Fatty acid composition and preservation of the Tyrolean Iceman and other mummies

In anthropology, objective parameters to adequately describe storage conditions and the preservation of mummies have yet to be identified. Considering that fatty acids degrade to stable products, we analysed their profile in human mummies and in control samples by gas chromatography coupled to mass spectrometry (GC/MS). Originating from different epochs and civilizations, samples of the Tyrolean Iceman, other glacier corpses, a freeze dried mummy, corpses from a permafrost region, a corpse mummified immersed in water, and a desert mummy were evaluated. Chemometric analysis based on the concentrations of 16 fatty acids revealed the degree of similarity between anthropologic and fresh corpse samples, which was mainly influenced by the content of palmitic acid, oleic acid, and 10-hydroxystearic acid. The presence of 10-hydroxystearic acid was associated with immersion in water, whereas dry mummification was accompanied by high contents of oleic acid. Samples of the Tyrolean Iceman clustered between fresh tissue and those of other glacier corpses indicating the good preservation of this mummy.Thus, environmental post-mortem conditions were associated with characteristic fatty acid patterns suggesting that chemometric analysis of fatty acid contents may add to our knowledge about post-mortem storage conditions and the preservation of human corpses. In anthropology, objective parameters to adequately describe storage conditions and the preservation of mummies have yet to be identified. Considering that fatty acids degrade to stable products, we analysed their profile in human mummies and in control samples by gas chromatography coupled to mass spectrometry (GC/MS). Originating from different epochs and civilizations, samples of the Tyrolean Iceman, other glacier corpses, a freeze dried mummy, corpses from a permafrost region, a corpse mummified immersed in water, and a desert mummy were evaluated. Chemometric analysis based on the concentrations of 16 fatty acids revealed the degree of similarity between anthropologic and fresh corpse samples, which was mainly influenced by the content of palmitic acid, oleic acid, and 10-hydroxystearic acid. The presence of 10-hydroxystearic acid was associated with immersion in water, whereas dry mummification was accompanied by high contents of oleic acid. Samples of the Tyrolean Iceman clustered between fresh tissue and those of other glacier corpses indicating the good preservation of this mummy. Thus, environmental post-mortem conditions were associated with characteristic fatty acid patterns suggesting that chemometric analysis of fatty acid contents may add to our knowledge about post-mortem storage conditions and the preservation of human corpses. In September 1991, an approximately 5,000-year-old frozen male mummy was found in the Similaun glacier of the Tyrolean Alps (1Seidler H. Bernhard W. Teschler-Nicola M. Platzer W. zur Nedden D. Henn R. Oberhauser A. Sjøvold T. Some anthropological aspects of the prehistoric Tyrolean Ice Man.Science. 1992; 258: 455-457Google Scholar). Interestingly, the subcutaneous tissue and the adipose tissue of the so-called Tyrolean Iceman appeared macroscopically better preserved than tissue of other corpses buried in glaciers for much shorter periods of time. Consecutive studies, however, showed an almost complete degradation of macromolecules (2Handt O. Richards M. Trommsdorff M. Kilger C. Simanainen J. Georgiev O. Bauer K. Stone A. Hedges R. Schaffner W. Utermann G. Sykes B. Pääbo S. Molecular genetic analyses of the Tyrolean Ice Man.Science. 1994; 264: 1775-1778Google Scholar). This indicated the need for more appropriate methods to adequately describe post-mortem alterations of anthropologic finds. Since fatty acids are small molecules with defined degradation products, the study of these components is more likely to reflect the influence of environmental conditions on post-mortem alterations (3Gülaçar F.O. Buchs A. Capillary gas chromatography-mass spectrometry and identification of substituted carboxylic acids in lipids extracted from a 4000-year-old Nubian burial.J. Chromatogr. 