Integrating cortisol and isotopic analyses of archaeological hair: Elucidating juvenile ante-mortem stress and behaviour

International Journal of Paleopathology 9 (2015) 28–37

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Integrating cortisol and isotopic analyses of archaeological hair: Elucidating juvenile ante-mortem stress and behaviour Emily C. Webb a,∗ , Christine D. White a , Stan Van Uum b , Fred J. Longstaffe c a b c

Department of Anthropology, The University of Western Ontario, London, Canada Department of Medicine, The University of Western Ontario, London, Canada Department of Earth Sciences, The University of Western Ontario, London, Canada

a r t i c l e

i n f o

Article history: Received 25 August 2014 Received in revised form 3 December 2014 Accepted 7 December 2014 Keywords: Mummified remains Cahuachi Juvenile health Weaning Childhood

a b s t r a c t Understanding childhood experiences of health and well-being is essential to reconstructing ancient lifeways. Here, archaeological hair samples from five juveniles from Cahuachi and near Huaca del Loro in the Nasca Region, Peru, are analyzed for their carbon- and nitrogen-isotope compositions and cortisol levels. Stable isotopic data are used to investigate dietary change and nitrogen metabolism, and cortisol levels are used to infer exposure to stress. Using a case study approach, we determined that juveniles have distinct, individualized experiences of diet and morbidity, which are, in some cases, similar to adult experiences in the same region. Overall, Nasca Region juveniles have high systemic cortisol levels (1444 ± 402 ng/g) compared to Peruvian adults (281 ± 35 ng/g; Webb et al., 2010). Younger juveniles have comparatively high ␦15 N values that decrease over several months, suggesting transition from breast milk to a weaning diet. Older juveniles exhibit patterns of dietary shifting similar to those determined for adults in the region, or suggestive of particular socioeconomic roles. This study demonstrates the value of applying biomolecular methods to juvenile mummified remains to better understand the life histories of children in archaeological contexts. © 2014 Elsevier Inc. All rights reserved.

1. Introduction In both contemporary and ancient societies, childhood is a critical period of biological and social growth and development, during which children are also susceptible to increased physiological stress, morbidity and mortality. Children are an important group within any society, and from a bioarchaeological perspective, reconstructing past life-ways based solely on adult experiences is lacking (Ardren, 2006; Baxter, 2005). The need to care for and support children may have considerable influence on the decisions and activities of those around them, and children also have the potential to be significant social and economic actors in their own right (Ardren, 2006; Baxter, 2005; Kamp, 2001; Sofaer Derevenski, 2000). Indeed, globally, ethnographic research points to the significance of children’s work, for example, in tending animals, participating in food acquisition and preparation, and through learning new skills or crafts (Baxter, 2008). Although children have been

∗ Corresponding author at: Organic Geochemistry Unit, School of Chemistry, Cantock’s Close, University of Bristol, Bristol BS8 1TS, United Kingdom. Tel.: +44 0117 3316795. E-mail address: [email protected] (E.C. Webb). http://dx.doi.org/10.1016/j.ijpp.2014.12.001 1879-9817/© 2014 Elsevier Inc. All rights reserved.

considered invisible or inaccessible in many archaeological contexts, bioarchaeological research methods can provide essential information on children and childhood through examination of juvenile skeletal remains. Juvenile remains are of particular relevance to the study of health in the past, because children are highly sensitive to indicators of biocultural change. Information about disease, weaning practices and subsistence strategies is likely to be recorded in the osteological or biomolecular archive of the human body (Buikstra and Ubelaker, 1994; Goodman and Armelagos, 1989). Juvenile experiences of morbidity and mortality reconstructed from juvenile skeletal remains will be distinct from an understanding generated by examining stressors on adult skeletons. A particular challenge of studying childhood in archaeological societies is an often poor understanding of what defines a child in a specific social context, and how a socially-defined childhood relates to biological age (Rothschild, 2002). In this paper, we use the term ‘juvenile’ to refer to skeletally immature individuals, and consider the concept of childhood to refer to a sociallyand biologically-constructed experience. Because juveniles do not appear to be depicted on Nasca ceramics (Proulx, 2007), nor are there a significant number of osteological or mortuary analyses reported for juvenile remains, remarkably little is known about children or childhood in the Nasca Region c. AD1-1000. Although

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Fig. 1. Map of the Rio Grande de Nasca drainage showing sites sampled.

we define juveniles using skeletal remains, by exploring individualized experiences of diet, metabolism and stress, we attempt to provide a conceptual space that allows some understanding of what could potentially constitute children’s experiences and behaviour for each individual included in this study. Here, hair samples from five juveniles excavated at Cahuachi and near Huaca del Loro in the Southern Nasca Region of Peru (SNR, c.AD1-1000; Fig. 1) were analyzed to determine cortisol levels and carbon- and nitrogen-isotope compositions (Webb, 2010). The objective was to assess the potential of combining cortisol and isotopic data to elucidate juvenile experiences of diet, health and stress, as well as frailty and risk. We use a case study approach to assess the interpretive potential of integrating biomolecular datasets to describe individual experiences of juveniles in the past. We suggest that by using systemic cortisol levels and isotopic data from hair, frailty and risk can be assessed in the months leading up to death and provide unique insight into juvenile behaviour and morbidity. We focus on “invisible” stress experiences (i.e., those not typically observable on the skeleton) and consider cortisol production in relation to diet and metabolic state during the arguably crucial period leading up to death. 2. Background 2.1. Archaeological context The Nasca polity inhabited the Rio Grande de Nasca Drainage, Peru, during the Early Intermediate period (AD1-750; Fig. 1). During the Early Nasca period (AD1-450), settlements emerged in the upper valleys of the drainage, and construction and use of the ceremonial centre, Cahuachi, increased (Silverman, 1993; Vaughn, 2009). Cahuachi is thought to have functioned primarily as a

