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    Applied Geochemistry,Vol. 7, pp. 145-158, 1992 0883-2927/92 $5 .00+ .00Printed in Gre at Britain Pergamon Press Lt d

    R e c o n s t r u c t i o n o f h u m a n d i e t f r o m 8 1 3C a n d ~ l SN i n c o n t em p o r a r yJ a p a n e s e h a i r : a s t o c h a s t i c m e t h o d f o r e s t i m a t i n g m u l t i - s o u r c ec o n t r i b u t i o n b y d o u b l e i s o to p i c t r a c e r s

    MASAO MINAGAWAMitsubishi Kasei Institute of Life Sciences, 11 Minami-Oya, Machida, Tokyo 194, Japan

    (Received 19N o v e m b e r 1990;accepted in revised fo rm 15N o v e m b e r 1991)Ab s t r a c t - - A stochastic method has been proposed for estimating contributions of multi-sources based ona simultaneous measurement of C and N stable isotope compositions of a mixture. Double isotope traceranalyses essentially allow a determination of the mixing proportion of up to three sources by mass balancecalculations. For more than three sources, a stochastic approach may provide a possible range of mixingproportions. In this work, a stochastic method using the Monte Carlo simulation was employed forreconstructing the dietary consumption of contemporary Japanese.13 15~The mean 6 C and 6 N values of contemporary Japanese scalp hair were found to be - 18.2 _ 0.4 and10.3 + 0.4%o relative to PDB and the atmospheric N2, respectively. A dietary model was constructed byusing mean isotope ratios of five major food groups and hair for modern Japanese. Based on this model, astochastic method was applied to simulate human feeding. A range of dietary patterns, consistent withreasonable energy/protein uptake ratio, was estimated for fitting C and N isotope distributions of hair.The estimated mean dietary pattern has protein contributions of 35, 9, 16, 14, 27% from C3 plants,legumes, C4 plants, land animal products, and fish products, respectively, in good agreement with theobserved food consumption in the National Statistics Report. The contribution of C4 type plant wasestimated to range from 10 to 20% in protein, indicating much higher consumption than the statisticalestimation.Thus, this stochastic method is useful in dietary analysis based on C and N isotope ratios. If the isotopiccompositions of source materials are sufficientlydifferent and each source is not isotopically reproducibleby mixing of others, the stochastic simulation may indicate accurately the different source contributions.This method is applicable also to many geochemical problems where mixing occurs.

    INTRODUCTIONNATURALLYoccurring stable isotopes of C and N havebeen often measured for tissues of humans and wildanimals (ScHOENINGER t a l . , 1983; DENIRO andEPSTEIN,1978). The purpose of such analyses is toestimate relative proportions of food materials indiets, as well as to search for un kn own pathways ofnutrient transport. The isotope composition is con-ventionally represen ted by the delta values, and isgoverned by the law of conservation of mass. Theisotope ratio of mixed materials can be calculated bya mass balance equati on based on the proportio ns ofsource materials and their isotope compositions.Hence, isotope ratio measurements have been suc-cessfully used to estimate the proportional contri-butions of source materials.

    Recent isotope studies on natural organisms havedepicted a variety of C an d N isotope distributions invarious ecosystems. Carbon and N isotope compo-sitions of living organisms are ultimately controlledby the isotope ratios of primary producers that areinitial suppliers of organic C a nd N in an ecosystem(PETERSON and FRY, 1987). There are three diffe rentpathways in photosynthesis; the most abu nda nt Cal-vin and Benson system (called the C3 type), Hatchand Slack system (C4 type), and crassulasian acidmetabo lism (CAM type ) (SMrrH and EPSTEIN, 1971;

    WHELANe t a l . , 1973). Because the C isotope fraction-ations associated with these three modes of photo-synthesis are different, these plant groups possesssignificantly different isotope ratios even though theyall obtai n their initial C from the same source, atmos-pheric CO 2. In contrast , the N isotope ratios of plantsare mostly controlled by the isotope ratios of theirsubstrates , such as NH 3 and NO3 in soil and water,and atmospheric di-nitrogen for leguminous plants(HOBNER, 1986).

    In addition to the isotopic variations in flora,feeding processes of animals modify both C and Nisotope ratios of their body tissues. Previous studieson many kinds of animals have revealed that theisotope fractionation during feeding and metabolismfalls in a limited range for both C and N. The Nisotope ratio of many animals is normally enriched3.5%o over that of dietary proteins (MINAGAWA ndWADA, 1984). T he C isotope fracti onati on seems tobe less than that of N (RooNlC~ and WtNXERBOORN,1986). This evidence indicates that the isotope frac-tionation during the human feeding and metabolismprocesses is approximately constant for most foodsources (MINAGAWAe t a l . , 1986). These biologicalprocesses in plants and animals finally determ ine theC and N isotope compositions of a given consumer inan ecosystem.

    Because human food has been produced from145

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    146 M. Minagawabiological resources in natural and agricultural eco-systems, the isotope composition of the hu man bodyis also controll ed by the same rules that apply to othe ranimals. Therefore, the hypothesis that the C and Nisotope compositions of animal tissues reflect thoseof diets is also the case for human food webs. This waspartially shown by a C isotope study of modernhu man hair analysis (NAKAMURA t a l . , 1982). Furtheruse of C and N isotope ratios for interpre ting modernAmerican human food web was reported bySCHOELLERet al . (1986). As human hair lengthens afew centimeters each month, the isotope ratios ofhair reflect the food digested through the last severalmonths. I n spite of this time lag, it was shown that theC and N isotope ratios of hair can be expl ained fairlywell by the nationa l food consum ption statistics andthe mean isotope ratios of major foods in U.S.A. Theapparen t discriminations between diet and h umanhair were estimated to be - 2 and 4%0 for C and N,respectively. These studies support the abovehypothesis in human food webs. On the basis of theseresults, the next question is how to reconstruct thedietary pattern of human s from isotope compositionsof food web samples.

    Dietary analysis has been used in the field ofarchaeology and anthropology, and many recon-structions of ancient human diets have beenreported. In such applications, the nu mbe r of dietarysources was usually restricted to two or three sources(e.g., CHISHOLM e t a l . , 1983; FA~NSWORTHe t a l . ,1985). For con temporar y humans, however, the foodweb is usually more complex than that of prehistorichuman s who depen ded solely on local food. This ispartly ascribed to the expansion of food trading in themodern world. Accordingly it is difficult to limitmajor foods to just two or three groups. From C andN isotope compositions, mode rn hu man food sourcesare characterized in at least four or five representa-tive groups (MINACAWAe t a l . , 1986). In this study,the C and N isotope compositions were analyzed forscalp hair and some food samples from contemp oraryJapanese food webs, and a new data analysis methodwas proposed to estimate a dietary pattern. Emphasiswas placed on an evaluation of a stochastic methodthat enabl es several food sources to be reconstructedby C and N isotope analysis.

    D I E T AR Y R E C O N S T R U C T I O N M O D E L

    The biological behavio rs of C and N isotopes havenot been studied closely in metabolic processes. Inparticular, it is still ambiguous as to how these iso-topes are distributed in animal tissues after themacromolecules in diet are metabolized. These pro-cesses are retained as a black box. However, arelatively constant isotope fractionation apparentlycan be seen between diets and animal tissues. In thepresent model, it is assumed that because the maj orcomponent of hair is protein, protein in diet is

    directly transferred to human hair and oth er macro-molecules do not participate. This assumption wasinvestigated in relation to the contribu tions of fat andcarbohydrates.The C and N isotope ratios of huma n tissues differin organs (Minagawa unp ubli shed data; LYON andB A X T E R , 1978, for C isotopes), but the isotopic trendsamong organs appear to be similar in other mam-mals. Therefore, the isotope ratios of hair may beused as representative of the human body for ahuma n feeding model.

