years. The two had eaten an identicaldiet over the 15 months prior to the hairbeing sampled. Long-term effects were ob-served by analysing hair from one of thecurrent vegan subjects. The individual hadkept a length of hair cut off approximately22 years earlier, when her diet was omnivo-rous and high in marine foods and meat. Thefirst sample is from 1972, while an omni-vore. The individual changed from an om-nivorous diet to a vegan diet in 1974, and thesecond sample is from 1994, while a vegan.

Analytical proceduresSample cleaning. Unlike most body pro-teins, hair is entirely exposed to the externalenvironment, so even modern samples areliable to contamination. Cleaning of the hairsamples prior to analysis is vital to removeany possible surface contaminants, such assebum lipids, shampoo residues, or particu-late matter. Samples were cleaned by soak-ing in a 2:1 mixture of methanol and chloro-form for about 2 h to remove any lipid orshampoo residue and then rinsed twice inwater. All reagents used were of analyticalgrade or above, and all water used wasdeionised and distilled. Organic solvents(acetone, methanol, and chloroform) havebeen used previously in such work (Katzen-berg and Krouse, 1989; Minagawa, 1992;Nakamura et al., 1982; Schoeller et al.,1986; Webb et al., 1980; Yoshinaga et al.,1996). Detergents were not used since thesehave been shown to be damaging to the hairsurface (Taylor et al., 1995).

Sample preparation and analysis. Iso-topic analyses were performed using anautomated carbon and nitrogen analyserand a continuous-flow isotope-ratio-monitor-ing mass spectrometer (cf-irm-ms) (ANCARoboprep coupled to a 20/20 mass spectrom-eter; Europa Crewe, UK). Typical replicatemeasurement errors are of the order of60.3‰ for d13C and 60.4‰ for d15N(O’Connell, 1996).

Hair samples were wrapped in aluminiumfoil and cut into sections for loading into thecf-irm-ms. The entire hair sample was placedlengthways onto a long strip of aluminium

foil approximately 15 mm wide; then thisstrip of foil was folded over twice to enclosethe hair sample. Problems with static elec-tricity were avoided by keeping the samplewet. The hair wrapped in foil was thensectioned into 1–2 cm lengths and driedovernight under vacuum to remove any re-maining water. After drying, the aluminiumlengths were rolled into balls and then loadedinto the carousel of the cf-irm-ms ready forcombustion.

Background signals of carbon and nitro-gen from the aluminium foil were negligible(O’Connell, 1996). The cf-irm-ms permittedthe analysis of hair samples between 600and 3,000 µg in mass.

Combustion, contamination, and C/N ra-tios. The theoretical carbon/nitrogen (C/N)atomic ratio of keratin is 3.4. From theanalyses performed, the measured C/N ra-tios in modern hair samples varied by upto 60.5 from the theoretical C/N value of 3.4(O’Connell, 1996). A range of measured C/Nratios was also seen between different sec-tions of one sample of hair from an indi-vidual, usually of the order of 0.25 but up toa maximum value of 0.6 in one individual.However, alongside this intraindividualvariation in C/N ratio, there was little con-current variation in isotopic values. There-fore, it must be concluded that the possiblerange of C/N ratios in modern hair is be-tween 2.9 and 3.8, and this wide variation isnot an artefact of the experimental analysis.Any samples with C/N ratios outside therange of 2.9–3.8 were deemed not to havebeen satisfactorily combusted and excludedfrom the data set.

For archaeological collagen isotopic analy-ses, the C/N ratio can be used to confirm thatthe sample is not contaminated with organicmaterials (DeNiro, 1985). Any variation out-side the range 2.9–3.6 is taken to indicatediagenetic contamination. The possibility ofusing the C/N ratios of hair keratin in asimilar way was considered. However, dueto the wide variation (2.9–3.8) found in theC/N ratio, this cannot be taken as a sensitiveindicator of contamination. A sample with akeratin C/N ratio of 3.1 could be contami-nated with 20% extraneous carbon (e.g.,with lipids that have a high carbon and low


nitrogen content) and still have an accept-able C/N ratio of 3.7. In summary, the C/Nratios of all samples should be calculatedbut can be used only as a very generalindicator of a sample’s quality.