pilgrimage centre with a small elite population described as a “centralized theocratic authority” (Orefici, 2006: 184). The transitional Middle Nasca period (AD450-550) is characterized by a dramatic re-working of iconography and the cessation of major construction at Cahuachi (Schreiber and Lancho Rojas, 2003; Silverman, 1993). During this period, the typically unstable Andean climate became even more unpredictable as the result of severe droughts (Thompson et al., 1985). From c.AD250 until the end of the Middle Horizon period (c.AD1000), climate in the Nasca Region became increasingly arid (Bird et al., 2011; Eitel et al., 2005; Eitel and Mächtle, 2009). The populace coalesced into a few large settlements during the Late Nasca period (AD550-750; Schreiber, 2005; Schreiber and Lancho Rojas, 2003). This population aggregation was concomitant with increased social complexity and greater representation of conflict-related themes on pottery. Towards the end of ˜ effects the Early Intermediate period, evidence of damaging El Nino has been observed, including flooding at Cahuachi (Beresford-Jones et al., 2009; Orefici and Drusini, 2003). The Wari polity expanded into Nasca c. AD750, marking the beginning of the Middle Horizon period (locally the Loro period, AD750-1000). Wari settlements were established throughout the Southern Nasca Region, and Wari ceramics are found in Nasca-affiliated cemeteries and at habitation sites along with the local Loro style (Schreiber, 2001, 2005; Strong, 1957). The local population of the northern river valleys decreased as Nasca people moved south to the Las Trancas river valley where a large local centre, Huaca del Loro, was established (Conlee and Schreiber, 2006; Schreiber, 2001). A recent archaeological survey (Edwards, 2010) further indicates that the Wari made important changes to the landscape of the upper and mid-valleys of the Southern Nasca Region, establishing several settlements and constructing roads that linked the headwaters of the Nasca river tributaries with the coastal plains.

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Bioarchaeological analysis of a skeletal sample excavated from the Las Trancas river valley cemeteries revealed an overall decline in health status from Early Nasca (c.AD1-450) to the Loro period (c.AD750-1000), but when compared to Pre-Hispanic archaeological skeletal samples from Central and South America, it appears that people living in the Nasca Region were not under excessive stress during any period (Kellner, 2002). Nasca Region inhabitants experienced physiological stress as juveniles (indicated by the presence of linear enamel hypoplasias), reactive new bone formation (i.e., osteoperiostitis), and anaemia (potentially caused by parasitic infection, infectious disease and/or chronic diarrhoea; Kellner, 2002). Increased population density often leads to a decline in health because of increased pathogen transmission and waste disposal problems. Moreover, elevated rates of accidental trauma and trauma associated with violence have been observed in skeletal samples from the Las Trancas and Palpa river valleys (Cagigao, 2009; Kellner, 2002). Combined data from these studies indicate that both physical and psychosocial stress caused by interpersonal violence and accidental trauma were likely significant during the Early Intermediate period and Middle Horizon times. Stress may be alleviated by improving socioeconomic conditions, but social development may generate stress as well (Goodman et al., 1988). Exposure to various stressors does not mean that there will be a harmful impact on an individual’s health, although there may be skeletal or biomolecular evidence preserved in the skeleton. Conversely, prolonged stress episodes, and many infectious agents and soft tissue diseases/injuries, will not leave clear skeletal evidence. Individuals with lesions may also be “paradoxically” healthier than those without, since, for skeletal traces to become evident, the individual must survive the stress episode and/or live for a significant amount of time with a disease (Wood et al., 1992). 2.2. Cortisol Cortisol, a glucocorticoid hormone, is produced in response to physical and emotional stress, and it plays a central role in mediating the body’s physiological stress response (Miller et al., 2007). In contemporary clinical contexts, cortisol in blood plasma, saliva or urine is typically measured at specific points over short experimental time periods, whereas hair, as a continuously growing tissue, can retain a retrospective record of cortisol levels and stress. Hair cortisol levels reflect stress-induced changes in systemic cortisol levels (Davenport et al., 2006; Kalra et al., 2007; Kirschbaum et al., 2009; van Uum et al., 2008), and biogenic patterns of hormone production can be preserved over archaeological timeframes (Webb et al., 2010). Cortisol production, mediated by the hypothalamicpituitary-adrenal axis, is an important mechanism through which experienced physiological and psychosocial stresses influence the physical body (Miller et al., 2007). The primary metabolic effect of cortisol is to promote gluconeogenesis, which mobilizes stored energy from adipose and proteins when dietary sources and glucose reserves are depleted. Cortisol also accelerates both proteolysis and lipolysis, and it modulates the immune system by directing the duration and magnitude of inflammatory responses and lymphocyte maturation (Sapolsky, 2004). Changes in cortisol production associated with malnutrition, trauma, and illness are particularly relevant to juvenile health and stress. Severe malnutrition and starvation may lead to higher plasma cortisol levels, most likely caused by increased cortisol production, a slowed metabolism and co-occurring infection or illness (Fichter et al., 1986; Johnstone et al., 2004; Manary et al., 2006; Støving et al., 1999). Clinical research has demonstrated that among juveniles with severe systemic infection, those with high cortisol levels were more likely to recover than those with “normal” or low cortisol production, suggesting that inadequate adrenal response may be an important indicator of frailty (den Brinker et al.,