    A n a l y t i c f e e d i n g m o d e lSuppose a person is living on an unchang ing diet of

    known isotope ratios, for either C or N. The isotoperatio of the bo dy tissues should be controlled by theisotope ratios of the food, and by the isotope fraction-ation between food and human tissue. In most ani-mals, including humans, the isotopic fractionation ofC and N appears to be nearly constant. If we knowthe isotope discrimination values (Ahuma,_diet) for acertain animal -diet system, the isotope compositionof the diet can be estimated by

    6 m = 6 h . . . - - m h . . . . d i el ' (1)where 6 m and 6 h u m a n a r e delta values of the diet andhuman tissue, and Ahuman_diet i s an offset due toisotope fractionation between diet and huma n tissue.In the case of a mixed diet composed of twodifferent food sources, the proportion of each sourcecan be calculated from the mass balance equation:

    6 m = f l 6 1 + ( 1 - - f l ) 6 2 , ( 2 )f l - - 6 m - - 6 2 ( 3 )61 - 62 'f 2 = 1 - f l , ( 4 )

    where fl and f2 are pro portions of source 1 and source2, respectively, and b 1, d2 and 6m indicate deltavalues of C or N, for source 1, source 2 and a mixeddiet, respectively. In this case, C and N isotope ratiosof source 1, source 2 and the mixed diet give anestimation of the p roportions of source 1 and source 2in the mixed diet.

    In the case of three sources, simultaneousmeasurements of both C and N are needed to esti-mate the contribu tion of each source. Using the massbalance equation, the proportion of one source isdet ermi ned analytically as follows:

    (d m - 63)(6 ~ - d~) - (d 2 - 63)(d m - d~) (5)fl = (61 63)(6~ d~) ( 6~ -- 6- ~ "' "f2 = 6m - d~ - fl (6] - 6~) (6)

    andf 3 = 1 - - f 2 - - f 3 , ( 7 )

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    Reconstruction of human diet from d13C and 615N in contemporary hair 147where 6 1 , 6 2 , 6 3 and 6 m represent C delta values offood sources 1, 2, 3 and mixed food, which is com-posed of food from each source in proportions )q, f2and f3, respectively. The 6~, 6~ and 6~ indica te N del tavalues in the same manne r.

    Thus, when the nu mbe r of food sources is limitedto three or less, the proportions of the food sourcesare unequiv ocally determ ined from the isotope com-positions of the sources and of a mixed diet.

    Stochastic feeding modelThis analysis can be extended to those cases with

    more than three food sources. Suppose that a personeats food from many sources which have significantlydifferent isotope ratios, and that the dietary intakeproportions are kept constant in the period sufficient-ly longer than the turn-ov er time of hair. The isotoperatios of body tissues should be controlled by theisotope ratios of each diet alternative and their mix-ing proportions, as given by the following mass bal-ance equation:

    dh .. . = Ah .. .. diet + f l dl ~" f262 +. . .+f nd n, (8)

    where,f1 ,f2 . . . . f, and 61, d 2 . .. d n are mass fractionof the diet alternatives and each isotope ratio, re-spectively, and Ahuman_die t is the isotope fractionationdue to the feeding process. Then, the isotope ratios ofthe averaged diets from the isotope data for humantissue and the isotope discrimination factor can beestimated. In this case, the dietary mixing pro-portions canno t be calculated exactly, as was done inthe analytical model.

    If C and N isotope compositions of every dietalternative and their consumpti on proportion can beobtained, it is possible to reconstruct the isotoperatios of the total food intake, i.e., the average dieteaten. The isotope ratios of the total diet do notsuggest the prop ortion of food consu mption directly,but it is useful to judge whether certain possiblemixing proportions produce the observed isotoperatios. To carry this out, the following stochasticapproach was developed; numerous hypotheticalpeople are served a variety of meals that are com-posed of varying amounts of defined food sources.The isotope ratios of C and N of such diets werecalculated. This operation was programmed in acomputer simulation, and run many times, usingrandomly generated food proportions. Where thecalculated isotope ratios were close to the observeddata, the food proportions yielding that result wererecorded as possible cases of dietary mixtures. Aftersufficient numbers of trial and error, the possibleranges of proportions were d etermin ed for each foodsource.

    With this program, it is possible to determinecombination s of multiple food sources which satisfy

    the observe d isotope values of human tissues. If thisprocess is repeated the above trial and error oper-ation for all proportions with eq ual possibilities, thedistribution of possible proportions that can satisfythe isotopic conditions can be obtained. For thispurpose, a special function that generates randomproportions having equivalent probabilities of occur-rence is necessary, because the sum of proportions ineach combi nation must be unity.

    For such a function, the Sweeping-out method isgenerally appropriate and is capable of generatingsuch homogeneous proportion frequencies. Mixingproportions are generated in series from 0 to 1 foreach food source using a constant interval for thecontrib ution proporti on of each source, then eachcombination is tested against the observed isotopiccondition. This method is suitable to find all pro-portion ranges with equivalent probabilities, but thefrequency distrib ution is discontinuous and it gener-ally takes a very long time computing until all pro-portion ranges are covered.

    Another technique is a Monte Carlo method(MC), that generates random mixing proportions,then checks if each case satisfies the isotopiccondition. This method is conven ient for an actualoperation, because computing time is saved. How-ever, it requires an appropriate function to generatehomogeneous random proportions. In this work, aprogram which generates random numbers underrestr icted condit ions using the algor ism of WAKIMOXO(1976) was used. The program was verified by run-ning problems of which results are known, and bycomparing the result obtained by the analyticalmethod.

    The result is initially given as a frequency distri-bution , then illustrated by a histogram or a box-hingeplot using the accumulated proportion of eachsource. The most likely contributio n pattern of eachfood source can be suggested by optimizing addi-tional factors in the human feeding process, such asprotein and energy demand.

    E X P E R IM E N T A L M E T H O D

    The C and N isotope ratios of human hair were analyzedfor 42 contemporary Japanese (23 males and 19 females),randomly chosen from volunteers of wide age range, livingin Tokyo, Okinawa, and Akita areas during 1984 and 1985.Another hair sample was the NIES No. 5 reference sample,which was powdered hair sample mixed from many contem-porary Japanese males in Tokyo, combined, and distributedfrom the National Institute of Environmental Sciences,Tsukuba, Japan, especially for the purpose of an inter-calibration of chemical analyses.Scalp hair specimens were cleaned by distilled water,methanol, chloroform and acetone. The solvents were eva-porated at room temperature, and then freeze dried. Hairwas cut from the scalp by stainless steel scissors. Theposition on the scalp and the length of hair from the skinwere not specified. Normal colored hair and gray from thesame old person did not result in significant differences inboth dl3C and blSN isotope ratios nor the content of C and

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    148 M. MinagawaN. Therefore, gray and partially gray hairs from olderpeople were analyzed in a similar manner.The food materials for isotope analysis were obtainedfrom several public markets in Tokyo during 1985. Theywere homogenized, treated with methanol-chloroform(1:2) to remove the solvent extractable lipid fraction, andthen were freeze dried. The food samples used in this work,therefore comprise both protein and carbohydrate frac-tions.Dried hair and food samples of -1 0 mg were combustedand converted into COE and N2 gas by the conventionalquartz combustion method described by MINAGAWA t al.(1984). Amounts of N and C in the samples were deter-mined volumetrically using a mercury manometer prior tocollection of the gases. The isotope ratios of carbon(13C/12C) and nitrogen (15N/14N) were analyzed by a FIN-NIGAN MAT 251 isotope ratio mass spectrometer. Isotoperatios were converted to delta values relative to the inter-national standards; PDB and atmospheric N2, respectively(CRAIG,1957; MARIOTTI,1983), by using the isotope ratios ofseveral laboratories working standards, such as reagents ofproline, histidine and valine. The overall analytical pre-cision of the measurement was _+0.08 for both 6]3C anddilSN in five replicate analyses of powdered human hair.

    RESULTS AND DISCUSSIONT h e 613C a n d 6 I5N o f c o n t e m p o r a r y J a p a n e s e h a ir

    There were no clear differences in isotope compo-sitions between males and females, nor dependenceon the age of individuals (Fig. 1, Fig. 2 and Table 1).The mean 613C and 615N of 42 individuals wereconsistent with those of the NIES composite hairsample. T he 613C of the examin ed hairs also agreedwell with the previous report for different Japanesegroups collected at least two years previously (NAKA-

    MUP.A e t a l . , 1982). These similarities suggest that theisotope ratios of the exam ined specimens are likely torepresent the mean isotope ratios of contemporaryJapanese hair.

    SCHOELLERe t a l . (1986), NAKAMURA t al . (1982)and MINAGAWA t al . (1986) rep ort ed tha t 613C valueof human hair was enriched about 1.4-2.0%o com-pared to that of the diets. For N, an enri chment in 15 Nof -4.3%o from diet to hair was found (MINAGAWAta l . , 1986; SCHOELLER t a l . , 1986). Acco rding to theseobservations, it appears that the 613C and 615Nvalues of th e average Japane se diet may be - 19.6 to-20.2%0 and 6%o, respectively. This estimate will bediscussed later.