Experiments addressing contaminationof keratin

Exogenous contamination is a problemwhen considering archaeological samples butis also a concern when using a modernsample material such as hair which is ex-posed to environmental alteration. Contami-nation may result from environmental pollu-tion, treatments such as shampoos, andother cosmetic treatments such as dyes.Other factors such as loss of pigment (grey-ing) may also affect the C/N ratios andisotopic values. Brief experiments were car-ried out to assess how prone hair is todifferent forms of contamination.

Shampooing and cleaning. The C/N ra-tios and isotopic values of horse hair (previ-ously untreated in any way) were unaffectedby soaking overnight in a protein-basedshampoo, by soaking in shampoo and thenwashing with an organic solvent (methanoland chloroform), or by washing only with asolvent (methanol and chloroform) (Table 1).It is surmised that the effect of shampoo onthe stable isotope ratios of hair is too smallto be significant in these experiments.

Dyes. Organic colourants such as henna, aplant-derived dye, could elevate the C/N

ratios and affect the isotopic values of hair ifsufficient quantities were adsorbed onto thehair shaft. Bleaching by hydrogen peroxide(a strong denaturing agent) and other suchcosmetic treatments could alter the C/Nratio and isotopic values of hair since suchprocesses are known to damage hair by theremoval of amino acids, such as cysteine(Baba et al., 1973). We found that the C/Nratios of human hair were unaffected bydyeing with henna but that bleaching byperoxide effected a small difference in C/Nratios and isotopic values (Table 2). The C/Nratio of hair after bleaching was lower thanthat of the untreated sample but was stillwell within the range of 2.9–3.8 found inother untreated samples analysed duringthis study. Similarly, the C/N ratios of hu-man hair samples from three individuals(not included in the diet study) who repeat-edly dyed their hair were also within thisrange of 2.9–3.8. Therefore, although it isnot necessary to automatically exclude dyedor treated samples, care should be takenwhen isotopically analysing modern hairsamples that have been frequently or aggres-sively treated with denaturing agents andother dyes.

Grey hair. There was no difference be-tween the C/N ratios or the isotopic values ofgrey and nongrey hair from members of thesame family with the same diet (Table 3),implying that lack of pigment in the hair hasno observable effect on its isotopic values.

TABLE 1. Effect of various cleaning treatments on horse hair keratin

TreatmentNumber of

samples

C/N ratio d13C (‰) d15N (‰)

Mean Std dev Mean Std dev Mean Std dev

Uncleaned 2 3.13 0.00 226.0 0.1 5.5 0.2Cleaned 3 3.08 0.12 226.0 0.4 5.6 0.1Shampooed but uncleaned 8 3.15 0.16 225.9 0.3 5.3 0.2Shampooed and cleaned 4 3.23 0.03 225.7 0.1 4.6 0.2

TABLE 2. Effect of various treatments on hair keratin

TreatmentNumber of

samples



Uncleaned 3 3.42 0.03 220.8 0.2 9.6 0.1Cleaned 17 3.53 0.08 220.7 0.4 9.3 0.1Hennaed 3 3.54 0.06 220.4 0.2 9.2 0.2Bleached 5 3.28 0.04 220.0 0.2 8.8 0.2


This is in agreement with Minagawa (1992)findings in Japan.

Environmental contamination. Therewere no statistically significant variationsbetween the isotopic values and C/N ratiosof different types of body hair (scalp, axil-lary, and pubic) from the same individual(Table 4). This is similar to the results ofDeAntonio et al. (1982), who found no differ-ence in the chromium concentrations inscalp and pubic hair from the same indi-vidual, implying that scalp hair was notpreferentially affected by chromium in theatmosphere. It is therefore assumed thatatmospheric contamination effects on theisotopic values of modern hair can be dis-counted.

