Applying the principles of isotope analysis in plant and animal ecology to forensic science in the Americas.

The heart of forensic science is application of the scientific method and analytical approaches to answer questions central to solving a crime: Who, What, When, Where, and How. Forensic practitioners use fundamentals of chemistry and physics to examine evidence and infer its origin. In this regard, ecological researchers have had a significant impact on forensic science through the development and application of a specialized measurement technique-isotope analysis-for examining evidence. Here, we review the utility of isotope analysis in forensic settings from an ecological perspective, concentrating on work from the Americas completed within the last three decades. Our primary focus is on combining plant and animal physiological models with isotope analyses for source inference. Examples of the forensic application of isotopes-including stable isotopes, radiogenic isotopes, and radioisotopes-span from cotton used in counterfeit bills to anthrax shipped through the U.S. Postal Service and from beer adulterated with cheap adjuncts to human remains discovered in shallow graves. Recent methodological developments and the generation of isotope landscapes, or isoscapes, for data interpretation promise that isotope analysis will be a useful tool in ecological and forensic studies for decades to come.

Evaluation of childhood nutrition by dietary survey and stable isotope analyses of hair and breath.

OBJECTIVES: The natural abundances of carbon, nitrogen, and sulfur stable isotopes in hair, and of carbon isotopes in breath serve as quantitative biomarkers of protein and carbohydrate sources, but applicability of isotopes for evaluating children's diet has not been demonstrated. In this study, we sought to describe the stable isotope patterns observed in the hair and breath of children and to assess dietary variations in relation to age and ethnicity, hypothesizing that these would reflect dietary differences across age and ethnic groups and would correlate with intake variables derived from a Food Frequency Questionnaire. METHODS: Data were obtained from a cross-sectional study of non-Hispanic white (N = 115) and Hispanic (N = 97) children, aged 9-16 years, in Salt Lake City, Utah. Sampling included a hair sample, breath samples (AM and PM), and a youth/adolescent food questionnaire (YAQ). Hair was analyzed for carbon (δ13 C), nitrogen (δ15 N), and sulfur (δ34 S) isotopes, and breath samples for δ13 CAM/PM of respired CO2 . RESULTS: Non-Hispanic whites had lower δ13 C, δ15 N, δ13 CAM , and δ13 CPM values than Hispanics. Hair δ13 C and δ15 N values were correlated with protein sources, particularly for non-Hispanics. Breath δ13 C values were correlated with carbohydrate sources, particularly for Hispanic students. Non-Hispanic white students reported greater intake of total protein, animal protein, dairy, and grain than Hispanic students. Hispanic students reported higher intake of carbohydrates, particularly sweetened beverages. CONCLUSION: While YAQ and stable isotope data reflected strong cultural influences in diet, no significant gender-based nor age-based differences were detected. Significant covariation between YAQ and isotopes existed and demonstrate the potential of stable isotopes for characterizing children's diet.

Hydrogen and oxygen isotope ratios in body water and hair: modeling isotope dynamics in nonhuman primates.

The stable isotopic composition of drinking water, diet, and atmospheric oxygen influence the isotopic composition of body water ((2)H/(1)H, (18)O/(16)O expressed as δ(2) H and δ(18)O). In turn, body water influences the isotopic composition of organic matter in tissues, such as hair and teeth, which are often used to reconstruct historical dietary and movement patterns of animals and humans. Here, we used a nonhuman primate system (Macaca fascicularis) to test the robustness of two different mechanistic stable isotope models: a model to predict the δ(2)H and δ(18)O values of body water and a second model to predict the δ(2)H and δ(18)O values of hair. In contrast to previous human-based studies, use of nonhuman primates fed controlled diets allowed us to further constrain model parameter values and evaluate model predictions. Both models reliably predicted the δ(2)H and δ(18)O values of body water and of hair. Moreover, the isotope data allowed us to better quantify values for two critical variables in the models: the δ(2)H and δ(18)O values of gut water and the (18)O isotope fractionation associated with a carbonyl oxygen-water interaction in the gut (α(ow)). Our modeling efforts indicated that better predictions for body water and hair isotope values were achieved by making the isotopic composition of gut water approached that of body water. Additionally, the value of α(ow) was 1.0164, in close agreement with the only other previously measured observation (microbial spore cell walls), suggesting robustness of this fractionation factor across different biological systems.