    T h e 6 i3C a n d 6 X N o f r e c e n t J a p a n e s e d i etCarbon and N isotope data of food available in the

    Tokyo area are listed in Table 2, and plot ted in Fig. 3.The results show that contemporary Japanese foodsfall into at least five groups based on C and N isotopecompositions: C3 plants including rice and mostvegetables; C4 plants such as corn and millet;N2-fixing plants (mostly leguminous plants); landanimal products including meat, eggs and dairy prod-ucts; and fish products (both marine and freshwater).

    The 613C of plants vary from - 35 to -10%o due tothe differences in photosynthesis. Consequently the613C of herbivo rous animals vary depending on theirfeeding patterns. As the animal feeding ecologycould change seasonally and locally, the isot ope com-position of food for humans may not be constant eve nfor the same items. In this work, the sample size and

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    Reconstruction of human diet from 613C and 615N in contemporary hair

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    FUG. 2. The relation between the C and N isotope compositions (613C) of hair and the age of Japanese,Open and closed circles indicate data from female and male Japanese, respectively.

    selection may be still insufficient to determine therepresentative isotope composition of Japanesefoods. However it is believed that the major foodgroups are represented. The average isotope compo-sitions of these food groups are probably not verydifferent from the results reported here.

    The measured isotope compositions for Japanesefoods overlap the distribution of Amer ican food datareported by SCHOELLER t al. (1986) as shown in Fig.3. This is partly because the carbon and nitrogenisotope compositions of contemporary food re-sources are mainly controlled by general factors, suchas C3 and C4 photosynthesis in terrestrial f ood chainsand marine food chains. In other words, the isotopedata on major food groups presented here may applyto wide areas of the world. In addition, many foodresources consumed in modern Japan are importsfrom the U.S.A., e.g. most of the soy beans and cornare imported from the U.S.A . The C isotope data onmeat (beef and poultry) also indicate that they werefed forage composed of corn imported from the

    U.S.A. However, dairy products in Japan, such asfresh milk, have quite negative 613C values sugges-ting that cows in Japan are fed dominantly C3 richforage.

    The isotope analysis of foods should be continuedto obtain a more complete picture of isotope values.In particular, the range in isotope composition of fishis still not well understood . This is import ant becausemodern commercial fish in Japanese markets arecollected from the world oceans. The habitat and thetrophic state of these new resources are not wellknown, so that their C and N isotope ratios may notbe in the range of fish customarily used in Japan.

    M e a n i s o to p e c o m p o s i t io n s o f C a n d N f o r J a p a n e s ef o o d

    Because the f ood items which are used in contem-porary Japanes e life are very diverse, it is difficult toprepare average dietary mixtures for chemical analy-

    Table 1. The C and N isotope compositions of Japanese scalp hair613C S.D. (No.) 615N S.D. (No.) Mean age (range)

    *Number of measurements of powdered hair.

    JapaneseFemale -18 .35 + 0.33 (19) 10.31 + 0.41 (19) 34 (3-83)Male -18 .04 + 0.45 (23) 10.36 + 0.48 (23) 43 (5-78)Total -18 .19 _+ 0.43 (42) 10.31 + 0.45 (42) 39 (3-83)Nies hair - 18.00 + 0.08 (5*) 10.54 + 0.08 (5*) Composite of male hairsJapanese -18.2 _+ 0.8 (15) - - NAKAMURA t al. (1982)

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    1 50 M . M i n a g a w as i s. I n p l a c e o f t h i s , t h e dl3c a n d 6 1 5N v a l u e s o f t h er e p r e s e n t a t i v e f o o d g r o u p s w e r e e s t i m a t e d f r o m t h ei s o t o p e d a t a o n i n d i v i d u a l f o o d s. T h e f o o d c o n s u m p -t i o n s t a t i s t i c s r e p r e s e n t a v e r a g e d i e t a r y p a t t e r n s o fm a j o r f o o d s f o r t y p i c a l J a p a n e s e . T h e 6 1 3 C a n d 6 1 5 Nv a l u e s o f m a j o r f o o d s c a n b e m e a s u r e d i n d iv i d u al l y .T h e 6 13 C a n d 6 1 5N v a l u e s o f a n a v e r a g e d i e t f o r at y p ic a l J a p a n e s e c a n t h e n b e e s t i m a t e d b y u s e o f am a s s b a l a n c e e q u a t i o n . T a b l e 3 s h ow s t h e n u t r i t i o n a ls ta t is t ic s o f J a p a n e s e f o o d c o n s u m p t i o n .

    T o c a l c u l a t e t h e a v e r a g e i s o t o p e r a t i o s o f f o o dg r o u p s , t h e a r i t h m e t i c m e a n i s i n a d e q u a t e , b e c a u s et h e C o r N e l e m e n t a l c o n t r i b u t i o n f r o m d i e t to h u m a nt is s ue is n o t u n i f o r m a m o n g f o o d g r o u p s . F o re x a m p l e , t h e N c o n t e n t o f oi l a n d s u g a r i s n e g l i g i b l e ,w h e r e a s t h e i r c o n s u m p t i o n i s s i g n i fi c a n t i n b o t hw e i g h t a n d c a lo r i es . T h e c o n s u m p t i o n r a t e o f e a c hf o o d i s i m p o r t a n t t o o b t a i n t h e r e l a t i v e w e i g h t i n g o fi s o t o p e s . M e a n w h i l e , t h e e f f ic i e n cy o f d i e t r e t e n t i o ni n h u m a n t i s s u e s i s n o t y e t c l e a r . I t m a y b e c o r r e c t

    t h a t p r o t e i n c o n s u m p t i o n r a t e s s h o u l d b e a m a j o rf a c t o r in u n d e r s t a n d i n g t h e N c o n t r i b u t i o n o f t h e d i e ta l t e r n a t i v e s , i n p r i n c i p a l , b e c a u s e N f r o m f o o d i sp r i m a r i l y f o u n d i n p r o t e i n s f r a c t io n s a n d m o s t N i nh u m a n t i ss u e s i s a l so f o u n d i n p r o t e i n s . H o w e v e r , f o rC , p r o t e i n i s n o t t h e o n l y p o t e n t i a l s o u r c e i n a n i m a lt i s s u e s .

    T h u s , i n o r d e r t o f in d r e a s o n a b l e f a c t o r s t o a v e r a g et h e i s o t o p e c o n t r i b u t i o n o f f o o d s , i t m a y b e n e c e s s a r yt o c o n s i d e r t w o c o m p o n e n t s , t h e c o n s u m p t i o n r a t ea n d t h e n u t r i t i o n a l fa c t o r o f e a c h f o o d g r o u p . T h r e ew e i g h t i n g f a c to r s w e r e c o n s i d e r e d i n t h e n u t r i t i o n a lf a c t o r : t h e u p t a k e r a t e s i n d i c a t e d b y f o o d w e i g h t ,e n e r g y a n d p r o t e i n . T a k i n g t h e s e c o m p o n e n t s a sw e i g h t i n g fa c t or s , t h e m e a n i s o t o pe c o m p o s i t i o n o f Ca n d N f o r a t y p i ca l J a p a n e s e c a n b e e s t i m a t e d . T h i s ise v a l u a t e d b y c o m p a r i n g t h e m w i t h t h e m e a n i s o t o p ec o m p o s i t i o n o f J a p a n e s e d i et s e s t im a t e d f r o m h a i ri s o t o p e o b s e r v a t i o n s ( T a b l e 4 ).