2005). Critical illness-related corticosteroid insufficiency (CIRCI) is a complex and controversial condition. Defined as inadequate corticosteroid activity for the severity of the illness, this syndrome is a result of both tissue resistance to corticosteroids and inadequate circulating levels of cortisol. Very high (pharmacological) doses of corticosteroids over a short period (i.e., 24 h) have not been found to improve patient outcomes and often increase health complications. In contrast, lower stress-doses of corticosteroids administered over longer periods (i.e., weeks) improved chances of survival considerably (Marik, 2009). Although CIRCI is not yet fully understood, the level of cortisol production during illness relative to baseline may influence the ultimate outcome. Systemic cortisol levels may also increase as a result of psychosocial or physical stress, including, for example, threats to physical integrity, stressful social interactions, and low controllability of stressors (Miller et al., 2007). 2.3. Isotopic analysis Stable isotope analysis entails measuring the ratio of heavy to light isotopes in a sample. This value, reported in delta (␦) notation, is an expression of the relative differences in isotope ratios between a known reference standard and the sample; for example ␦13 C =

13 C/12 C

sample − 13 C/12 C

13 C/12 C

standard

standard

The reconstruction of palaeodiet using carbon- and nitrogenisotope analysis is based on the assumption that tissue isotopic compositions reflect the isotopic composition of consumed foods (Ambrose, 1993). There are systematic differences in isotopic composition between tissue and food, and among different tissues within the body. These diet-tissue and tissue-tissue offsets are a result of fractionation, the differential partitioning of isotopes between phases in a reaction caused by the slight mass differences among isotopes of the same element (e.g., 13 C, 12 C and 15 N, 14 N). Isotopic data are used to assess relative contributions of isotopically distinct foods to diet by comparing tissue isotopic compositions to a food web model that describes the natural variability in isotopic compositions of local food resources. The carbon-isotope composition of hair keratin reflects dietary protein, when diet is protein-sufficient, as it is derived from plants at the base of the food web, i.e., C3 plants (e.g., quinoa, tubers, fruits and vegetables) vs. maize, a C4 plant (Ambrose, 1993; Sandford and Kissling, 1993). Typically, C3 plants have lower (i.e., more negative) ␦13 C values (∼−26.5‰), whereas C4 plants have higher (i.e., more positive) ␦13 C values, with an average of ∼−12.5‰. Plant ␦13 C values can be impacted by local environmental factors, such as temperature, altitude, aridity and light intensity, so local plant sources are preferred to global averages when assessing diet. Marine plants obtain carbon from dissolved bicarbonate (∼0‰), which is 13 Cenriched compared to the terrestrial carbon source, atmospheric carbon dioxide (∼−8‰). Marine C3 plant carbon-isotope compositions may thus be indistinguishable from those of terrestrial C4 plants (Ambrose, 1993; Heaton, 1999; Tieszen et al., 1979). Stable nitrogen-isotope analysis is typically used to investigate dietary protein source and to assess trophic level, or relative position in the food web. Nitrogen-isotope compositions vary among different food sources, but the consumer is always 15 Nenriched relative to its food source by 3–4‰ per trophic level (DeNiro and Epstein, 1981; Schoeninger, 1985). Bioarchaeological, environmental and biomedical research has demonstrated that nitrogen-isotope compositions can also be significantly influenced by the environment (e.g., aridity), anthropogenic soil modification (e.g., salinity and application of fertilizers), metabolic and physiological stress (e.g., malnutrition or pregnancy) and pathological conditions, including disease, illness and trauma