Intrauterine growth restriction affects hippocampal dual specificity phosphatase 5 gene expression and epigenetic characteristics.

Intrauterine growth retardation (IUGR) predisposes humans toward hippocampal morbidities, such as impaired learning and memory. Hippocampal dual specificity phosphatase 5 (DUSP5) may be involved in these morbidities because DUSP5 regulates extracellular signal-regulated kinase phosphorylation (Erk). In the rat, IUGR causes postnatal changes in hippocampal gene expression and epigenetic characteristics. However, the impact of IUGR upon hippocampal DUSP5 expression and epigenetic characteristics is not known. We therefore hypothesized that IUGR affects hippocampal 1) DUSP5 expression, DNA CpG methylation, and histone code, and 2) erk1/2 phosphorylation in a well-characterized rat model of IUGR. We found that IUGR significantly decreased DUSP5 expression in the day of life (DOL) 0 and 21 male rat, while decreasing only DUSP5 protein levels in the DOL21 female rat. Fluorescent in situ hybridization and immunohistochemistry analyses localized the changes in DUSP5 mRNA and protein, many of which occurred in the dentate gyrus. IUGR also caused sex-specific differences in DNA CpG methylation and histone code in two sites of the hippocampal DUSP5 gene, a 5'-flanking specificity protein-1 (SP1) site and exon 2. Finally, when IUGR decreased DUSP5 protein levels, Erk phosphorylation increased. We conclude that IUGR affects hippocampal DUSP5 expression and epigenetic characteristics in a sex-specific manner.

Spatial distributions of carbon, nitrogen and sulfur isotope ratios in human hair across the central United States.

We present data on the carbon (δ(13)C), nitrogen (δ(15)N) and sulfur (δ(34)S) isotope ratios of human hair collected in the central portions of the USA. These elements are incorporated into hair from the diet and thus provide a record of dietary inputs that may also document geospatial patterns. We detected regional differences in hair δ(34)S values across the USA, with the lowest values in the northern Great Plains and increasing values towards the east, west and south. In contrast, no statistically significant patterns were detected in the spatial variation of human hair δ(13)C and δ(15)N values. Using δ(34)S values and a Geographic Information System approach, we created a map ('sulfur isoscape'). The accuracy of the map was tested using hair samples not included in its generation. We conclude that sulfur isotope analysis may represent a new tool to investigate the movements and/or region-of-origin of humans.

Variations in human body water isotope composition across the United States.

Accurate human provenancing using stable isotopes depends directly on solid understandings of the geographic and individual factors affecting isotope variability and incorporation into human tissues. Transfer of isotopic, and therefore spatial, information between environmental water and biological tissues is mediated by the isotopic composition of body water. Thus, there is a need to study body water isotope ratios at a population level and over a large isotopic and geographic range. We evaluated oxygen (δ18Obw) and hydrogen (δ2Hbw) isotope values of body water from 72 volunteers in 10 different cities across the US, and over a 5-10-day period. We analyzed covariates (e.g., water intake, physical activity, biometrics, gender) that might explain individual stable isotope ratio variations and tested a predictive model that incorporates the δ-values of drinking water, food, and O2 as well as individual variables to predict the δ-values of body water. The individual variability in body water isotope values overtime (mean 0.3‰ for δ18Obw and 2.3‰ for δ2Hbw) was lower than the intra-city variability (mean 0.9‰ for δ18Obw and 6.9‰ for δ2Hbw). Body water isotope values differed among cities (ANOVA: δ18ObwF = 97.2, p < 0.001; δ2HbwF = 176.2, p < 0.001). However, significant overlap among some cities with different drinking water was discovered. We detected significant covariation of measured drinking water and human body water isotope values (both isotope systems R2 ≥ 0.89, p < 0.001) and small but significant effects of the average daily exercise and amount of fluid intake. The differences between measured and model-predicted body water values (mean 0.12 ± 1.2‰ for Δδ18O and -1.2 ± 8.2‰ for Δδ2H) were statistically indistinguishable from zero (Δδ18O t = -0.751, p = 0.45; Δδ2H t = 1.133, p = 0.26). Here we show that community level variation exists in the δ18Obw and δ2Hbw values and the primary drivers are the regional differences in drinking water isotopes. Consistency of the body water isotope composition over the study period suggests that tissues would incorporate a stable isotope signal over time. The amount of drinking water and physical activity influence body water values, while the variation in the isotopic values of food may contribute to regional level variability, but that still remains to be assessed further. The human body water model provides accurate estimates for measured values, capturing and reproducing the main features of the body water isotope variation across space.