    A s n o t e d b e f o r e , t h e p r o t e i n c o n s u m p t i o n r a t e

    T a b l e 2 . T h e C a n d N i s o t o p e c o m p o s i t i o n s o f J a p a n e s e f o o d a v a i l a b l e in T o k y oF o o d (n ) 6 13 C 6 15 N I s o to p e c a t e g o ryCer ea l 2 -2 4 . 7 _+ 2 .1 4 .4 _+ 0 .3 C3 p lan tR i c e - 2 6 . 8 4 . 7W h e a t - 2 2 . 6 4 .1M il le t 2 -1 0 . 8 _+ 0 .4 1 .7 _+ 2 .8 C4 p lan t

    B u c k w h e a t fo x ta i l m i l l e t - 1 1 .2 4 . 5C o r n - 1 0 . 4 - 1 . 1L e g u m e 1 -2 5 . 5 + 0 . 8 2 . 0 4,- 0 . 5 N 2 f ixe rC o w p e a s - 2 6 . 2 2 . 4S o y b e a n - 2 4 . 7 1 .5Ve ge ta b le 4 -2 5 . 8 _+ 1 .6 3 .5_+ 1 .1 C3 p lan tW e l s h o n i o n - 2 7 . 0S w e e t p e p p e r - 2 9 . 0 4 . 6L e t t u c e - 2 5 . 4U d o - 2 5 . 0 2 . 4P o t a t o 1 - 2 5 . 4 0 . 2 C 3 p l a n tY a m - 2 5 . 4 0 .2Fru i t 2 -2 6 . 9 _+ 0 .4 2 .3 +_ 1 .6 C3 p lan tB a n a n a - 2 7 . 3 3 .9A p p l e - 2 6 . 5 0 .7M e a t 5 -1 6 . 3 _+ 1 . 7 6 . 4 _+ 1 . 0 L a n d a n im a lB e e f - 1 5 . 8 7 .7B e e f - 1 9 . 5 7 .0Por k - 15 .9 6 .6Ch icke n - 16 .1 6 .0Ch icke n - 14 .3 4 .9E g g 3 -1 4 . 2 + 0 . 6 6 . 1 _+ 1 .2 L a n d a n im a lY o l k - 1 3 . 5 7 . 7W hite - 14 .9 5 .7W h o le - 1 4 .3 4 . 9D a i r y 1 - 2 1 . 1 6 . 6 L a n d a n i m a lM i l k - 2 1 . 1 6 . 6Fish 4 -1 7 . 6 _+ 1 .1 12 .4 _+ 3 .8 M arin e an im alSkip jac k - 18 .2 10 .3T u n a - 1 6 . 8 1 9.0

    Pu ffer - 16.1 12. lC a r p - 1 8 . 9 9 . 4Sa l mo n - 17 .8 11 .4She l l f ish 4 -1 8 . 0 _+ 3 .1 8 .1 _+ 1 .1 M arin e an im alCla m - 17 .3 7 .2S h o r tn e e k e d c l a m - 1 5 .2 9 . 6S c a lp - 1 6 . 7 7 . 8C o r b s h e ll - 2 3 . 3 9 . 7 ( B r a c k i s h - w a t e r a n i m a l )

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    Reconstruction of human diet from (~13C and ~15N in contemporary hair 1512 0

    1 5

    1 0

    - 3 !

    C 3 PL A N TLEGUME

    I I- 3 0 - 2 5

    F

    MEAT,EG G& DAIRY

    I I I- 2 0 - 1 5 - 1 06 1 " a C */,,

    PLANT

    - 5

    Fie. 3. Carbo n and N isotope compositions of contemporary human food. O pen circles show the Japanes efood ob tain ed in this work; c losed circles were the data of U. S.A . food f rom SCHOELLER t a l . (1986).

    Table 3. Nutritional data of contemporary Japanese food groupsConsumption rat er(/d/person) Content (/g)

    Weight Calorie Protein Calorie ProteinFood Type* (g) (cal) (g) (cal) (g)RatioCalorie/protein(cal/g)

    Cerea l 309 986 21 3.2 0.068Rice C3 216 763 15 3.53 0.068Barley C3 0.6 2.1 0.1 3.5 0.167Whe at C3 91.3 217.1 6.3 2.38 0.069Millet C4 0.9 3.7 0.1 4.1 0.111Legume N2 66.6 94.1 6.5 1.41 0.098Vegeta ble C3 73.9 18.7 1.5 0.25 0.02Gre en vegetable C3 178.1 42.7 2.1 0.24 0.012Oil 17.7 144.2 0.1 8.15 0.006Sugar C3/C4 11.2 42.4 0.0 3.78 0.000Potato C3 63.2 51.0 1.1 0.81 0.017Fruit C3 140.6 71.8 0.7 0.51 0.005Meat LA 71.7 167.6 12.7 2.34 0.177Beef LA 16.2 33.5 3.1 2.07 0.191Pork LA 27.5 73.5 4.7 2.67 0.171Poul try LA 16.9 34.9 3.2 2.07 0.189Whale MA 0.7 0.9 0.2 1.29 0.286Other LA 10.5 24.9 1.5 2.37 0.143Egg LA 40.3 65.2 5.0 1.62 0.124Dairy LA 116.7 81.8 3.8 3.1 0.033Milk LA 108.0 63.7 3.1 0.59 0.029Other LA 8.7 18.1 3.7 2.08 0.425Fish MA 90.0 141.4 18.7 1.57 0.208

    47.052.321.034.53714.512.520.3

    46.4102.613.210.815.610.94.516.613.021.520.34.97.6

    * C3, C3 plan t; CA, C4 plant; N2, di-nitrogen fixing plant ; LM, te rrestr ial animal; MA, marine an imal.tD at a from MlNIs ~v OF HEALTHA N D W E L F A R E , JAPAN (1987).

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    152 M . M i n a g a w aTab le 4 . Carb o n an d N i so to p e co n t r i b u t io n s o f av erag e Jap an ese fo o d s

    Co n t r i b u t i o n (% )* We ig h t ed 13C W eighted 15N13C 6 1 5 N W e i g h t C a l o r ie P r o t e in W e i g h t C a l o r ie P r o t e in W e i g h t C a l o r ie P r o te i n

    Cerea l -2 4 .7 4 .7 2 6 .2 5 1 .6 2 8 .6 -6 . 5 -1 2 .7 -7 .1 1 .2 2 .4 1 .3Mi l l e t - 1 0 .8 1 .7 0 .1 0 .2 0 .1 -0 .0 -0 .0 -0 .0 0 .0 0 .0 0 .0Leg u m e -2 5 .5 2 .0 5 .7 4 .9 8 .9 -1 .5 -1 .2 -2 .3 0 .1 0 .1 0 .2Veg e t ab l e -2 6 .6 3 .5 2 1 .4 3 .2 4 .9 -5 .7 -0 .9 -1 . 3 0 .7 0 .1 0 .2P o t a to -2 5 .4 0 .2 5.4 2 .7 1 .5 -1 .4 -0 .7 -0 .4 0 .0 0 .0 0 .0Oi l+ -2 6 .9 - - 1 .5 7 .5 0 .1 -0 .4 -2 .0 - - 0 .0 0 .0 - -S u g ar - 1 2 . 0 - - 0 . 9 2 .2 0 . 0 - 0 . i - 0 . 3 - - 0 .0 0 .0 - -F ru i t - 2 6 .9 2 .3 1 1 .9 3 .8 1 .0 -3 .2 -1 .0 -0 .3 0 .3 0 .1 0 .0Mea t -1 6 .3 6 .4 6.1 8 .8 1 7.3 -1 .0 -1 .4 -2 .8 0 .4 0 .6 1 .1Eg g -1 4 .2 6 .1 3 .4 3 .4 6 .8 -0 .5 -0 .5 -1 .0 0 .2 0 .2 0 .4Dai ry -2 1 .1 6 .6 9 .9 4 .3 5 .2 -2 .1 -0 .9 -1 . 1 0 .7 0 .3 0 .3F i sh -1 7 .8 1 0 .7 7 .6 7 .4 2 5 .5 -1 .4 -1 .3 -4 .5 0 .8 0 .8 2 .7To ta l 1 00 1 00 1 00 -2 3 .6 -2 3 .0 -2 0 .7 4 .4 4 .6 6 .3

    * Ca l cu l a t ed f ro m d a t a i n Tab l e 2 .t l so to p e d a t a w ere q u o t ed f ro m NAKAM URA t al. (1982).

    f r o m e a c h f o o d g r o u p s h o u l d b e u s e d a s th e w e i g h t i n gf a c t o r f o r t h e i s o t o p e c o n t r i b u t io n s o f N . A s s h o w n i nT a b l e 4 , t h e 6 ~ 3 C e s t i m a t e b a s e d o n t h e p r o t e i nf r a c t i o n i s 6 .3 % 0 f o r t h e g r o s s J a p a n e s e d i e t . T h i sv a l u e i s s u p p o r t e d f o r th e m e a n d i e t , b e c a u s e t h e 1 5Ne n r i c h m e n t f r o m d i e t t o h u m a n h a i r h a s b e e nr e p o r t e d t o b e - 4 . 3 % o w h i c h w il l g i v e a h u m a n h a i r6 1 5 N n e a r t h e o b s e r v e d h a i r i s o t o p e d a t a ( 1 0 . 3 +0.5%0).

