(The FASEB Journal. 1999;13:559-562.)
© 1999 FASEB
The Ice Man's diet as reflected by the stable nitrogen and carbon isotopic composition of his hair
S. A. MACKOa ,
G. LUBECb , 1 ,
M. TESCHLER-NICOLAc ,
V. ANDRUSEVICHd and
M. H. ENGELd
a Department of Environmental Sciences, University of Virginia, Charlottesville, Virginia 22903; USA;
b Department of Pediatrics, University of Vienna, Vienna, Austria;
c Department of Anthropology, Vienna, Austria; and
d School of Geology and Geophysics, University of Oklahoma, Norman, Oklahoma 73019, USA
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ABSTRACT
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Establishing the diets of ancient human populations is an integral
component of most archaeological studies. Stable isotope analysis of
well-preserved bone collagen is the most direct approach for a general
assessment of paleodiet. However, this method has been limited by the
scarcity of well-preserved skeletal materials for this type of
destructive analysis. Hair is preserved in many burials, but is often
overlooked as an alternative material for isotopic analysis. Here we
report that the stable carbon and nitrogen isotope values for the hair
of the 5200 year-old Ice Man indicates a primarily vegetarian diet, in
agreement with his dental wear pattern. Whereas previous investigations
have focused on bone collagen, the stable isotope composition of hair
may prove to be a more reliable proxy for paleodiet reconstruction,
particularly when skeletal remains are not well preserved and
additional archaeological artifacts are unavailable.—Macko, S. A., Lubec, G., Teschler-Nicola, M., Andusevich, V., Engel, M. H.
The Ice Man's diet as reflected by the stable nitrogen and carbon
isotopic composition of his hair.
Key Words: stable isotopes • amino acids • paleodiet • neolithic
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INTRODUCTION
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TRADITIONAL METHODS FOR the determination of
paleodiets are often based on incomplete and sometimes misleading
archaeological records of, for example, faunal and floral remains,
artifacts or other cultural evidence, and dental records of ancient
populations. Subsequent to early reports that the stable isotope
composition of heterotrophs tend to reflect the isotope compositions of
their respective diets 1; 2)
, there have been numerous
attempts to use this approach for paleodietary reconstructions,
including ancient human populations (e.g., refs 3; 4
) and
even dinosaurs (5)
. Work on ancient populations has
focused on the stable isotopic analysis of bone collagen, the
assumption being that the preservation of bone collagen is sufficient
to retain the original isotopic signal of the organism. However, even
under the best of circumstances, bone collagen tends to be altered by
diagenetic reactions such as hydrolysis, decarboxylation, and
deamination (6)
. Thus, how closely the stable isotope
composition of residual collagen is likely to reflect that of the
original biomolecule has been the topic of much debate (e.g., ref 7
).
An alternative proteinaceous material that is commonly encountered in
ancient human burials is hair. Consisting of 
-keratins, which
include hair and fingernails as well as horn, skin, and wool
(8)
, hair is composed of hydrophobic proteins that are not
easily degraded (9)
. The microbial degradation of these
insoluble macromolecules depends on the secretion of extracellular
enzymes with the ability to act on the surface. Keratins have a
mechanical stability dependent on the tight packing of the -helix,
and can be degraded by a few species of parasitic and saprotrophic
fungi as well as thermophilic anaerobic bacteria (10, 11).
Minagawa (12)
recently demonstrated that the stable carbon
and nitrogen isotope compositions of modern human hair are a reliable
indicator of the diets of these individuals. Based on these and our own
isotopic studies of modern hair (S. A. Macko and M. H. Engel,
unpublished results), we hypothesize that it should be possible to
estimate the paleodiets of ancient humans based on the stable isotope
composition of their hair. Hair is often observed at archeological
sites and potentially could be used to show not only dietary
differences, but also short time scale variation in diet, perhaps on a
seasonal basis. Collagen, on the other hand, represents a material that
is on a longer time scale integration of dietary preferences. By using
several hairs at different distances from the roots, hair can similarly
be used in an integrative fashion. The relationships between the
isotopic compositions of collagen and hair have not been well
established, although the isotopic composition of hair appears to
exhibit the previously observed 3
shift with trophic level (13, 14)
Here we report a first test of this hypothesis, focusing on
reconstruction of the paleodiet of Ice Man, who was preserved in a
glacier of the Oetztaler Alps during the neolithic period, ~5200
years ago. Farming was already widespread in Europe at that time, and
meat and crops were readily available (15)
. Plant and
pollen material found in association with the Ice Man confirm the
availability of cereals (16)
, and suggest that these
materials could have been a portion of his diet. Furthermore, the
pronounced dental attrition/abrasion of the Ice Man's teeth
(16)
point to a predominantly herbivorous diet.