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(Fuller et al., 2004, 2005; Heaton et al., 1986; Hobson et al., 1993; Katzenberg and Lovell, 1999; Mekota et al., 2006, 2009; Olsen et al., 2014; Petzke et al., 2006; Schwarcz et al., 1999; White and Armelagos, 1997; Williams et al., 2011). As well, breastfeeding infants are believed to exhibit a trophic-like effect, in which infant tissue ␦15 N values are consistently higher than those of their mothers, eventually equilibrating to a weaning or adult diet (Fogel et al., 1989). The nitrogen-isotope compositions of fast-growing tissues like hair keratin can also be used to elucidate nitrogen metabolism. In healthy individuals, the rate of protein synthesis equals the rate of protein breakdown and loss (Waterlow, 1999). This body nitrogen balance will change in response to amino acid availability in the metabolic amino acid pool, which is composed of amino acids from dietary protein and catabolised body protein. Health status and stress (e.g., tissue growth and repair, infection, pregnancy, inadequate diet, etc) can push the body into a state of negative or positive nitrogen balance, which can influence the nitrogen-isotope composition of bodily tissues. The body is in positive nitrogen balance when protein synthesis exceeds protein breakdown and loss, i.e., during periods of rapid growth or tissue repair. Newly synthesized tissues will be 15 N-depleted relative to dietary nitrogen. Negative nitrogen balance, induced by stress, causes progressive 15 Nenrichment of the metabolic amino acid pool; this occurs because nitrogen from catabolised body proteins is 15 N-enriched relative to diet. Isotopic fractionation is thought to occur during deamination and transamination reactions as nitrogen is eliminated from the body. Isotopically-light nitrogen (14 N) is favoured for excretion in the form of urea, resulting in an overall enrichment of the metabolic amino acid pool (Hobson et al., 1993; Schoeller, 1999). 2.4. Hair growth Hair grows at a rate of approximately 0.35 mm/day and is metabolically inactive, retaining a sequential archive of isotopic information (O’Connell and Hedges, 1999; Saitoh et al., 1969). There is a fourteen-day lag between hair formation and emergence of the mature hair from the scalp, and this creates a blind spot covering the two weeks immediately preceding death (Nakamura et al., 1982; O’Connell and Hedges, 1999). Hairs that have completed growth are periodically shed, and subsequently replaced by new hairs. Hair grows during the anagen phase (lasting ∼two to three years), and in the context of biomolecular analyses, it is during this phase that new information is incorporated. The anagen phase is followed by the catagen, or quiescent, phase, which lasts from one to four weeks, and the telogen phase, which lasts from one to three months. The hair is finally shed when a new anagen phase begins in the follicle, and the old hair is pushed out by the new hair. Typically, 80–85% of scalp hair is in anagen phase, 1–2% in catagen phase and 10–20% in telogen phase, but these proportions may change seasonally and with illness (Harding and Rogers, 1999; Schwertl et al., 2003). In a mixed-phase hair sample, temporal trends may be obscured and inter-segment variation in isotopic composition will be dampened. The impact of a change in diet can be detected in the isotopic composition of the body, including hair keratin, within days (Nakamura et al., 1982), but isotopic equilibration of the body protein pool with this new diet takes longer. The amino acid requirements for day-to-day protein synthesis for tissue growth or repair greatly exceed the amount of protein that can be consumed daily, to the extent that about three-fourths of the amino acids needed must be drawn from the body’s protein pool or through breakdown of other body proteins. The protein pool acts as a buffer, reducing the effect of short-term fluctuations in isotopic composition, and amino acids accessed through the breakdown of stored body proteins will further slow the equilibration process. Based on

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controlled research with human scalp hair, it has been determined that complete equilibration with a new diet may take up to 12 months for carbon-isotope compositions, and approximately five months for nitrogen-isotope compositions (O’Connell and Hedges, 1999). 3. Methodology 3.1. Sampling Hair samples were collected at the State Collection for Anthropology and Palaeoanatomy, Munich, Germany in 2008. The remains were collected during Heinrich Ubbelohde-Doering’s 1932 field season, during which he excavated approximately fifty graves in the Rio Grande de Nasca drainage (Ubbelohde Doering, 1958, 1966). Hair sampled from five juveniles from Cahuachi (CAH) and from cemeteries south of Huaca del Loro in the Las Trancas river valley (LTR) was analyzed for cortisol levels and carbon- and nitrogenisotope compositions. Two of the juveniles (I; CAH 560 and LTR 639) are three years of age or less, and three juveniles are somewhat older, between 3 and 12 years of age (II; CAH 529, CAH 579 and LTR 618). Hair samples were collected with care taken to maintain orientation and alignment of individual hair strands within each sample bundle. For each individual, a large bundle of hair was sectioned into 1 cm segments, with each segment representing approximately one month of growth based on the average growth rate of 0.35 mm/day for scalp hair. The quantity of hair required for cortisol and isotopic analyses is large (ideally, >12 mg per 1 cm segment); thus, both tissue availability and preservation were limiting factors with regard to sample size and selection for this study. Age estimates are based primarily on dental eruption using whichever dental elements were available for inspection (Schaefer et al., 2009; Ubelaker, 1979). 3.2. Laboratory procedures For carbon- and nitrogen-isotope analyses, each hair sample bundle was cleaned of loose particulate matter by wiping gently with ethanol. Hair bundles were then sectioned and each segment sample was placed in a glass vial. Lipids and decomposition fluid residues were removed by soaking hair segments in 2:1 (v/v) chloroform: methanol solution for 24 h. Each segment sample was then rinsed with fresh solution and, when necessary, this process was repeated. Segment samples were air-dried for 12–24 h, and then finely minced and weighed (510 ± 10 ␮g) into tin capsules for isotopic analysis. To assess the significance of growth phase error (Williams et al., 2011), three individuals with well-preserved scalp tissue (CAH 560, CAH 579 and LTR 639) were selected for microscopic separation of actively growing (anagen) hair. Small samples of scalp with attached hair were soaked in dimethyl sulfoxide (100 ml, 5% solution) for 36–48 h, washed twice with distilled water and once with a phosphate buffer (pH 7.4). Individual hairs were withdrawn from the rehydrated tissue and the follicle was examined using an Olympus SZX9 stereoscope to determine hair growth phase. Between 30 and 50 full length anagen phase hairs were collected for each individual, sectioned into 1 cm segments, and sample preparation proceeded as above. Isotopic analysis of keratin was performed using a Costech Elemental Analyzer interfaced with a Thermo Finnigan DeltaPlus XL mass spectrometer. The ␦13 C values were calibrated to VPDB (Coplen, 1994, 1996) using ANU-Sucrose and IAEA NBS-22, and the ␦15 N values were calibrated to AIR (Hoefs, 2004; Mariotti, 1983) using IAEA-N1 and IAEA-N2. Analytical precision was determined through duplicate analyses of samples, and was ±0.2‰ for