    O t h e r p o s s i b l e f a c t o r s s u c h a s f o o d w e i g h t a n dc a l o r ie c o n s u m p t i o n r a t e w e r e a l so e x a m i n e d a ss h o w n i n T a b l e 4 , a n d g a v e 4 . 4 a n d 4 .6 % 0 a s t h e m e a ni s o t o p e r a t i o f o r g r o s s N u p t a k e , r e s p e c t i v e l y . T h i sr a n g e i s t o o s m a l l to i n t e r p r e t t h e h u m a n 6 1S N as d u et o th e i s o t o p e e n r i c h m e n t o n f e e d i n g p ro c e s s . H e n c ei t w o u l d b e m o r e r e l i a b le t o c o n s i d e r t h e p r o t e i nf r a c t i o n a s t h e m a i n f a c t o r c o n t r o l l i n g 6 ~s N i n h u m a nf o o d w e b s , a n d n o t c a l o r i e c o n t e n t o r f o o d w e i g h t .

    T h e C s o u r c e s f o r h u m a n t is s u es a r e n o t a s s i m p l ea s i s t h e c a s e f o r N . T h e C c h a i n s o f s o m e a m i n o a c i d sm a y h a v e f o r m e d t h r o u gh t h e T C A c y c le , a nd c o n s e -q u e n t l y p r o d u c e a m i n o a c id s o f d i f f e r e n t i s o to p ec o m p o s i t i o n s t h a n f o u n d i n p r o t e i n ( GA L IM O V , 1 9 85 ) .H e n c e d i e t a ry p r o t e i n m a y n o t b e th e o n l y s o u r c et h a t c a n a l t e r t h e 6 J 3 C o f a n i m a l t i s s u e s , a l t h o u g h t h ec o n t r i b u t i o n o f C f r o m c a r b o h y d r a t e a n d l i p id to t h ep r o t ei n c o m p o n e n t is u n k n o w n .

    F o o d w e i g h t i n d ic a t e s t h e a m o u n t o f f o o d i n th ep a r t i c u la r d i e ts , i n c l u d i n g e v e r y m a c r o n u t r i e n t .C a l o r i c c o n t e n t c a n b e a n i n d i c a t o r o f u p t a k e o fe n e r g y t h a t s h o u l d b e d i r e c t l y r e l a t e d t o r e s p i r a t i o ni n t h e T C A c y c le . R e s u l t s o b t a i n e d w i t h t h e s e d if f e r -e n t f a c t o r s a r e s h o w n i n T a b l e 4 . T h e 6 13 C v a l u e s o ft h e p r o p o s e d J a p a n e s e d i e t , e s t i m a t e d u s i n g t h e s ed i f f e r e n t f a c t o r s w e r e - 2 3 . 6 , - 2 3 . 0 a n d - 2 0 . 7 % o ,b a s e d o n w e i g h t , c a lo r i c a n d p r o t e i n c o n t e n t , r e -s p e c t i ve l y . I n p r e v i o u s s tu d i e s , t h e a p p a r e n t e n r i c h -m e n t f a c t o r s fo r 61 3C in m o d e r n h u m a n s w e r er e p o r t e d i n t h e r a n g e f r o m 1 . 4 t o 2.0 % o (ScHOELLER tal . , 1 9 8 6 ; M I N A G A W A e t a l . , 1 9 8 6 ) . T a k i n g t h e s e

    b i a s i n g f a c t o r s in t o a c c o u n t , t h e e s t i m a t i o n b a s e d o nt h e p r o t e i n c o n t r i b u t i o n s e e m s t o b e t h e m o s t r e a s o n -a b l e , p a r t ic u l a r ly w h e n c o m p a r e d w i t h o b s e r v e d h a i r6 13 C v a l u e s o f - 1 8 . 4 % o .

    T h u s , t h e f o o d i s o t o p e d a t a s u g g e s t t h a t t h e i s o -t o p e c o m p o s i t i o n o f t h e J a p a n e s e d i e t s h o u l d b e- 2 0 . 7 a n d 6 .3 %o f o r 6 ~3 C a n d 6 1 5 N , r e s p e c t i v e l y .T h e s e e s t i m a t e s w e r e c o n s i s t e n t w i t h t h e i s o t o p ec o m p o s i t i o n o f h u m a n h a i r , w h e n t h e i s o t o p e f ra c -t i o n a t i o n b e t w e e n d i e t a n d h a i r w e r e t a k e n i n t oa c c o u n t .

    M C s i m u l a t i o n t o r e c o n s tr u c t J a p a n e s e d i e t a r yp a t te r n f r o m h u m a n i s o to p e d a ta

    T h e s t o c ha s t ic m o d e l w a s u s e d t o e s t im a t e d i e t a r yp a t t e r n s f r o m t h e 6 13 C a n d 6 1 5 N o b s e r v a t i o n s o fJ a p a n e s e h a i r . A s d e s c r i b e d b e f o r e , t h e 6 13 C a n d6 15 N v a l u e s o f t h e d i e t c a n b e e s t i m a t e d b y s u b t r a c t -i n g t h e i s o t o p e f r a c t i o n a t i o n f a c t o r s d u e t o f e e d i n g .F i v e f o o d s o u r c e s w e r e s e l e c t e d o n t h e b a si s o f f o o di s o t o p e d a t a a n d t h e 6 13 C a n d 6 15 N v a l u e s o f e a c hf o o d g r o u p w e r e c o m p i l e d f ro m m e a s u r e d d a t a a ss h o w n i n T a b l e 5 .

    B a r s d r a w n a s li g h t t o n e i n F ig . 4 s h o w t h e r e s u l t o ft h e M C s i m u l a t i o n u s i n g t h e i s o t o p e d a t a f r o mm o d e r n J a p a n e s e . I n t h e s i m u l a t io n , 2 0 0 0 p o s s ib l ec o m b i n a t i o n s o f f iv e f o o d g r o u p s a r e r e c o r d e d , f r o m> 2 3 2 , 0 0 0 t r i a l s. T h e f r e q u e n c y p a t t e r n o f p r o -p o r t i o n s w h i c h c a n r e p r o d u c e t h e s a m e i s o t o p e c o m -p o s i t i o n a s th e d i e t i s s h o w n b y o p e n b a r s i n th ef i g u re . B e c a u s e t h e i s o t o p e d a t a o n d i e t w a s o r i g -i n a ll y e s t i m a t e d f r o m i s o t o p i c d a t a o n h u m a n h a i r( = p r o t e i n ) , t h is d ie t a r y p a t t e r n s h o w s t h e p r o t e i nc o n t r i b u t i o n f r o m f o o d g r o u p s . T h e b a r h e i g h t i n t h eg r a p h s h o w s t h e n u m b e r o f c a s e s t h a t w e r e a c c e p t e da t e a c h p r o t e i n d e p e n d e n c e l e v e l ( p r e s e n t e d i n p e rc e n t ) . T h e h i s t o g r a m p r e s e n t e d b y t h e l i g h t- t o n e db a r s s h o w s t h e f r e q u e n c y p a t t e r n t h a t s a t i s f i e s t h e

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    Reconstruction of human diet from blJC and bISN in contemporary hair 153

    l--Z

    o 9(.3

    LU

    (.9ii i

    100

    5 0

    0

    100

    5 0

    50 100

    h

    isotope condition of the hair. The p robable contri-bution of fish was restricted to a narrow range from12.1 to 42,0% of the total protein uptake, and thecontributions of C4 plants and leguminous plantswere estimated to be

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    154 M. MinagawaTable 5. Isotope and nutritional data used for the MC simulation for the contemporaryJapanese food

    613C 615N Protein content* Energy content*Group (%0) (%0) (g/g) (kcal/100 g)C3-plant -25 3.5 0.11 3.4N2-fixer -25 1.0 0.1 1.41(leguminous plant)C4-plant - 11 3.0 0.11 4.11Land animal - 15 6.0 0.14 1.76(meat, egg etc.)Marine animal - 18 13.0 0.21 1.65(~sh)*Mean values weighted by real consumption statistics (see text).