RESULTSDifferent dietary groups

Isotopic analyses of all hair samples col-lected for dietary analysis were judged to bevalid. The C/N ratios of all samples werewithin the range 3.0–3.7. The standard de-viations of the analyses of separate sectionswithin a whole sample from an individualwere less than 0.5‰ in d13C and 0.4‰ ind15N, implying that each individual had arelatively constant diet as represented by thelength of hair analysed. Results are presentedin Tables 5 and 6 and Figures 1 and 2.

Nitrogen data. A comparison of the meannitrogen isotopic values for all subjects (Table6; Fig. 2) shows that there is a statisticallysignificant distinction between the nitrogenisotopic values of hair from vegans and fromthe two other groups (Student’s t-test: com-paring vegans and ovo-lacto-vegetarians, t 57.49, P t 5 9.55, P 0.0001, 18 degrees offreedom). The hair from vegans is 2‰ lowerin d15N than that from omnivores or ovo-lacto-vegetarians.

There is no statistical difference in haird15N between omnivores and ovo-lacto-vegetarians (Student’s t-test, t 5 0.41, P 50.69, 10 degrees of freedom). As mentionedpreviously, nitrogen isotopic values of foodin the literature show that all animal-derived protein from the same individual,including meat, eggs, and dairy products,are approximately isotopically equivalentand can be classed as animal protein for thesake of isotopic dietary reconstruction (Kat-zenberg and Krouse, 1989; Minagawa, 1992;Schoeller et al., 1986). Consequently, thereis no fundamental difference between thediets of omnivores and ovo-lacto-vegetar-ians; the diets have components of similarisotopic composition, and the quantity ofconsumption is the only variable.

When the results are examined in moredetail, within the two groups of omnivoresand ovo-lacto-vegetarians there is a correla-tion between an increasing amount of meator animal protein reported eaten and anincreasing d15N (Table 7; Fig. 3). Thoseomnivores or ovo-lacto-vegetarians who re-ported an infrequent animal protein intake(once or twice weekly) had d15N of around8.4‰, those eating animal protein moreoften had higher d15N values (8.6‰), whilethose individuals with d15N values of over9.2‰ ate meat and dairy products once dailyor more frequently. Ovo-lacto-vegetarianshad a maximum d15N of 9.4‰, while foromnivores the maximum d15N was 9.6‰(Table 8).

Some of the omnivores in this study re-corded that they ate marine foods on aver-age once weekly. Marine foods have a higherd15N and d13C than terrestrial C3 foods, and,although the proportion of dietary proteincoming directly from marine foods was esti-mated to be less than 10% for these individu-als, consumption of marine foods could havecontributed to a slightly higher d15N in thoseindividuals. However, the similarity (differ-ence of 0.2‰) in the maximum d15N ob-served in ovo-lacto-vegetarians (eating nomarine foods at all) and omnivores (someeating marine foods once a week) suggeststhat the consumption of marine foods hadlittle observable effect on the d15N of thisstudy population.

TABLE 3. Variability in the measured C/N ratiosof grey and pigmented hair

Subject

Samplelength(cm)

Number ofsections

C/N ratio

Mean Std dev

Grey 1 12 6 3.48 0.07Grey 2 12 6 3.36 0.06Grey 3 8 4 3.39 0.06Nongrey 1 8 4 3.51 0.08Nongrey 2 12 6 3.35 0.03


Carbon data. Given the mean d13C ofeach of the three groups, there was nostatistically significant shift in hair d13Cvalues between vegans and ovo-lacto-veg-etarians (Student’s t-test, t 5 0.29, P 5 0.78,9 degrees of freedom) or vegans and omni-vores (Student’s t-test, t 5 2.13, P 5 0.06, 12degrees of freedom). However, omnivores

and ovo-lacto-vegetarians appear to be statis-tically slightly different (Student’s t-test, t 53.79, P 5 0.002, 17 degrees of freedom). Incomparison, Webb et al.’s (1980) work foundthat it was not possible to differentiatebetween ovo-lacto-vegetarians and omni-vores using d13C values alone. There wasalso no correlation between the amount of