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␦13 Cker , and ±0.1‰ for ␦15 Nker . Accuracy was assessed using a laboratory keratin standard, which gave an average ␦13 C value of −24.06 ± 0.05‰, and an average ␦15 N value of +6.26 ± 0.09‰, which compare well with its accepted values (−24.04‰ and +6.36‰, respectively). Isotopic analyses were performed at The University of Western Ontario, London, Canada in the Laboratory for Stable Isotope Science. The methodology used to measure cortisol abundance in hair is described in detail in van Uum et al. (2008). At least 10 mg of hair per segment is required for cortisol analysis.1 Hair segments were placed in glass vials and the hair was very finely minced with surgical scissors. 1 ml of methanol (>98%) was added to extract the steroid. The vials were then sealed and incubated for 16 h, while shaking at 100 rpm and heating at 50 ◦ C. The methanol was then removed from the vial, transferred to test tubes and evaporated by heating to 40 ◦ C under a nitrogen stream. The residue was reconstituted in 250 ␮l of phosphate-buffered saline (PBS) at pH 8.0. The PBS mixture was then analyzed using a commercially available salivary enzyme immunoassay kit (ELISA, ALPCO Diagnostics). All measurements were done in duplicate. Crossreactivities of steroids in the salivary ELISA kit are as follows: cortisol 100%, corticosterone 31%, progesterone < 2%, deoxycortisol < 2%, dexamethasone < 2%, estriol < 0.001%, estrone < 0.001%, and testosterone < 0.001%. The lower limit of detection for cortisol is 1.14 ng/ml (information from manufacturer), and the absolute cortisol extraction recovery is 88% in a 100 ng/ml standard, and 87% for a 2 ng/ml standard (van Uum et al., 2008). Cortisol analyses were performed at The University of Western Ontario, London, Canada. 4. Results Cortisol and isotopic results are presented in Table 1 and Figs. 2 and 3. The juvenile mean cortisol level is 1444 ± 402 ng/g for the five juveniles included in this study. There is a statistically significant difference between younger (1172 ± 225 ng/g) and older (1800 ± 441 ng/g) juveniles (Mann–Whitney, p = 0.0002 ). The juvenile mean cortisol level is also higher than the adult mean cortisol level previously determined for Peruvian adults (281 ± 35 ng/g; Webb et al., 2010) and statistically different from adults from the same sites in the Nasca Region (522 ± 368 ng/g, Mann–Whitney, p = 0.003; Webb, 2010). One possible explanation for this trend is that hair cortisol levels of juveniles are inherently higher than adult levels. Juvenile salivary cortisol levels are, however, somewhat lower than adult levels, although the change in cortisol production during and after exposure to a stressor is essentially the same (Kudielka et al., 2004). Further, children’s hair has been used in clinical contexts to investigate stress associated with, for example, new experiences (e.g., going to school) or health conditions, such as obesity (Groeneveld et al., 2013; Vanaelst et al., 2013; Veldhorst et al., 2014). It is therefore more likely that the high cortisol levels in hair observed in this study are instead reflective of health and stress for these individuals. There are several factors that can impact the detection of biogenic cortisol levels, such as leaching, contamination or modification of biogenic cortisol. External contamination from the burial environment is unlikely, since cortisol is only produced by mammals. A possible source of exogenous cortisol may be animal

1 Cortisol levels were not compared for anagen vs. mixed phase hair because of the large volume of hair required (≥10 mg per segment). 2 For all statistical comparisons, all data are pooled and tested, i.e., each individual is represented by all of his or her hair data rather than an average value. Although this method risks over-representing juveniles with longer hair samples, it provides enough data to apply statistical testing. Hair sample lengths do not range widely, i.e., from two to nine segments, compared to previous work (2–34 segments; Webb et al., 2013).