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MATERIALS AND METHODS
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Hair samples
Hair samples from the Ice Man were provided by Dr. G. Wortmann,
Deutsches Wollforschungs Institut, Aachen, Germany. For comparison,
modern hair samples were also analyzed from two vegans (individuals who
reported that they consumed no animal protein) and from nine other
individuals who either described themselves as vegetarians (deriving
nutrition from plants, eggs, or milk products: ovo-lacto-vegetarians)
or subsisted on an omnivorous diet (one of the authors, S.A.M.). The
hair samples, consisting of several strands of hair of different
lengths, were cleaned with distilled ethanol to remove any possible
superficial debris and minimize the levels of surface lipids on the
hair. Although the keratin structure is a protein, the presence of
lipids could strongly bias the carbon isotope signal if they were not
removed. In addition we examined samples of the goat fur that was found
with the Ice Man. Finally, stable isotope analyses were also performed
on samples of a grass-like plant, a possible dietary source of the goat
recovered in association with the Ice Man. With these samples, we hoped
to allow for a representation of the potential diets available to the
Ice Man—the goat being a meat source, and the grass allowing for the
isotope signals of plants to be suggested. These samples are in all
likelihood the best ones for comparison, since they represent materials
of precisely the same age and reduce the potential for variation due to
geography, climate, and the effects of modern fertilizers and fossil
fuel on the carbon pools.
Amino acid distributions and stereochemistry
Given the expectation that even well-preserved ancient hair
keratin will likely have undergone alteration to some minor extent
(17)
, samples of the author's (S.A.M) hair and that of
the Ice Man were analyzed for their respective amino acid distributions
as a first approximation of relative preservation. The individual hair
samples were hydrolyzed (6N HCl, 24 h, 100°C) and the resultant
hydrolysates were analyzed for their amino acid distributions by high
performance liquid chromatography (postcolumn derivatization with
o-phthaldialdehyde) using the method of Hare et al.
(18)
. Whereas proline has been reported to be a component
of keratin, it could not be detected by the high-performance liquid
chromatographic method used in this study. Proline, however, was
observed in the analyses done by gas chromatography (GC)2
but was not quantified. Aliquots of the hydrolysates of the Ice Man's
hair and of the modern hair (S.A.M.) were derivatized and analyzed by
GC using an optically active stationary phase (Chirasil-Val) to
determine the extent to which amino acids may have been racemized.
Details of the GC method are reported elsewhere (19)
.
Stable isotope analyses
Stable carbon and nitrogen isotope analyses of the individual
hair samples, the grass-like plant, and the goat fur were performed
using a Carlo Erba elemental analyzer interfaced to a Micromass Optima
isotope ratio mass spectrometer. Details of the method are reported
elsewhere (20)
. Stable carbon and nitrogen isotope values
are presented by using the standard convention:
where N is the heavier stable isotope of the element E, and R is
the abundance ratio of the heavy to light isotopes of the element
(i.e., 13C/12C or
15N/14N). The reference for 
15N
is air (0%); values have a standard deviation of 0.2. The reference
for 13C is NBS-22 (-29.8) relative to PDB carbonate
(0%); values have a standard deviation of 0.2.
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RESULTS
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Compared to modern hair, the Ice Man exhibited, as expected,
slight decreases in the absolute abundances (Fig. 1A
) of several unstable amino acids (e.g., serine, threonine).
However, the relative percentages of individual amino acids that
comprise the modern hair and that of the Ice Man were remarkably
similar (Fig. 1B
). The elevated percentages of hydrophobic
amino acids (e.g., leucine, alanine, glycine, valine, isoleucine) in
the Ice Man hair sample are consistent with the distribution observed
for the modern hair keratin, and may in part account for its resistance
to hydrolysis subsequent to interment. The fact that none of the amino
acid constituents of the Ice Man's hair exhibited any indication of
partial racemization above background also supports excellent
preservation of this ancient hair keratin. In addition, the low C/N
seen in the hair (3.2 to 3.4) is consistent with that of pure proteins.