    72 kcal/g. The mixing proportions estimated in theprevious MC simulation include some cases outsidethis range. It is therefore reasonable to omit theseimprobable combinatio ns from the initial results ofthe MC simulation.

    The protein and energy content of contemporaryfoods have been reported in the Standard Tables ofFood Composition in Japan (RESOURCECOUNCILS C I E N C E A N D T E C H N O L O G Y A G E N C Y , 1982). Usingthese data, the protein and energy conten t was calcu-lated for five food groups (Table 5). The protein orcaloric content of food varies within the same foodcategory. Therefore, to calculate the representativecontent of these nutritional components for eachgroup, only the edible parts were used by weightingthe actual co nsumpti on rate of maj or foods in 1985.For example, milk and bee f are classified in the sameanimal food group, b ut their p rotein con tent is differ-ent; the protein content is 19 g/100 g for beef, but2.9 g/100 g for raw milk. On the o ther hand , based oncurrent statistics, the protein dependence on milkand beef is only 15% each of all animal products

    ingested. To estimate the average protein conten t ofall animal food, the protein c ontent of beef an d milkwas weighted by 0.15. By calcula ting other animalfoods, using similar weighting corrections, the meanprotein co ntent for land animal food is estimated tobe 13.9 g/100 g. In the same manner, the meanprotein conten t and m ean caloric conten t of all otherfood groups were calculated, based on food n utritiondata and the actual consumption rate of food items(Table 5). The energy/protein ratio of each foodgroup was calcula ted using these results.

    Applying these energy/protein ratios for foodgroups to the mixing proportion obtain ed by the MCsimulation, the energy/pro tein ratios for each combi-nation were calculated for all recorded proportions.The densely toned bars in Fig. 5 show the frequencydistribution of proportions whose energy/proteinratio are in the range for current Japanese consump-tion (12-72 kcal/g). Hence, the distrib ution indicatesthe possible range of protein depend ence which cansatisfy both iso tope condit ions (~3C and 15N content)and the nutritional condition (protein and energy

    C 3 p l a n t

    L e g u m e

    C 4 p l a n t

    P r o t e i n c o n t r i b u t io n ( * ,4 , )0 2 0 4 0 6 0 8 0

    I

    m

    - - - . r ~ m

    M e a t I I J

    F i s h ~ [ ' - - ~ Ai t i I I t

    : M H W r e p o r t ( 19 88 )F A O r e p o r t ( 19 84 )

    F1G. 5. Box hinge plot of dietary pattern as protein contribution obtained by the MC simulation forJapanese hair.

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    Reconstruction of human diet from 613Cand 615N in contemporary hair 155Table 6. Classification of the isotope category of food groups and statisticresults of food consumption rate as a protein source for the average Japanese

    Annual consumptionAnnual supply of protein (%)of protein (%) (MINISTRYF HEALTH IsotopeFood FAO (1983) ANn WELFARE,JAPAN 1987) categoryCereal 24.2 21.0 C3 plantMillet 0.6 0.1 C4 plantPotato 1.0 1.1 C3 plantLegume 9.9 6.5 C3 N2 fixerFruit 0.9 0.7 C3 plantVegetable 4.0 3.6 C3 plantMeat 10.5 12.7 Land animalEggs 5.2 5.0 Land animalMilk 5.5 3.8 Land animalFish 25.2 18.7 Marine animalOther 6.1 - -

    demand ) for mixed diets. This should provide morereliable reconstructions of the contemporary Japa-nese dietary pattern.

    Comparison between MC simulation result and foodconsumption statisticsThe average food cons umption for Japanese is

    reported in two different sources: a nationwideobservation based on the questionnaires adminis-tered by the MINISTRY OF HEALTHAND WELFARE,JAPAN (1987) (MH W), and the food balance sheetsrepo rt ed by the FOOD AND AGRICULTUREORGANIZ-ATIONOF THE UNITED NATIONS(1984) (FA D). Tabl e 6shows protein consumption reported by MHW andprotein supply reported by FAD of 11 food groupsbased on the food statistic report space (1979-1981).Because the FAD data were estimated from foodstatistics based on the annual balance of consump-tion, stock, import and export, the supply rate doesnot completely indicate the real consumption offood. On the other hand, the food consumptionreport from the MHW is based on a questionnairegiven to abo ut 20,000 people from 300 areas through-out Japan and represents an actual food consump-tion. However, the data were gathered during threedays in November, every two years. This may causeerrors due to seasonal differences in food cons ump-tion. Thus, b oth estimates may not accurately reportthe actual con sumptio n, but only reflect the generalpattern. Further, these reports give poor infor mationabout the rate of C4 plant use, because only milletand corn are distinguished as being C4. It is notknown if the vegetables and fruit category includesother C4 plants. This also applies to the category of"other foods" which includes cooked foods (Table 6).Hence, the food statistics tend to give an underesti-mation for the C4 plant contributio n in the contem-porary diet. To compare these observational datawith the estimation by the isotope-MC method, anisotopic category was classified by the major fooditem in each group.

    The food consu mption patterns obtained from theMC simulation are shown in a box-hinge graph (Fig.5). The box and the vertical line in the figure indicatethe range from the 25th to the 75th percentile ofaccepted cases and the median of the protein depen-dence, respectively. The solid triangles represent themean value from the MHW observations, and theprotein supply rate by the F AO report were figuredby open triangles. These results clearly show that allstatistical data (MHW and FAO data) fall in therange indicated by the MC simulation. Further, theconsumption rate of all food groups, except C4plants, are distributed between the 25th and the 75thpercentile. In particular, the MC simulation resultagreed well with FAO and MHW data for fish, C3plants, and legumes.

    The estimate based on the modern food modelsuggested a p rotein contrib ution of C4 plant possiblyranging from 0 to 25% for conte mporary Japanese,whereas the M HW data suggests a contribution of1% or less. As noted before, the MHW data on C4plants may give a minimu m estimate of the actual C4contribution.

    Estimates of food group consump tion from differ-ent methods are listed in Table 7. For the MCmethod, the medi an values optmized by the energy/protein ratio agree well with the observations of theMHW and FAO, except for C4 plants. In addition,the estimate of the amo unt of animal food in the dietwas also consistent with the MH W and FA O resultsfor both protein and energy.

    Thus, it has been demonstrate d that the MC simu-lation has produced quite reasonable estimates of theproportional protein consumption rate in the modernJapanese food web.

    Implications of frequency pattern in a stochasticsimulationThe frequency pattern obtai ned by the MC method

    represents the range of the proportio n of each food

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    156 M. Minagawasource in the total diet. The frequency patterns ofeach food group show different distributions; someare symmetric with a single peak, others not sym-metric (Fig. 4). Are these distributions related to theactual frequency pattern of human feeding? To ob-tain the frequency pattern it was postulated thatthousands of people ate mixed diets with randomproportions of food groups. Hence, it is unlikely thatthe generated frequency pattern reflects the realfrequency pattern of feeding, because normally ahuman does not choose his diet randomly. Variousfactors, such as economic, religious and culturalreasons may influence dietary selection and mayconstrain it in a particular direction. If the exa minedhuman po pulatio n does not have strong preferencesin food choice, a stochastic model will provide afrequency pattern indicating the distribution of indi-vidual feeding patterns in the pop ulation. Such a casemay be expected for pop ulations facing food short-age, for ins tance, for wild animals in natura l field andprobably for prehistoric human populations whodepended o n natural food.

    Figure 5 shows that the mean or median value ofthe c ontribu tions of C3 plants, legumes and fish isclose to each observed contribut ion reported in FA Oand MHW statistics, but it is different for meat (landanimal food). One of the possible reasons for thisdisagreement may be no n-ra ndom food selection. Areason for the differing MC result is that the increas-ing palatability of meat products might affect currentJapanese feeding habits. This tendency agrees withthe fact that the c onsump tion rate of meat and milkincreased annually following World War II(MINISTRY OF HEALTH AND W ELFARE, JAPAN, 1987).

    Evaluation o f result obtained in the modelThe average uptake rate of protein for the Japa-

    nese was -79 g/d based on the national observation

    in 198 4 (MINISTRY OF HEALTH AND WELFARE, JAPAN,1987). Assuming that the dietary pattern shown inFig. 5 is true, this protein uptake rate introduces1706 kcal/d per person, because the mean energy/protein ratio is to be 21.6 based on the nutrientcondition of the mean Japanese diet presented inTable 3. This energy uptake rate is -3 00 kcal higherthan the actual observed daily energy uptake.