Table 1 Cortisol levels and isotopic data. Sample (age)1

Cortisol (ng/g)2

␦13 C (‰, VPDB)

␦15 N (‰, AIR)

CAH 529 [II] 1 2 3 4

1563 ± 365 1995 1109 1515 1631

−15.2 ± 0.9 −14.3 −14.9 −15.3 −16.4

+9.9 ± 0.6 +10.7 +10.3 +9.6 +9.2

CAH 560 [I] 1 2 3 4 5 6 7 8

1123 ± 249 757 862 1011 1123 1356 1486 1300 1086

−13.2 ± 0.5 −13.8 −13.5 −13.6 −13.6 −13.2 −12.7 −12.6 −12.5

+11.6 ± 0.5 +11.1 +10.8 +11.1 +11.6 +12.2 +12.3 +11.9 +11.9

CAH 579 [II] 1 2 3

1235 1107 1363

−17.4 ± 0.9 −16.5 −17.4 −18.3

+7.4 ± 0.3 +7.7 +7.1 +7.4

LTR 618 [II] 1 2 3 4 5 6 7 8

2096 ± 264 2368 2507 1830 2040 2169 1923 1838

−13.0 ± 2.4 −9.8 −12.4 −16.2 −16.1 −15.1 −12.1 −10.9 −11.4

+7.8 ± 0.7 +8.0 +7.5 +6.7 +7.2 +7.5 +8.8 +8.6 +8.2

LTR 639 [I] 1 2 3 4 5 6 7 8 9

1216 ± 207 1041 908 1297 1273 1202 1368 952 1404 1496

−15.0 ± 0.6 −15.4 −15.5 −15.6 −15.7 −15.3 −14.4 −14.3 −14.2 −14.6

+9.0 ± 1.1 +8.2 +7.7 +7.8 +7.9 +8.9 +9.7 +10.0 +10.2 +10.3

1 2

I: less than 3 years of age; II: between 3 and 12 years of age. Values in bold are reported as average ± 1 when n > 2.

fats used in the manufacture of soaps or shampoos, but, if this were occurring, a systematic distribution of contaminant cortisol along the hair shaft or obliteration of inter-segmental differences would be expected. The most likely form of diagenetic alteration in an archaeological context is leaching. Cortisol is most effectively removed from hair by repeated or prolonged exposure to a solvent (e.g., water or alcohol, Davenport et al., 2006; Novak et al., 2013; S. Thomson, pers. comm.), particularly at elevated temperatures. In modern studies, for example, the amount of cortisol retained in hair further than three cm from the scalp decreases sharply, which was attributed to loss of cortisol through washing with alcohol-containing shampoos (Kirschbaum et al., 2009). Compared to modern clinical levels of cortisol in hair, the amount of cortisol in these archaeological hair samples is high, suggesting good retention post-burial. Thus, the changing levels of cortisol along individual hair bundles determined herein most likely reflect biogenic patterns of cortisol production, even if the absolute amount of cortisol may be somewhat lower due to post-mortem loss. The hair keratin mean C/N ratio for samples analyzed in this study was 3.8 (range 3.6–4.0), which indicates that diagenetic modification was minimal. The theoretical C/N ratio of keratin protein is 3.4, higher than that of bone collagen, and the range of C/N ratios found in modern hair (2.9–3.9) compares well with the range reported here (O’Connell and Hedges, 1999). The mean carbonisotope composition for all hair samples (calculated as an average of each individual’s mean ␦13 C value) is −14.8 ± 1.8‰, and the mean

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Fig. 2. Cortisol and isotopic results for younger children (CAH 560 and LTR 639). For cortisol data, segment 1 is closest to the scalp. For isotopic data, the open point designates segment 1.

Fig. 3. Cortisol and isotopic results for older children (CAH 529, CAH 579 and LTR 618). For cortisol data, segment 1 is closest to the scalp. For isotopic data, the open point designates segment 1.

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Fig. 4. Carbon and nitrogen isotopic data for anagen vs. mixed-phase samples from LTR 639. Errors bars represent analytical error and are ±0.2‰ and ±0.1‰ for carbon- and nitrogen-isotope compositions, respectively.

nitrogen-isotope composition is +9.1 ± 1.7‰. There is no statistically significant difference between younger and older juveniles for carbon isotopic data (Mann–Whitney, p = 0.455), but the average nitrogen-isotope compositions are different (+10.3 ± 2.7‰ for young juveniles vs. +8.4 ± 1.4‰ for older juveniles; Mann–Whitney, p = 0.001). The adult carbon isotopic data from the same sites are similar to both younger and older juveniles (−14.9 ± 1.3‰ for Cahuachi and −14.4 ± 1.4‰ for Las Trancas). At both sites, nitrogen-isotope compositions of younger juvenile’s hair keratin are 15 N-enriched relative to adult nitrogen-isotope compositions, which are +8.4 ± 1.6‰ for Cahuachi and +9.2 ± 1.1‰ for Las Trancas (Webb et al., 2013). A comparison of mixed phase and actively growing anagen phase hair samples reveals minimal differences in isotopic composition for both carbon and nitrogen (e.g., LTR 639; Fig. 4). The 13 Canagen-mixed difference ranges from −0.7 to +0.7‰, with an average value of ±0.3 ± 0.2‰. Similarly, the 15 Nanagen-mixed difference ranges from −0.6 to +0.6‰, with an average value of ±0.3 ± 0.2‰. The standard deviations are also moderately greater for carbon and nitrogen in anagen phase hair, indicating greater variability in isotopic composition along the hair sample when anagen phase hair is analyzed. This result supports the inference that the use of mixed phase hair attenuates inter-segmental variation in isotopic composition. Using mixed phase hair therefore provides a more conservative estimate of dietary change along the hair sample. Although the differences are minor, anagen phase hair samples will thus likely provide more accurate information (sensu Williams et al., 2011) and are used for CAH 560, CAH 579 and LTR 639 for the remainder of this paper. 5. Discussion For CAH 560 (age birth to 3 years), hair selected for isotopic analysis was in anagen phase, which increases the resolution of these