1989; 479: 61-72Google Scholar, 4Mayer B.X. Reiter C. Bereuter T. Investigation of the triacylglycerol composition of iceman's mummified tissue by high-temperature gas chromatography.J. Chromatogr. B. 1997; 692: 1-6Google Scholar). Post-mortem, body fat is converted into adipocere under humid and microaerobic conditions. Adipocere is a lipid mixture of wax-like consistency and greyish-white color consisting mainly of free saturated fatty acids with even numbers of carbon atoms and eventually hydroxy-fatty acids. The formation of the latter has been attributed to biotic as well as to abiotic processes (5Takatori T. Yamaoka A. The mechanism of adipocere formation 1. identification and chemical properties of hydroxy fatty acids in adipocere.Forensic Sci. 1977; 9: 63-73Google Scholar, 6Takatori T. Investigations on the mechanism of adipocere formation and its relation to other biochemical reactions.Forensic Sci. Int. 1996; 80: 49-61Google Scholar). On the other hand, air circulation and/or elevated temperatures lead to mummification of human tissue by means of desiccation. Under these conditions, the epidermis becomes tanned thus protecting the tissue underneath. A rapid desiccation process is often associated with macroscopically well-preserved tissue. To learn more about the millennial preservation of the Tyrolean Iceman, we analysed the fatty acid composition of Iceman's tissue specimens by gas chromatography coupled to mass spectrometry (GC/MS). Using chemometric methods, these data were compared with those of other well-preserved mummies from different epochs and civilisations exposed to defined climatic conditions. Specimens obtained from the Tyrolean Iceman, two other corpses found in glaciers nearby, a body permanently immersed in an Austrian mountain lake over 50 years, two Scythian corpses buried in the permafrost of Siberia, a freeze dried Inca mummy from the Peruvian Andes, and, finally, a mummy buried in the Peruvian desert were evaluated (Table 1). As reference, the fatty acid profile of fresh tissue samples from three recently deceased control subjects were evaluated (17 specimens including skin, muscle, bone marrow, lung, and liver).TABLE 1Origin, storage conditions, burial time, and specimens of the evaluated human mummies and control subjectsIDOrigin and Storage ConditionsBurial TimeProbable Age at DeathSpecimen (Weight)aBecause of the unique nature of ancient specimens, it was not possible to obtain larger or multiple samples.yearsmgA“Tyrolean Iceman”, glacier Similaun, South Tyrol, Italy; 3,200 m altitude; male corpse probably exposed to weather immediately after death, then stored in ice, found in melting ice in 1991.∼5,20035–40A1: trabecular bone (11), A2: nasal cavity (8.8); A3: paranasal sinus (8.2); A4: skin left hip (13).BGlacier Madatschferner, Austria; 2,800 m altitude; female corpse initially buried in the glacier, after melting of ice likely to be immersed in water for several months, found uncovered on ground free of snow in 1952. 2928B1: muscle right calf (15); B2: skin right thigh (17); B3: left lung surfacial adipocere (9.4); B4: liver surfacial tissue (10); B5: liver internal tissue (12).CGlacier Sulztalferner, Austria; 2,700 m altitude; male corpse partially buried in the glacier, likely to be immersed in melting ice for several months, found uncovered on ice in 1991. 5762C1: muscle upper left arm (17); C2: liver internal tissue (13); C3: skin abdomen (15); C4: skin with fat and muscle radial section of upper arm (19).DMountain lake Achensee, Austria; female corpse found in 50 m depth in 1989. 5030D1: left lung (17); D2: cardiac muscle (12); D3: muscle left thigh (14).EAltai Mountains, Siberia, Russia; 2,500 m altitude; male corpse buried in a permafrost zone, excavated completely enclosed in ice in 1995.∼2,200Adult; detail information missingE: skin abdomen (13).FAltai Mountains, Siberia, Russia; 2,500 m altitude; female skeleton with residual tissue found buried in a permafrost zone in 1993.∼2,500Adult; detail information missingF: tissue from the pelvic region (9.7).GMount Ampato, Andes, Peru; 6,000 m altitude; female corpse dry frozen by mountain winds in a zone of eternal ice, found in 1995. ∼5008–10G1: skin left temple (11); G2: hair left temple (9.1).HIlo, Peru; male corpse mummified in a desert without rainfalls in the last millennium, found in 1988.∼1,000Adult; detail information missingH: muscle left lower leg (16).I–KThree fresh corpses as reference (two females and one male).47, 72, and 90, respectivelyI1: muscle abdomen (17); I2: muscle lower leg (23); I3: liver (15); I4: lung (18); I5: bone marrow (13); I6: skin with fat thigh (21); I7: skin with fat abdomen (19); J1: liver (13); J2: lung (23); J3: muscle abdomen (27); J4: skin with fat thigh (21); J5: skin with fat abdomen (19); K1: liver (22); K2: lung (15); K3: muscle abdomen (25); K4: skin with fat thigh (28); K5: skin with fat abdomen (21).a Because of the unique nature of ancient specimens, it was not possible to obtain larger or multiple samples. Open table in a new tab In order to saponify the lipid material, tissue samples were homogenised and treated for 30 min at 100°C with 1 ml of a mixture of 7.5 N sodium hydroxide (Merck, Darmstadt, Germany) and methanol (1:1, v/v; Merck). The sodium salts of the free fatty acids were converted to their methyl esters by adding 2 ml of a mixture of methanol and 6 N hydrochloric acid (4.6:5.4, v/v; Merck) and heating for 10 min at a temperature of 80°C. Fatty acid methyl esters were then transferred from the acidic aqueous phase to an organic