TABLE 4. Isotopic values of hair from different areas of the body

Hairtype

Number ofsections



Scalp 8 3.29 0.10 219.9 0.4 9.5 0.3Axillary 3 3.23 0.13 219.7 0.6 9.6 0.2Pubic 3 3.33 0.07 219.9 0.7 9.5 0.1

TABLE 5. Isotopic analyses of all individuals, grouped by dietary preference

Subject type

Number ofsample

sections1MeanC/N

d13C (‰) d15N (‰)

Mean Std dev Mean Std dev

VegansV1 4 3.20 222.3 0.3 7.5 0.3V2 4 3.39 221.1 0.5 6.9 0.1V3 4 3.29 220.8 0.2 7.1 0.2V4 4 3.12 220.3 0.1 6.8 0.2V5 4 3.07 220.7 0.1 6.3 0.1V6 4 3.07 221.1 0.0 7.3 0.3V7 4 3.17 219.6 0.1 6.9 0.3V8 3 3.04 221.0 0.2 6.4 0.1

Ovo-lacto-vegetariansOLV1 4 3.24 221.3 0.3 8.4 0.1OLV2 4 3.61 221.0 0.2 8.4 0.2OLV3 4 3.21 220.7 0.2 9.4 0.2OLV4 4 3.16 221.1 0.1 9.2 0.2OLV5 4 3.10 220.6 0.1 8.6 0.1OLV6 4 3.14 221.0 0.1 8.1 0.3

OmnivoresO1 4 3.11 221.2 0.4 9.5 0.1O2 4 3.31 219.9 0.5 9.6 0.2O3 4 3.45 219.8 0.3 8.6 0.1O4 4 3.28 220.2 0.5 8.4 0.1O5 4 3.51 220.7 0.3 8.7 0.2O6 4 3.49 221.2 0.3 8.7 0.1O7 4 3.39 221.0 0.4 8.6 0.2O8 4 3.39 219.2 0.1 8.0 0.1O9 4 3.36 219.7 0.2 8.1 0.1O10 4 3.27 219.5 0.4 8.5 0.1O11 4 3.34 220.0 0.4 9.4 0.1O12 4 3.26 220.6 0.4 8.9 0.2O13 4 3.06 219.7 0.3 8.5 0.4O14 4 3.02 219.8 0.1 9.6 0.2

1 Data from only the first four sections of each hair sample closest to the scalp were used to calculate the isotopic values of eachindividual. Subject V8’s values were calculated from three samples, as the hair sample was only 3 cm long.

TABLE 6. Mean isotopic analyses of each dietary preference group

Subject typeNumber of

subjects

d13C (‰) d15N (‰)

Mean Std dev Mean Std dev

Vegans 8 220.9 0.8 6.9 0.5Ovo-lacto-vegetarians 6 221.0 0.3 8.7 0.5Omnivores 14 220.2 0.7 8.8 0.6


meat or animal protein reported eaten andd13C values.

These results indicate that there is littledifference in the plant base of the threetypes of diets and suggests that it is notpossible to differentiate the levels of dietaryanimal protein in this population from d13C.A possible reason for the small enrichmentof omnivores relative to ovo-lacto-vegetar-ians could have been the consumption ofmarine foods, but a consideration of the d15N(as discussed in the previous section) showedthat this was not likely.

The carbon isotopic values of all bodyproteins are related to the carbon isotopicvalues of the dietary protein, and it has beenshown that hair keratin is enriched relativeto diet by 12–3‰ in d13C values. When thecarbon isotopic values of all subjects aretaken as a single sample population, therange observed of -22.6 to -19.0‰ in the

Fig. 1. Mean isotopic analyses from the hair samplesof each individual. The mean was calculated using onlythe first four sections of each hair sample.