data and the interpretive significance of any change in ␦-values (Williams et al., 2011). There are two increases in ␦15 N values of 0.4‰, but the nitrogen-isotope compositions decrease overall by 1.5‰. The ␦13 C values also decrease gradually (by 1.3‰ overall). The average cortisol level is 1109 ± 236 ng/g. Cortisol levels are high during the sixth month, and decrease from the fifth to the final month before death. The small 15 N-enrichment and high cortisol production during month six suggest illness or stress. The increase in the ␦15 N values during the final month ante-mortem suggests another similar stress episode without a co-occurring increase in cortisol levels. It is possible that the overall change in ␦15 N values is caused by weaning, i.e., transition from breast milk to a childhood, or perhaps a more adult-like, diet. The magnitude of the shift in nitrogen-isotope compositions is suggestive of a change in the trophic level of consumed protein. Clinical research has demonstrated that, among severely ill juveniles, those with appropriately high cortisol levels were more likely to recover than those with “normal” or low cortisol production (den Brinker et al., 2005). Therefore, this individual may have been more ill or less able to cope with stress than these data initially indicate. The variation in the ␦13 C and ␦15 N values for LTR 639 (age birth to 3 years) suggests a dietary transition over a small carbon isotope range, and a fairly large nitrogen isotope range (1.5‰ for carbon, 2.5‰ for nitrogen). Both carbon- and nitrogen-isotope compositions generally decrease from the ninth to the final month before death. As with CAH 560, hair selected for isotopic analysis was anagen-phase, so the 0.4‰ increase in ␦15 N values in the final month is likely meaningful, indicating a brief period of negative nitrogen balance before death. The average cortisol level is 1216 ± 207 ng/g, and it fluctuates considerably during the ninemonth period represented. The gradual decrease in ␦15 N values from +10.3‰ to +7.7‰ over the course of eight months (months nine to two), combined with fluctuating cortisol production, may indicate transition from breast milk to weaning diet.

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The weaning process entails reduced intake of breast milk and the introduction of other foods. The impact of this dietary change includes loss of the passive immunity associated with breastfeeding, as well as consumption of new, possibly less nutritious foods and liquids, both of which are potential sources of infection (Katzenberg et al., 1996; Larsen, 2003). Isotopic analysis of bone collagen has been used to identify the weaning process in both archaeological and clinical studies (Dupras and Schwarcz, 2001; Fogel et al., 1989; Katzenberg et al., 1996). The expectation is that breastfeeding infants will have ␦15 Ncol values that are enriched by 1.5–3‰ relative to maternal isotopic compositions. A 15 N-enrichment of juvenile bone collagen relative to female bone collagen of +2.6‰ has been observed for younger juveniles from this region (CAH 560 and CAH 567; Webb, 2010). This finding is consistent with the magnitude of the change in ␦15 Nker values observed for LTR 639 and CAH 560. Current knowledge of Nasca weaning practices is minimal, and female ␦15 Nker and ␦15 Ncol values are highly variable (Kellner and Schoeninger, 2008; Webb et al., 2013). As a result, it is not possible to be certain what ␦15 N value would constitute breastfeeding-related tissue 15 N-enrichment or how long the weaning process might be expected to last. Variability in cortisol production associated with weaning stress is also currently unknown. If these individuals were ill and thus in negative nitrogen balance, the ␦15 N values would be expected to increase (as was determined for the final month ante-mortem). Similarly, the high cortisol levels preclude the possibility that they are in positive nitrogen balance (i.e., a period of tissue growth while consuming a nutritionally adequate diet), because they suggest higher-thanusual stress. Attributing the decreasing ␦15 N values and fluctuating cortisol levels to dietary change and physiological stress or illness associated with weaning is somewhat speculative, but this pattern does raise an interesting possibility for future applications of this methodology. Although the hair samples CAH 529 and 579 are short, some inferences are still possible. For CAH 529 (representing four months), the observed change in ␦-values may represent a seasonal shift in diet (range in ␦13 C of 2.1‰, range in ␦15 N of 1.4‰), suggesting participation in adult consumption patterns (Webb et al., 2013). The average cortisol level is 1563 ± 365 ng/g. Cortisol levels drop from the fourth to the second month before death and then rise to a maximum value of 1995 ng/g in the final month, indicating increasing ante-mortem stress. These cortisol data suggest repeated exposure to stress, culminating in death. Paradoxically, survival of stress episodes indicates that the individual was initially healthy before eventually succumbing to illness or injury, and the high cortisol levels support this, suggesting an appropriate HPA axis response to experienced stress. As with CAH 529, the hair sample from CAH 579 is short, with only three months of isotopic data and two months of cortisol data. The changes in ␦13 C values may indicate a seasonal shift in diet (range in ␦13 C of 1.8‰), but the ␦15 N values are essentially stable (range in ␦15 N of 0.6‰). The average cortisol level is 1235 ± 181 ng/g, and decreases from the second to the final month before death. For LTR 618, dietary shifting suggests a rapidly changing diet (range in ␦13 C of 6.4‰, range in ␦15 N of 2.1‰), which is similar to patterns of dietary shifting observed for adults from this region that were attributed to mobility across local production zones (Webb et al., 2013). The strong positive correlation between nitrogenand carbon-isotope compositions (r = 0.79, p = 0.021) indicates that minimal non-dietary change has occurred. Absolute cortisol levels are high compared to both adults and other juveniles (average 2096 ± 264 ng/g), and cortisol peaks in the fifth and second month ante-mortem correspond to periods when average monthly change in ␦13 C values was highest, suggesting interaction between stress and mobility. The evidence for mobility through dietary change raises the possibility that this individual may have been engaged