Intrauterine growth retardation affects expression and epigenetic characteristics of the rat hippocampal glucocorticoid receptor gene.

Studies in humans and rats suggest that intrauterine growth retardation (IUGR) permanently resets the hypothalamic-pituitary-adrenal (HPA) axis. HPA axis reprogramming may involve persistently altered expression of the hippocampal glucocorticoid receptor (hpGR), an important regulator of HPA axis reactivity. Persistent alteration of gene expression, long after the inciting event, is thought to be mediated by epigenetic mechanisms that affect mRNA and mRNA variant expression. GR mRNA variants in both humans and rats include eleven 5'-end variants and GRalpha, the predominant 3'-end variant. The 3'-end variants associated with glucocorticoid resistance in humans (GRbeta, GRgamma, GRA, and GRP) have not been reported in rats. We hypothesized that in the rat hippocampus IUGR would decrease total GR mRNA, increase GRbeta, GRgamma, GRA, and GRP, and affect epigenetics of the GR gene at birth (D0) and at 21 days of life (D21). IUGR increased hpGR and exon 1.7 hpGR mRNA in males at D0 and D21, associated with increased trimethyl H3/K4 at exon 1.7 at both time points. IUGR also increased hpGRgamma in males at D0 and D21, associated with increased acetyl H3/K9 at exon 3 at both time points. hpGRA increased in female IUGR rats at D0 and D21. In addition, our data support the existence of hpGRbeta and hpGRP in the rat. IUGR has sex-specific, persistent effects on GR expression and its histone code. We speculate that postnatal changes in hippocampal GR variant and total mRNA expression may underlie IUGR-associated HPA axis reprogramming.

Correlating diet and digestive tract specialization: examples from the lizard family Liolaemidae.

A range of digestive tract specializations were compared among dietary categories in the family Liolaemidae to test the hypothesis that herbivores require greater gut complexity to process plant matter. Additionally, the hypothesis that herbivory favors the evolution of larger body size was tested. Lastly, the association between diet and hindgut nematodes was explored. Herbivorous liolaemids were larger relative to omnivorous and insectivorous congeners and consequently had larger guts. In addition, small intestine length of herbivorous liolaemids was disproportionately longer than that of congeners. Significant interaction effects between diet and body size among organ dimensions indicate that increases in organ size occur to a greater extent in herbivores than other diet categories. For species with plant matter in their guts, there was a significant positive correlation between the percentage of plant matter consumed and small intestine length. Herbivorous liolaemids examined in this study lacked the gross morphological specializations (cecum and colonic valves) found in herbivores in the families Iguanidae and Agamidae. A significantly greater percentage of herbivorous species had nematodes in their gut. Of the species with nematodes, over 95% of herbivores had nematodes only in the hindgut. Prevalence of nematodes in the hindgut of herbivores was 2 x that of omnivores and 4 x that of insectivores.