    Still not considered is the contribu tion of oil fat andsugar in the dietary model, because their proteincon tent is effectively zero. These foods are no rmallyutilized as energy sources, but do not participate inthe C and N metabolism of tissues composed ofprotein. Con sequently, these carbohydrate and lipidcompounds should not affect the isotope compo-sitions of human tissues, such as hair, muscle andbone. Further, to obtain the isotope composition ofprotein, the lipid fraction in hair and food sampleswas extracted prior to isotope analysis. Thus thedietary proportion estimated by the MC simulationrepresents the proportion based on protein. Recon-struction of the energy balance was based mainly onthe contr ibution of protein. The c ontrib ution ofcarbohydrate is not completely included and fat, oiland sugar were excluded in the calculation. Thus, thepresent model should estimate the min imum amou ntof energy. The c onsumpt ion rate of such C sourcesfor average Japanese were reported to be 144.2 and42.4 kcal/d, respectively. Adding these to the totalenergy uptake estimated from the protein intakeresults in an energy uptake for average Japanese of1900 kcal. This is still -100 kcal less than the ob-served average energy consumption rate. The dis-agreement between these is partly due to the mis-match between the actual feeding pattern thatsimulated the model, which was based on the r andomfood selection. Real food selection by contemporaryJapanese may tend toward food yielding greaterenergy when compared to a diet made up throughrandom selection.

    Table 7. Comparison of Japanese food consumption (% in protein) betweenthe MC model and the statistical reportsMC method (mean)

    After nutrition MHW FAOFood group Initial consideration re port * report ~-C3 plant (%) 23.6 32.0 33.4 33.2Legume (%) 18.4 11.3 8.5 12.0C4 plant (%) 11.0 13.8 0.1 0.8Land animal (%) 19.3 16.2 27.9 23.9Fish (%) 27.7 26.7 23.7 28.4Other - - - - 7.0 1.7Calorie/protein ratio 21.6 26.4 32.8(18.0-31.4)Animal protein (%) 40.5 50.8 51.1Animal energy (%) 17.8 22.3 19.3

    *National nutrition observation report, 1984from the M|NISTRYOF HEALTHAND WELFARE,JAPAN (1987).t 1979--81 Food balance sheets report by FOODAND AGRICULTUREORGANIZ-ATIONOF THE UNITED NATIONS(1984).

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    Reconstruction of human diet from ~13Cand 615N n contemporary hair 157Limitat ion o f reconstruct ion

    The isotopic conditions of all source materials caninfluence the limitation of reconstrucion of a dietmixture. If the isotope compositions of sourcematerials are sufficiently differen t, it is expected thatthe stochastic method can specify the pro bable distri-bution of the diet compon ents within a reliable range.The isotope differences among source groups shouldbe larger than the isotope variations within each foodgroup.

    In addition, if a source material has the sameisotope composition as can arise with mixtures ofother food sources, the frequency distributions canbe affected. This might be ha ppening to the estimatesfor land animal use in the Japanese food web modelpresented above. Recent cattle and poultry in Japanare fed on a mixed forage of corn and millet. Becauseof this C4 plant con tributio n and the 15N enrichm entassociated with feeding, a mixture of marine food, C4plants and C3 plants can produce a quasi food mix-ture which has the same isotope composition as landanimals. This quasi mixing value might fall in thefrequency distribution obtained by the MC simu-lation. This may be a reaso n for the disagree ment onthe contribution of land animals in the diet as esti-mated by the MC method and the MHW and FAOstastical data.

    The number of sources chosen for a model is alsoan important factor affecting the reconstruction. Ifthe model includes an improbable source, erron eousresults will be obtain ed. In order to obtai n accurateestimations, the nu mbe r of sources should be mini-mized. On the other hand, it is more difficult todetermine the mean value of one group when it iscombined with other food groups to minimizesources, because it requires knowledge of the pro-portion of these c ombine d food sources in advance.In most cases, this information is what is needed.Thus, the deter minati on of source groups should bedone carefully.

    Isotope data on diet are still limited. Further analy-sis of diet should be continued to revise the isotopecomposition of diet in future. Further, the dietarymodel involves several hypothetical concepts on thebehavior of isotopes, which are partly related to thebiochemical and physiological problems with C andN isotopes. In particular, the isotope fractionation ofC and N must be confirmed. Regardless of what isrevealed in future work, this approach can be appliedas a general method to find the probable range ofmixing proportions of known sources.

    S U M M A R Y

    A new data analysis method for estimating mixingproportions of multiple food sources from a doubleisotope tracer analysis has been proposed. If themixing is governed by stochastic processes and the

    law of conservation of mass holds for stable isotopesin the system, the presen t metho d is useful for analyz-ing the range of possible mixing proportions ofknown diet compon ents. The tracer used for analysisis not limited to only stable isotopes, but otherchemical components including radioactive tracersmay be applicable. The number of sources must beidentified prior to the stochastic analysis, and theconcentration of tracers in each source must bemeasured. The lack of any of these data makesreasonable estimates unlikely. Additional infor-mation characterizing the system, such as energybalance, can be used to increase the degree of cer-tainty of the results. If the pro portion of any sourcecan be estimated by other methods, it helps to de-crease the num ber of unkn own sources. Because thenum ber of sources includ ed in a model is critical todetermin ing the range of possible proportions accu-rately, it is important to minimize the number ofunknown sources which are analyzed. Unnecessarychoices Of sources makes the estimate vague.

    The human food web model may not be the mostappropriate application of this physical mixingmodel, because the food selection by humans maynot always be performed in a stochastic manner.Moreover, the consistency of the isotope fraction-ation for each food source must be ascertained inmore detail, whereas it was assumed to be constantfor the foods examined in this study. In spite of thesedisadvantages, the human food web is still uniquebecause the food consump tion data have bee nrecorded in detail. The present estimate showedgood consistency in comparison to the publisheddata.

    Based on the hypothesis that the C and N isotopecompositions of hu man hair are essentially controlledby the mixing proportions of diets, and of theirisotope compositions, it will be possible to reproducethe huma n isotope composition. If this mixing pro-cess can be described in an experimental model, itmay be possible to judge if a proportion of mixedfoods can be shown to agree with observed humanisotope compositions. In this work, a Monte Carlosimulation was designed and employed for perform-ing such an e xperiment.

    The C and N isotope compositions of contempor-ary Japanese scalp hair were measured, and gavevalues of - 18.1 0.4 and 10.4 + 0.4%0 on average for613C and 615N, respectively. The MC s imulat ionapplied to these isotope data has generated a possiblefood dependence pattern. Other possible combi-nations of foods were selected by adding furtherconditions, on energy/p rotein uptake ratios in addi-tion to the isotope conditions. It has been shown thatthe estimated proportions of five food groups agreewell with the observed food co nsumptio n rate basedon other reported data.In conclusion, the present method is useful foranalyzing a dietary mixing model, involving multi-sources of foods, using a small numb er of chemical or