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in food acquisition and travel in the Nasca Region, which would have exposed him or her to experiences and stressors similar to adult members of the group at a comparatively young biological age (i.e., age 3–12 years). 6. Conclusion In archaeological explorations of ancient societies, children’s lives have, in the past, often been hidden or ignored. Moreover, morbidity and mortality in a population are often related to juvenile experiences of health and stress, but they are typically understood through adult remains, i.e., the individuals who survived. Here, following a growing trend in bioarchaeological and archaeological research, we explore childhood experiences from the perspective of deceased juveniles. We were able to assess morbidity, diet, and stress using isotopic and cortisol data from mummified hair, and to some extent, we have exposed hidden frailty and risk by providing a history of stress exposure and responses before death. In doing so, we have revealed considerable potential for a deeper understanding of uniquely juvenile experiences, such as weaning (CAH 560, LTR 639), and we were also able to identify participation in ‘adult’ socioeconomic activities, in this case, seasonal dietary change (CAH 529), travel and food acquisition (LTR 618). Further research is, however, required to refine our understanding of stress, diet, and the biomolecular archive recorded in hair, particularly the relationship between nitrogen metabolism and cortisol production in a clinical context. Although biomolecular analysis of archaeological hair is contingent upon excellent soft tissue and juvenile skeletal preservation, we believe that this methodology is a promising new tool for exploring childhood experiences in the months before death. Acknowledgements We thank Dr. Gisela Grupe, Dr. George McGlynn and the State Collection for Anthropology and Palaeoanatomy, Munich, Germany for providing archaeological samples for analysis, and Dr. Karyn Olsen for collecting samples. We also thank Rachel Gow for analytical support. This research was supported by the Social Sciences and Humanities Research Council of Canada, the Natural Sciences and Engineering Research Council of Canada, the Canada Research Chairs Programme and the Physicians’ Services Incorporated Foundation, and utilized infrastructure made possible by the Canada Foundation for Innovation and Ontario Research Fund. This is Laboratory for Stable Isotope Science contribution # 319. References Ambrose, S.K., 1993. Isotopic analysis of paleodiets: methodological and interpretive considerations. In: Sandford, M.K. (Ed.), Investigations of Ancient Human Tissue: Chemical Analyses in Anthropology. Gordon and Breach Science Publishers, Langhorne, PA, pp. 59–130. Ardren, T., 2006. Setting the table: why children and childhood are important in an understanding of ancient Mesoamerica. In: Ardren, T., Hutson, S. (Eds.), The Social Experience of Childhood in Ancient Mesoamerica. University of Colorado Press, Boulder, CO, pp. 3–24. Baxter, J.E., 2005. The Archaeology of Childhood: Children, Gender and Material Culture. Alta Mira, Walnut Creek, CA. Baxter, J.E., 2008. The archaeology of childhood. Annu. Rev. Anthropol. 37, 159–175. Beresford-Jones, D.G., Torres, S.A., Whaley, O.Q., Chepstow-Lusty, A.J., 2009. The role of Prosopis in ecological and landscape change in the Samaca Basin, Lower Ica Valley, South Coast Peru from the early horizon to the Late intermediate period. Lat. Am. Antiq. 20 (2), 303–332. Bird, B.W., Abbott, M.B., Vuille, M., Rodbell, D.T., Stansell, N.D., Rosenmeier, M.F., 2011. A 2300-year-long annually resolved record of the South American summer monsoon from the Peruvian Andes. Proc. Natl. Acad. Sci. U. S. A. 108, 8583–8588. Buikstra, J.E., Ubelaker, D.H., 1994. Standards for Data Collection from Human Skeletal Remains. Arkansas Archaeological Survey Report No. 44, Fayetteville, AR. Cagigao, E.T., 2009. Talking bones: bioarchaeological analysis of individuals from Palpa. In: Reindel, M., Wagner, G. (Eds.), New Technologies for Archaeology. Springer-Verlag, Berlin, pp. 141–158.

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