phase by liquid-liquid extraction using 1.25 ml of a mixture of n-hexane and t-butylethylether (1:1, v/v; Merck). Finally, cleanup of the organic extract was performed by liquid-liquid extraction using 3 ml of a 0.3 N sodium hydroxide solution. All reagents were of analytical grade. This extraction protocol—first described by Sasser (7Sasser M. MIS whole cell fatty acid analysis by gas chromatography. Technical note #101. MIDI Inc., Newark, DE1990Google Scholar)—allows for the analysis of the whole fatty acid content of tissue specimens including bound and unbound fatty acids, and also those originating from sources other than lipids (e.g., lipoproteins). Considering the small amount of the available ancient samples, this approach may maximize the recovery of fatty acids. The extracts were subjected to qualitative and quantitative analysis twice by gas-liquid chromatography (Hewlett Packard 5890, Agilent Technologies, Waldbronn, Germany) using a capillary column (Hewlett Packard Ultra 2; 25 m × 0.2 mm × 0.33 μm film thickness with 5% phenyl methyl silicone as stationary phase) coupled to a mass spectrometer (Finnigan 8200, Bremen, Germany). Species resolved by gas chromatography were identified by mass spectrometry using the database system MassLib (Max-Planck Institut für Kohlenforschung, Mülheim an der Ruhr, Germany). In order to visualize the relationships within the fatty acid data set, principal component analysis (PCA) was applied (8Massart D.L. Vandeginste B.G.M. Buydens L.M.C. De Jong S. Lewi P.J. Smeyers-Verbeke J. Handbook of chemometrics and qualimetrics: Part A. Elsevier, Amsterdam, The Netherlands1997: 519-556Google Scholar). This standard technique of exploratory multivariate data analysis was chosen due to the composition of the available data set that, because of the small number of samples and the lack of replicates, can not be evaluated by elementary statistical tests. More importantly, this advantageous multivariate approach uses a set of variables instead of a single variable for the description of similarities between samples. The concentrations of 16 selected fatty acids (threshold: 1% of total fatty acids) were used as features to characterize a sample. In order to eliminate the influence of absolute concentration values, the features were autoscaled (to a mean of zero and a variance of 1) before PCA. The resulting first and second principal component scores (each a linear combination of the concentrations of the 16 fatty acids) were used as coordinates for a scatter plot with a point for each sample. Visual inspection of this score plot shows clustering according to the similarity of samples. In a loading plot, the principal component loadings were used as coordinates for points that correspond to the features (fatty acids). A fatty acid, which for instance is located in the upper right hand corner, is characteristic for samples located in the same region of the score plot. Fatty acids with a large distance from the origin of the coordinate system possess highest influence on the data set. The results obtained from PCA were confirmed by cluster analysis using dendrograms, and by k-nearest neighbour classifications. These methods extract relevant information from a data matrix and are useful in the interpretation of results. However, the small data set did not allow for the estimation of statistical validity. The software used was SCAN (Minitab Inc., State College, PA). As shown in Fig. 1A, unsaturated fatty acids and palmitic acid dominated the fatty acid profile of fresh tissue. The concentration of unsaturated fatty acids, predominantly oleic acid (18:1), was higher in specimens of the Tyrolean Iceman (Fig. 1B; Table 2, line A) than in samples from the other corpses found in glaciers nearby (Fig. 1C; Table 2, lines B and C). With concentrations up to 49%, the amount of hydroxy stearic acid (18:0 10OH) was similar in both the Tyrolean Iceman and the other two glacier corpses (Fig. 1B, C; Table 2).TABLE 2Concentrations of the main components of the fatty acids in the evaluated human mummies and control subjectsHuman Mummies and Control SubjectsFatty AcidsABCDEFGHIJKMyristic acid (14:0)2.8–7.03.2–8.52.8–6.2 15–216.05.53.3–4.24.1<1–5.03.1–6.41.8–4.7Palmitoleic acid (16:1)0–5.0n.d.0–2.7n.d.1.1n.d.4.0–4.5192.7–103.5–7.62.9–10Palmitic acid (16:0) 18–36 21–37 36–50 35–462555 30–3322 17–30 22–32 25–30Linoleic acid (18:2)0–2.3n.d.n.d.n.d.n.d.n.d.n.d.1.28.6–18 12–15 13–15Oleic acid (18:1)8.7–221.4–4.22.9–82.9–4.3107.1 19–2544 27–45 30–46 27–4210-Hydroxypalmitic acid (16:0 10OH)0–3.53.5–8.72–3.4n.d.n.d.n.d.n.d.n.d.n.d.n.d.n.d.Stearic acid (18:0)5.0–17 3–193.4–7.44.4–9.96.3268.7–141.82.5–112.5–7.1 2.2–10Arachidonic acid (20:4)n.d.n.d.n.d.n.d.n.d.n.d.n.d.n.d.