δ2H and δ18O of human body water: a GIS model to distinguish residents from non-residents in the contiguous USA.

An understanding of the factors influencing the isotopic composition of body water is important to determine the isotopic composition of tissues that are used to reconstruct movement patterns of humans. The δ(2)H and δ(18)O values of body water (δ(2)H(bw) and δ(18)O(bw)) are related to the δ(2)H and δ(18)O values of drinking water (δ(2)H(dw) and δ(18)O(dw)), but clearly distinct because of other factors including the composition of food. Here, we develop a mechanistic geographical information system (GIS) model to produce spatial projections of δ(2)H(bw) and δ(18)O(bw) values for the USA. We investigate the influence of gender, food, and drinking water on the predicted values by comparing them with the published values. The strongest influence on the predicted values was related to the source of δ(2)H(dw) and δ(18)O(dw) values. We combine the model with equations that describe the rate of turnover to produce estimates for the time required for a non-resident to reach an isotopic equilibrium with a resident population.

Accuracy and precision of a laser-spectroscopy approach to the analysis of δ²H and δ¹8O in human urine.

The isotope ratio analysis of body water often involves large sample numbers and lengthy sample processing. Here we demonstrate the ability of isotope ratio infrared spectroscopy (IRIS) to rapidly and accurately analyse the isotope ratios of water in urine. We analysed water extracted from human urine using traditional isotope ratio mass spectrometry (IRMS) and compared those values with IRIS-analysed extracted water and un-extracted urine. Regression analyses for δ²H and δ¹8O values between (1) extracted water analysed via IRMS and IRIS and (2) urine and extracted water analysed via IRIS were significant (R²=0.99). These results indicate that cryogenic distillation of urine was not required for an accurate estimate of the isotopic composition of urine when using IRIS.

Links between purchase location and stable isotope ratios of bottled water, soda, and beer in the United States.

This study investigated the impact of purchase location on the stable isotope ratios of beverages by measuring the delta(2)H and delta(18)O values of bottled water, soda, beer, and tap water collected across the contiguous United States. Measured beverage delta(2)H and delta(18)O values generally fit the Global Meteoric Water Line (GMWL), suggesting region-of-origin information is recorded in beverage water. Tap water delta(2)H and delta(18)O values were strongly correlated with the stable isotope ratios of bottled water and soda purchased in the same location. Beer water delta(2)H and delta(18)O values were also correlated with tap water, although not as strongly. Variability in delta(2)H and delta(18)O values among beverages purchased at a single location ranged from 2 to 41 per thousand and from 0.3 to 5.2 per thousand, respectively, but was generally moderate in most locations. It was concluded that the isotopic composition of local tap water is a reasonable proxy for consumers' fluid intake in most U.S. cities.

Hydrogen and oxygen stable isotope ratios of milk in the United States.

Models of hydrogen and oxygen incorporation in human tissues recognize the impact of geographic location on the isotopic composition of fluid intake, but inputs can include nonlocal beverages, such as milk. Milk and cow drinking water were collected from dairies, and commercially available milk was purchased from supermarkets and fast food restaurants. It was hypothesized that milk water delta(2)H and delta(18)O values record geographic location information. Correlations between milk water isotope ratios and purchase location tap water were significant. However, the amount of variation in milk delta(2)H and delta(18)O values explained by tap water was low, suggesting a single estimation of fluid input isotope ratios may not always be adequate in studies. The delta(2)H and delta(18)O values of paired milk and cow drinking water were related, suggesting potential for geographical origin assignment using stable isotope analysis. As an application example, milk water delta(18)O values were used to predict possible regions of origin for restaurant samples.

Intrauterine growth restriction alters hippocampal expression and chromatin structure of Cyp19a1 variants.