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Figure 1. A) Amino acid concentrations in the Ice Man and modern hair.
B) Relative distribution of amino acids in the Ice Man and
modern hair.
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The stable carbon and nitrogen isotope values for the Ice Man's hair
and the associated remains of goat fur and grass-like plants
(Table 1
) provide some significant clues with respect to his diet.
Depending on the complexity of a food web, 13C values of
an organism's protein may show slight to moderate enrichment in
13C relative to diet (6)
. However, the key to
distinguishing trophic levels of heterotrophs is the stable nitrogen
isotope composition of their proteins. This is because with increasing
progression in a food chain, the 15N values of protein
of organisms tend to become from 2 to 3 enriched relative to the
preceding (lower) food chain level (6)
because most of the
animal's nitrogen is derived from the protein of its food (21, 22). As
will be discussed below, a 15N value of 7.0 for the Ice
Man clearly points to a primarily vegetarian diet, consistent with the
tooth abrasion observed for this specimen (Fig. 2
).
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DISCUSSION
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With the remains of goat fur found on this site, one might
intuitively suggest that goat meat was a significant component of the
Ice Man's diet. However, if this were the case, the stable nitrogen
isotope composition of his hair should have been 3 enriched relative
to the goat 15N value, and clearly this is not so.
Rather, the 15N value for the Ice Man's hair is
approximately 3 enriched relative to the 15N values
of plants, indicating that plants were likely a primary component of
his diet at the time of his death. The stable nitrogen isotope
composition of the Ice Man is consistent with that observed for modern
vegetarians with low levels of animal protein in their diet (Table 1)
and who are depleted in 15N by several per milligram
relative to modern humans on omnivorous diets. The goat should reflect
the vegan diet, being enriched by 3 over primary production. In all
likelihood it does, but represents an integration of browsing on a
variety of terrestrial plants, some of which may be more depleted in
15N owing to nitrogen fixation. The fact that the
13C value of the Ice Man was depleted in 13C
reflects a high percentage of grains in the diet that can be attributed
to plants that utilized the C-3 pathway for photosynthesis (Table 1)
.
In general, C-3 plants (e.g., wheat, rice, legumes) are depleted in
13C by 5 to 15% relative to C-4 plants (e.g., corn) due to
differences in enzymes used for the primary fixation of carbon (e.g.,
ref 23
). A similar preference for C-3 plants (or for organisms that
subsisted on C-3 plants) was reflected in the diets of the other
omnivores, all of which are expected to be enriched in 13C
by a few per milligram relative to their respective C-3 sources. Strict
adherence to only vegetation is not suggested, however. Small amounts
of animal protein in the diet, as well as variation in the
isotopic signals of potential plant and animal foods, could be masked
by the consumption of large amounts of plant proteins.
In summary, stable isotopes provide a powerful probe for the
reconstruction of paleodiets of organisms. Our initial results indicate
that when hair is well preserved, under burial conditions such as
mummification, its stable isotope composition can be used to help
reconstruct the paleodiet of the individual by making use of very small
amounts (milligram quantities) of material. Our stable isotope results
confirm the initial indication of a vegetarian diet for the Ice Man
that was based on the abrasion of his teeth.
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ACKNOWLEDGMENTS
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We thank Dr. G. Wortmann and the British Museum for providing the
hair samples. S.A.M. and M.H.E. acknowledge the National Science
Foundation (Geology and Paleontology) for support of this research. We
also are grateful for the efforts of the two anonymous reviewers. G.L.
is highly indebted to the Red Bull Company, Salzburg, Austria, for
generous financial support.
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FOOTNOTES
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1 Correspondence: University of Vienna, Dept. of Pediatrics, Waehringer Guertel 18, A 1090 Vienna, Austria. E-mail: GERT.LUBEC{at}AKH-WIEN.AC.AT 
2 Abbreviation: GC, gas chromatography.
Received for publication March 23, 1998.
Revision received October 29, 1998.
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ABSTRACT
INTRODUCTION