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    1 58 M . M i n a g a w a

    i s o t o p e t r a c e r s . T h i s is a p p l i c a b l e t o p r o b l e m s i n v o l v -i n g s i m p l e m i x i n g p r o c e s s e s o r s t o c h a s t ic p h e n o m -e n a . H u m a n f e e d i n g b e h a v i o r s e em s t o b e d e s c r i b e db y a st o c h a s ti c p r o c e ss , e v e n f o r m o d e r n h u m a n s .H e n c e , t h i s a n a l y s i s s h o u l d b e v e r y u s e f u l f o r e s t i m a t -i n g t h e f e e d i n g h a b i t s o f p r e h is t o r i c h u m a n s , w h ow o u l d d e p e n d o n n a t u r a l r e s o u r c e s w i t h l es s s e le c -t iv i ty t h a n m o d e r n p e o p l e , a n d u s i n g C a n d N i s o t o p ea n a l y s is o f f o s si l b o n e t o p r o v i d e t h e d a t a . T h ef e e d i n g e c o l o g y o f w i l d a n i m a l s a n d f i s h w h o s e d i e t isc o m p l i c a t e d b y m a n y f o o d s o u r c e s s h o u l d a ls o b ee l u c i d a t e d b y t h i s d i e t a r y a n a l y s is .A c k n o w l e d g e m e n t s - - T h e a u t h o r t h a n k s a l l v o l u n t e e r s fo rp r o v i d i n g h a i r s a m p l e s, a n d D r B . C h i s h o l m o f th e D e p a r t -m e n t o f A n t h r o p o l o g y i n t h e U n i v e r s i t y o f B r i t i sh C o l u m b i aa n d D r E . W a d a o f M i t s u b i s h i K a s e i I n s t i t u t e o f L i f eS c ie n c e s fo r t h e i r c r i t i c a l r e a d in g s to im p ro v e th i s a r t i c l e .M r s K y o k o K a r a s a w a - T s u r u a n d M i s s Y u k o K a b a y a i nMi t s u b i s h i K a s e i In s t i t u t e o f L i fe S c ie n c e s h e lp e d w i thc h e m i c a l p r e p a r a t i o n s o f h a i r a n d f o o d s a m p l e s . T h e a u t h o ri s g re a t ly in d e b t e d to r e v ie w e rs , e s p e c ia l ly P ro fe s s o r H . R .K ro u s e , fo r t h e i r c r i t i c a l c o m m e n t s . T h i s w o rk w a s p a r t lys u p p o r t e d b y t h e G r a n t - i n - A i d f o r S c ie n ti f ic R e s e a r c h o nP r i o r it y A r e a s , M i n i s t r y o f E d u c a t i o n , S c i e nc e a n d C u l t u r e .E d i to r ia l h a n d l in g : P. Fr i tz .

    R E F E R E N C E S

    CHISHOLM B., NELSON D. E. an d SCHWARCZ H. P. (1983)S ta b le c a rb o n i s o to p e r a t io s a s a m e a s u re o f m a r in ev e r s u s t e r r e s t r i a l p ro te in in a n c ie n t d i e t s . Sc ience 216 ,1131-1132 .C RA IG H . (1 9 5 7 ) I s o to p ic s t a n d a rd s fo r c a rb o n a n d o x y g e na n d c o r re c t io n f a c to r s fo r m a s s - s p e c t ro m e t r i c a n a ly s i s o fc a rb o n d io x id e . G e o c h i m . c o s m o c h i m . A c t a 12 , 133-149 ,DENIRO M. J . and EPSTEIN S . (1978 ) C arb on iso top ic ev i-d e n c e fo r d i f f e re n t f e e d in g p a t t e rn s in tw o h y ra x s p e c ie so c c u p y in g th e s a m e h a b i t a t . Sc ience 2 0 1 , 9 0 6 - 9 0 8 .FARNSWORTH P ., BRADYJ. E . , DENIRO M . J . an d MACNEISHR . S . (1 9 8 5 ) A re -e v a lu a t io n o f t h e i s o to p ic a n d a rc h a e o -lo g ic a l r e c o n s t ru c t io n s o f d i e t i n th e T e h u a c a n v a l l e y .A m . A n t i q . 50 , 102-116 .FOOD AND AGRICULTURE ORGANIZATION OF THE UNITE DNATIONS (1984) Food Ba lanc e Shee ts , 1979-81 avera ge ,R o m e .GALIMOV E. M . (1985) T h e B io lo g ic a l F r a c t io n a t io n o fIso topes . A c a d e m i c P r e ss .

    H O aN E R H . (1 9 8 6 ) I s o to p e e f f e c t s o f n i t ro g e n in th e s o i l a n db i o s p h e r e . I n H a n d b o o k o f E n v i r o n m e n t a l I s o t o p e G e o -c h e m is t r y (ed s P. FRITZ an d J . CH. FONTES), pp. 361--426.E l s e v ie r .LYON T. D. B. an d BAXTER M. S. (1 9 7 8 ) S ta b le c a rb o ni s o to p e s in h u m a n t i s s u e s. N a tu r e 273 , 750-751 .MA RIO TT I A . (1 9 8 3 ) A t m o s p h e r i c n i t r o g e n i s a r e l i a b les t a n d a r d f o r n a t u r a l 6 1 5 N a b u n d a n c e m e a s u r e m e n t s .N a tu r e 3 0 3 , 6 8 5 - 6 8 7 .MINAGAWA M., KARASAWA K. and KABAYA Y. (1 9 8 6 ) C a r -b o n a n d n i t r o g e n i s o t o p e a b u n d a n c e s i n h u m a n f e e d i n ge c o s y s t e m . G e o c h e m is t r y 2 0 , 7 9 -8 8 ( in J a p a n e s e w i thE n g l i s h a b s t r a c t ).MINAGAWA M. an d WADA E. (1 9 8 4 ) S te p w is e e n r i c h m e n t so f 1 5 N a lo n g fo o d c h a in s ; fu r th e r e v id e n c e a n d th e r e -l a t io n b e tw e e n 1 5 N a n d a n im a l a g e . G e o c h i m , c o s m o -c h im . A c ta 48, 1135-1140.MINAGAWA M. , WINTER D. A. an d KAPLAN I. R. (1984)C o m p a r i s o n o f K j e l d h a l a n d c o m b u s t i o n m e t h o d f o rm e a s u r e m e n t o f n i t r o g e n i s o to p e c o m p o s i t i o n i n n a t u r a lo r g a n i c m a t t e r s . A n a l . C h e m . 56, 1859-1861.MINISTRY OF HEALTH AND WELFARE, JAPAN (1987) K o k u m i ne iy o u n o g e n jy o u (N a t io n a l S ta tu s o f N u t r i t i o n ) , pp . 148 .D a i i t i -s y u p p a n T o k y o ( i n J a p a n e s e ) .NAKAMURA K. , SCHOELLER D. A . , WINKLER F, J. andSCHMIDTH H.-L . (1982) G eog rap h ica l va r ia t ion s in thec a r b o n i s o t o p e c o m p o s i t i o n o f t h e d i e t a n d h a i r i n c o n -t e m p o r a r y m a n . B io m e d . M a s s S p e c . 9 , 390-394 .PETERSON B. J . a nd FRY B. (1987 ) Sta ble i sot ope s in ecosy s-t e m s tu d ie s . A n n . R e v . E c o l . S y s t . 1 8 1 , 2 9 3 -3 2 0 .RESOURCE COUNCIL SCIENCE AND TECHNOLOGY AGENCY(1982) S ta n d a r d T a b le s o f F o o d C o m p o s i t io n in J a p a n , p .7 02 . O k u r a s h o u - I n s a t u k y o k u , T o k y o ( i n J a p a n e s e ) .ROUNICK J. S. an d WINTERBOURN M. J. (1 9 8 6 ) S ta b le c a rb o ni s o to p e s a n d c a rb o n f lo w in e c o s y s te m s . B io S c i . 3 6 , 1 7 1 -

    177.SCHOELLER D. A., MINAGAWAM. , SEATER R. an d KAPLAN I.R . (1 9 8 6 ) S ta b le i s o to p e s o f c a rb o n , n i t ro g e n a n d h y d ro -g e n i n th e c o n t e m p o r a r y N o r t h A m e r i c a n h u m a n f o o dw e b . E c o l . F o o d N u t r i t i o n 18, 159-170.SCHOENINGER M. J. , DENIRO M. J. and TAUBER H. (1983)1 5N /1 4N ra t io s o f b o n e c o l l a g e n r e f l e c t m a r in e a n d t e r r e s -t r i al c o m p o n e n t s o f p r e h i s t o r i c h u m a n d i e t. Sc ience 220 ,1381-1383.SMITH B. N. and EPSTEIN S . (1971) Tw o ca tego ries o f

    13Cl12C r a t io s fo r h ig h e r p l a n t s . P la n t P h y s io l. 4 7 , 3 8 0 -384.WAK IMOTO K . (1 9 7 6 ) A lg o r i th m fo r g e n e ra t in g a r a n d o mv e c t o r w i t h r e s t r ic t e d i n t e g e r c o m p o n e n t s a n d t h e i r e x -t e n s io n to m a t r ix . E s s a y s in P ro b a b i l i t y a n d S ta t i s t i cs ,O g a w a .WHELAN T., SAKETT W. an d BENEDICT C. (1 9 7 3 ) E n z y m a t i cf r a c t i o n a ti o n o f c a r b o n i s o to p e s b y p h o s p h o e n o l p y r u v a t ec a rb o x y la s e . P la n t P h y s io l . 51, 1051-1054.