Fig. 2. Mean isotopic analyses for each dietary pref-erence group.

TABLE 7. Levels of animal protein consumptionand the nitrogen isotopic values of an individual

SubjectFrequency ofconsumption1 d15N (‰)

Ovo-lacto-vegetariansOLV6 Intermediate 8.1OLV1 Intermediate 8.4OLV2 Intermediate 8.4OLV5 Frequent 8.6OLV4 Frequent 9.2OLV3 Daily 9.4

OmnivoresO8 Intermediate 8.0O9 Intermediate 8.1O4 Intermediate 8.4O10 Intermediate 8.5O13 Intermediate 8.5O3 Frequent 8.6O6 Frequent 8.7O7 Frequent 8.6O5 Frequent 8.7O12 Frequent 8.9O11 Daily 9.4O1 Daily 9.5O2 Daily 9.6O14 Daily 9.6

1 Frequency of consumption defined as daily (once or more a day),frequent (more than twice a week), intermediate (once or twiceweekly), or rarely (less than once a week).

Fig. 3. Dependence of mean hair nitrogen isotopicvalues on the frequency of animal protein consumptionfor each dietary group.


subjects’ hair keratin is typical of a primar-ily C3 ecosystem, such as northwest Europe.Most of the subjects recorded that they atesome C4 plant-derived products such assweet corn, corn oil, and cane sugar, al-though such foods were not major dietarycomponents. After we correct for the shiftbetween keratin and collagen d13C (approxi-mately 12‰) and the industrial revolution‘‘fossil fuel effect’’ (modern d13C values are-1‰ relative to pre-1800 d13C values [De-Niro, 1987]), the range measured in modernhuman d13C is slightly enriched (< 12‰)relative to that observed in several inlandBritish prehistoric and Iron Age (farming)populations of about -21 to -19‰ in bonecollagen (Richards and van Klinken, 1997;Richards et al., 1998). This enrichment rela-tive to a population that would not have hadaccess to C4 plants suggests that C4 foods dosupply some proportion of the dietary pro-tein of the individuals studied here.

Effects of a change in dietShort-term change in diet. In the studyof short-term change in diet, we observed adramatic effect on the isotopic values of anindividual’s hair following a change from anomnivorous to a vegan diet 15 months priorto sampling. The data presented are that ofBG, a woman who changed from an omnivo-rous to a vegan diet (not included in themain study) and of her husband, PG, avegan for 8 years (Figs. 4, 5). The change indiet for BG also roughly coincided with amove from Texas, USA, to Oxford, UK—hence the enriched initial d13C values of herhair samples (USA residents are more en-riched in d13C than northwest Europeans(Schoeller et al., 1986) because of the in-creased consumption of C4 plants, both di-rectly and as animal feed; see later discus-sion). In the first couple of months after thedietary change (1cm of hair length approxi-mates to 1 month of growth), both carbonand nitrogen isotopic values became moredepleted (lines marked on the graphs inFigs. 4 and 5 as BG). After 5 months, thenitrogen isotopic values were approachingequilibrium, while the carbon isotopic val-ues had changed two-thirds of the way to-wards the isotopic values expected from thenew diet (as seen in the subject’s husband’shair, marked PG in Figs. 4, 5). This initialperiod of change is followed by a moregradual but steady shift in d13C towards the

Fig. 4. Effect of a short-term change in diet on thecarbon isotopic values of hair keratin.

Fig. 5. Effect of a short-term change in diet on thenitrogen isotopic values of hair keratin.