We evaluated the impact of uteroplacental insufficiency (UPI), and subsequent intrauterine growth restriction (IUGR), on serum testosterone and hippocampal expression of Cyp19a1 variants and aromatase in rats. Additionally, we determined UPI induced histone modification of the promoter regions of Cyp19a1 variants using chromatin immunoprecipitation. Cyp19a1 is the gene encoding the protein aromatase, that catalyzes the biosynthesis of estrogens from androgens and is necessary for masculinization of the brain. IUGR was induced via bilateral uterine artery. UPI increased serum testosterone in day of life 0 (D(0)) and day of life 21 (D(21)) IUGR males to 224% and 299% of control values, respectively. While there was no significant impact of UPI on testosterone in D(0) females, testosterone in D(21) IUGR females was 187% of controls. Cyp19a1 variant 1.f and variant II are expressed in the rat hippocampus at D(0) and D(21). UPI significantly reduced expression of Cyp19a1 variant 1.f in D(0) males, with no impact in females. Similarly at D(0), UPI reduced expression of aromatase, the protein encoded by Cyp19a1, in males. Dimethylation of H3K4 was increased in the promoter region of variant 1.f (P1.f) and trimethylation of H3K4 was decreased in the promoter region of variant II (PII). At D(21), dimethylation of H3K4 is significantly reduced in PII of IUGR males. We conclude that UPI increases serum testosterone and reduces Cyp19a1 variant 1.f expression in the hippocampus of D(0) IUGR males. Additionally, UPI alters the chromatin structure of CYP19a1 at both D(0) and D(21).

Aberrant water homeostasis detected by stable isotope analysis.

While isotopes are frequently used as tracers in investigations of disease physiology (i.e., 14C labeled glucose), few studies have examined the impact that disease, and disease-related alterations in metabolism, may have on stable isotope ratios at natural abundance levels. The isotopic composition of body water is heavily influenced by water metabolism and dietary patterns and may provide a platform for disease detection. By utilizing a model of streptozotocin (STZ)-induced diabetes as an index case of aberrant water homeostasis, we demonstrate that untreated diabetes mellitus results in distinct combinations, or signatures, of the hydrogen (delta2H) and oxygen (delta18O) isotope ratios in body water. Additionally, we show that the delta2H and delta18O values of body water are correlated with increased water flux, suggesting altered blood osmolality, due to hyperglycemia, as the mechanism behind this correlation. Further, we present a mathematical model describing the impact of water flux on the isotopic composition of body water and compare model predicted values with actual values. These data highlight the importance of factors such as water flux and energy expenditure on predictive models of body water and additionally provide a framework for using naturally occurring stable isotope ratios to monitor diseases that impact water homeostasis.

Isotopic insight into host-endosymbiont relationships in Liolaemid lizards.

Nitrogen isotopes have been widely used to investigate trophic levels in ecological systems. Isotopic enrichment of 2-5 per thousand occurs with trophic level increases in food webs. Host-parasite relationships deviate from traditional food webs in that parasites are minimally enriched relative to their hosts. Although this host-parasite enrichment pattern has been shown in multiple systems, few studies have used isotopic relationships to examine other potential symbioses. We examined the relationship between two gut-nematodes and their lizard hosts. One species, Physaloptera retusa, is a documented parasite in the stomach, whereas the relationship of the other species, Parapharyngodon riojensis (pinworms), to the host is putatively commensalistic or mutualistic. Based on the established trophic enrichments, we predicted that, relative to host tissue, parasitic nematodes would be minimally enriched (0-1 per thousand), whereas pinworms, either as commensals or mutualists, would be significantly enriched by 2-5 per thousand. We measured the (15)N values of food, digesta, gut tissue, and nematodes of eight lizard species in the family Liolaemidae. Parasitic worms were enriched 1+/-0.2 per thousand relative to host tissue, while the average enrichment value for pinworms relative to gut tissue was 6.7+/-0.2 per thousand. The results support previous findings that isotopic fractionation in a host-parasite system is lower than traditional food webs. Additionally, the larger enrichment of pinworms relative to known parasites suggests that they are not parasitic and may be several trophic levels beyond the host.