TABLE 8. Variation in mean nitrogen isotopic valueswith the frequency of animal protein consumption

for each dietary group

Dietary group

Frequency ofconsumption ofanimal protein

d15N(‰)

Vegans Never 6.9Ovo-lacto-vegetarians Intermediate 8.3

Frequent 8.9Daily 9.4

Omnivores Intermediate 8.3Frequent 8.7Daily 9.5


carbon isotopic values of PG’s hair. After7–12 months, both carbon and nitrogen iso-topic values are close to equilibrium. It isinteresting that after a year the values ofthe husband and wife on the same diet for 12months still have not converged. This maybe a result of BG still not having reachedequilibrium, or it may reflect small indi-vidual preferences in diet; it is not possibleto say which is true from the limited dietaryinformation recorded by both subjects.

Long-term change in diet. The long-term effect of a change from an omnivorousto a vegan diet on the isotopic values of hairfrom individual AB gave similar results tothose produced from the isotopic analysis ofthe different dietary groups (Fig. 6). A com-parison of nitrogen isotopic values from thetwo samples shows a difference of 2.5‰ be-tween hair grown when an omnivore (1972,aged 20–22) and that grown whilst a vegan(1994, aged 44). Since there is no evidence sofar to suggest a dependence of isotopic val-ues of the body tissues of a healthy adult onage (Katzenberg et al., 1993; Lovell et al.,1986; White and Schwarcz, 1989), this de-crease in d15N in the samples from oneindividual can be ascribed to a decrease inmeat or animal protein consumption withinthat one individual’s diet. The change of 2.5‰is greater than the mean difference betweenomnivores and vegans in this study (2‰);

however, after 21 years the individual ABwas unable to assess adequately the retro-spective composition of her diet; it is pos-sible that marine foods were a significantproportion of her diet, which would result inelevated d15N relative to omnivores who didnot consume much marine fish. The strikingchange in carbon isotopic values is lesssimple to explain than the nitrogen isotopicvalues. However, the change is consistentwith a change from a diet high in meat andparticularly marine foods (both with ele-vated d13C values) to a vegan C3-based diet.

DISCUSSION

Diet and hair d15N

This study has showed that hair nitrogenisotopic values can be related to certainaspects of the individual’s diet, specificallythe amount of animal protein consumed.Taking each dietary group as a whole, we seethat vegans have lower nitrogen isotopicvalues, by 2‰, than omnivores and ovo-lacto-vegetarians, whereas the nitrogen isotopicvalues of omnivores and ovo-lacto-vegetar-ians are the same. This similarity betweenomnivores and ovo-lacto-vegetarians re-flects the observation that all animal-de-rived protein from the same source (meat,milk, eggs) has similar isotopic values. At amore detailed level, there is a systematicrelationship between an increasing fre-quency of animal protein intake and anincrease in d15N of the individual (Fig. 3).

Diet and hair d13C

We found no correlation between haircarbon isotopic values and aspects of diet:although ovo-lacto-vegetarians and omni-vores were statistically different in d13C,there was no significant difference betweenvegans and the other two groups in d13C.

The modern British humans measuredhere are more enriched in d13C than theirprehistoric counterparts, which, given theavailability and ubiquity of C4 plants in themodern world, is unsurprising. However, itmight have been expected that omnivoresand ovo-lacto-vegetarians might have sub-stantially different carbon isotopic valuesthan vegans. In addition to the direct con-

Fig. 6. Effect of a long-term change in diet on theisotopic values of hair keratin.


sumption of C4 plants, the diet of omnivoresand ovo-lacto-vegetarians can contain a hid-den indirect C4 signal, in that maize (corn)-fed chickens and their eggs, together withmeat and dairy products from herds eatingC4-derived feed, will have enriched d13Cvalues that will be transferred up the foodchain, enriching the isotopic signal of theconsumers. However, as there was no differ-ence in d13C between vegans and ovo-lacto-vegetarians/omnivores, we must infer thateither the hidden signal of C4 animal feed inthe diet of omnivores and ovo-lacto-vegetar-ians is balanced by a concurrent increase ofC4 plant protein consumption by vegans orthat all three groups ate a similar propor-tion of C4 plant protein; it is not possible tosay which.

Effects of a change in diet composition

The shifts in isotopic values of hair in theindividual BG immediately following herchange from an omnivorous to a vegan dietallows us to examine the equilibration timeof the body to a diet of a different isotopiccomposition (Figs. 4, 5). The pattern seen inthe isotopic values of the individual BGfollowing a change in diet is consistent withthe initial fast turnover of the majority ofthe tissues in the body protein pool followedby the slow turnover of other proteins.

Within the first 5 months after the changeof diet, most of the body tissue proteins(such as kidney, liver, and muscle) have beenbroken down and reabsorbed (Tieszen et al.,1983; Waterlow et al., 1978). The rapidchange in the isotopic values of the hairreflects this fast turnover of amino acids.After this initial period, the hair keratin ismainly composed of amino acids consumedafter the diet change.

However, 5–12 months after the change indiet, the continued slow modification of thehair keratin d13C indicates that there weresome amino acids being reabsorbed into thekeratin that had been released from pro-teins synthesised prior to the diet change.This indicates the continuing breakdown ofproteins such as bone collagen. These re-sults suggest that, for this individual, thebody takes at least 7–12 months to equili-brate isotopically with a new diet.

It appears that the d13C of the body takeslonger to equilibrate after a change in dietthan the d15N values. This may be a result ofthe separate and different ways that carbonand nitrogen from amino acids are processedin the body. De- and trans-amination mayresult in amino groups equilibrating fasterthan the carbon skeletons of the aminoacids; this differing turnover time has notpreviously been studied.

The measurement of the isotopic values ofthe two samples of the individual AB’s hairmarks the first time that changes in theisotopic values of adult human body tissueshave been observed over a long period oftime following a change in diet (Fig. 6). Theanalyses show that 20 years after the di-etary change the body is in complete isotopicequilibrium with the new diet.

Variations in d15N within one trophic level

The use of nitrogen isotopic values todistinguish varying levels of animal proteinin the diet is an attempt to investigatesmall-scale isotopic variation. Given the widerange of observed d15N in plants and ani-mals, the accuracy with which the d15N ofhuman body tissues can reflect such smalldifferences must be considered.

Nitrogen isotopic values within and be-tween ecosystems are know to vary widely;terrestrial, marine, and freshwater plantscan have d15N from -2 to 110‰ (Katzenbergand Krouse, 1989; Minagawa, 1992;Schoeninger and DeNiro, 1984; Yoshinaga etal., 1991). One trophic level is taken to beapproximately 3‰; consequently, for the as-sessment of levels of animal protein in thediet from complete herbivory to completecarnivory, there must be fine yet observablegradations within a range of approximately3‰.

In this study, the individuals were se-lected at random, and all ate widely differ-ing diets. Yet the change of d15N betweenthose with no animal protein and those witha high animal protein intake is within arange of only 2.5‰, and the d15N variationwithin each group was much less (Table 5;the standard deviation of the omnivore groupwas 0.6‰, and for ovo-lacto-vegetarians andvegans it was 0.5‰). This suggests that,despite the extensive variety of foods avail-


able to north-west Europeans, the averagediet for most individuals within each dietarygroup is isotopically very similar.

In addition, the d15N variation along thehair length for each individual was small,with a maximum standard deviation of 0.4‰.Such small variation must be ascribed to theeffect of the body protein pool acting as anisotopic buffer, as discussed previously. As aresult, despite small possible isotopic fluctua-tions in diet, the isotopic values of bodytissues of each individual are representativeof the average of their long-term dietaryintake.

We conclude that the isotopic values ofbody tissues can reflect small mean isotopicdifferences (d15N changes of 1–3‰) in thediet, even in those tissues that have a rela-tively fast growth rate or turnover time,such as hair.

Magnitude of the trophic level effect

This study demonstrates that the hypoth-esis outlined earlier in the paper is correct:higher animal protein consumption does re-sult in higher d15N values in the body of theconsumer. We can use these results to con-sider the magnitude of the trophic leveleffect by expressing the hypothesis in theform of the following equations.

First, there is an enrichment in d15Nbetween the diet protein and body tissues:

d15Nbody 5 d15Ndiet protein 1 DN, (1)

where DN 5 D15N(body - diet protein)Secondly, that d15Ndiet protein is dependent

on the proportion of animal protein in thediet:

d15Ndiet protein 5 (AP 3 M) 1 ([1 2 AP] 3 V),

where M is the mean d15N of animal proteinconsumed, V is the mean d15N of plantprotein consumed, and AP is the proportionof dietary nitrogen from animal protein (0 ,AP , 1) (i.e., d15Ndiet protein corresponds to theweighted average of the two sources of di-etary protein). This equation can be re-arranged to give

d15Ndiet protein 5 V 1 AP 3 (M 2 V). (2)

Now we can consider the magnitude of thetrophic level effect as long as we assume thatthe enrichment of body tissues relative to

diet (DN) is a constant factor, that d15Nbody isa linear function of the proportion of animalprotein in the diet, and that the meannitrogen isotopic values of the plant andanimal proteins consumed are the same foreach individual.

Combining equations 1 and 2, we see that

d15Nbody 5 V 1 AP 3 (M 2 V) 1 DN, (3)

indicating that the nitrogen isotopic valuesof the body tissues are composed of contribu-tions from the d15N of dietary plant andanimal protein (V and M), dependent on theproportions in which they were consumed,together with an enrichment factor, DN,that results from the incorporation of di-etary components into the body tissues.

Omnivores resident in the United King-dom typically consume 63% of their dietaryprotein as animal protein (Davidson andPassmore, 1979). If we make the assump-tion that the mean isotopic values of allomnivores analysed in this study approxi-mate to the isotopic values expected from atypical omnivore in the United Kingdom,then, from the results of this study, thed15Nbody of vegans (where AP 5 0) is 6.9‰,and the d15Nbody of omnivores (where AP 50.63) is 8.8‰ (Table 6). Using these values inequation 3, for vegans 6.9 5 V 1 DN, and foromnivores 8.8 5 V 1 0.63 (M - V) 1 DN.Therefore, M 2 V 5 3.0‰. Therefore, thedifference between the mean d15N values ofthe animal dietary protein and plant dietaryprotein is an estimated 13.0‰. Substitutingthis value back into equation 3, we canevaluate a difference of 3‰ between thebody tissues of an individual consuming noanimal protein (vegan or complete herbi-vore) and those of an individual consuming100% of his or her dietary protein as animalprotein (complete carnivore).

The transition up the food chain fromherbivore to carnivore is equivalent to onetrophic level. Therefore, this estimate of13‰ is a measure of the isotopic shiftbetween one trophic level and the next, orthe trophic level effect. The value derivedhere is equal to the observed enrichmentbetween trophic levels in a variety of eco-systems (Minagawa and Wada, 1984;Schoeninger and DeNiro, 1984).


CONCLUSIONS

We conclude that in living humans a diethigh in animal protein correlates to rela-tively higher nitrogen isotopic values in hairand one low in animal protein correlates torelatively lower nitrogen isotopic values inhair, demonstrating that the d15N values ofhuman body tissues, like those of othermammals, are dependent on the dietaryintake of meat or animal protein. We foundno such dependence in the carbon isotopicvalues.

Hair isotopic values reflect diet isotopicvalues, and if the diet is changed the isotopicvalues of hair will also change to reflect thenew diet. Human hair keratin takes at least7–12 months to approach equilibrium aftera dietary isotopic change.

The expected difference in the nitrogenisotopic values of an individual eating noanimal protein (herbivore) and one eating100% of his or her dietary protein as animalprotein (carnivore) is approximately 3‰,which can be equated to one trophic level.

ACKNOWLEDGMENTS

This study would not have been possiblewithout the willing participation of manyvolunteers; we thank them all. This workwas supported by a SERC/NERC student-ship for T.C.O’C.

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Documents

Investigations into the effect of diet on modern human hair isotopic values