<1–9.4<1–7.7<1–1210-Hydroxystearic acid (18:0 10OH) 15–48 21–497.5–439.8–3445n.d. 0–1.21.2n.d.n.d.n.d.Data represent the concentration range of the respective fatty acid. Results are calculated as percentage of the total fatty acid concentration (n.d., not detected). Letters A–K identify the human mummies and control subjects evaluated as indicated in Table 1. Open table in a new tab Data represent the concentration range of the respective fatty acid. Results are calculated as percentage of the total fatty acid concentration (n.d., not detected). Letters A–K identify the human mummies and control subjects evaluated as indicated in Table 1. The influence of continuous immersion into cold water on post-mortem degradation was studied by analysing the fatty wax of a body recovered 50 years after death from a mountain lake in a depth of 50 meters. The fatty acid composition of samples obtained from this corpse (Table 2, line D) differed from the recently buried glacier corpses mainly in the higher concentrations of myristic acid (range: 15–21% of total fatty acids vs. 2.8–8.5% in glacier corpses). The fatty acid profiles obtained from two corpses buried in the Altai mountains differed between each other (Pazyryk culture in Siberia). One of them, a Scythian warrior, was excavated as a frozen mummy in a wooden coffin filled with ice. The fatty acid composition of this specimen was similar to that observed in specimens of glacier corpses with 10-hydroxystearic acid as the dominant component (45% of total fatty acids; Table 2, line E). After burial, the other corpse decayed in the permanent frost without being enclosed in ice. A specimen from the pelvic region was one of the few tissue samples conserved on this skeleton. In contrast to the specimen obtained from the Scythian warrior, the fatty acid profile of this sample consisted predominantly of saturated fatty acids (Table 2, line F). Two specimens originating from a freeze-dried mummy found in a cavern of the Peruvian Andes were also evaluated. by the for years this well preserved mummy of a was by winds at an of 6,000 A high concentration of unsaturated fatty acids was observed and of total fatty whereas hydroxy stearic acid was completely (Table 2, line in these samples was a fatty acid with carbon atoms and of the total fatty acids). under the complete of has been by analysing a tissue sample from a burial in the Peruvian desert years In this has been for of This specimen showed almost of fresh in the concentration of acid vs. of total fatty acids) accompanied by an concentration of acid vs. of total fatty acids; Table 2, line Chemometric data analysis by principal component analysis was performed to similarities samples based on their fatty acids a multivariate approach may samples, even single variables not The score plot (Fig. from the first and second principal component shows which can be attributed to different sample The consisted of the fresh tissue samples from three different corpses clustering different specimens as skin, muscle, bone marrow, and lung This was not because the statistical analysis of the lead palmitic acid, oleic acid, and acid showed a of of to control samples Samples obtained from the desert corpse and the Inca mummy and were to the of fresh to this samples of the glacier corpses B and a second With of the skin specimen the samples of the Tyrolean Iceman were between these two As by the loading plot (Fig. the clustering of samples is mainly influenced by three oleic acid, 10-hydroxystearic acid, and palmitic acid. Considering these components in the Tyrolean Iceman, the skin the exposed region of the differed from the other three samples acid, vs. 10-hydroxystearic acid, vs. palmitic acid, vs. component analysis (PCA) of the autoscaled fatty acid is the first principal component of total and is the second principal component of total and the sample identification as defined in Table of is linear in score plot clustering of the loading plot the fatty acids that are characteristic for the in the score The number of available and defined specimens may be This may the of recovered tissue as well as the amount of sample. As a for the performed in the one to the of different tissue As shown in Fig. the consisted of specimens from three recently deceased subjects indicating that the of tissue a influence with to their on the score plot Thus, the of a sample on the score plot is mainly due to the environmental conditions it has been exposed to than to the of the The samples from the two glacier corpses as well as those from the corpse recovered from a lake a second (Fig. samples The specimens of the Tyrolean Iceman were between these two (Fig. samples This was and indicated the good preservation of this mummy in to the corpses, which were buried in glaciers for much shorter periods and years, The of the samples from the Tyrolean Iceman is likely due to the environmental conditions to which the different body were exposed the millennial to higher concentrations of 10-hydroxystearic acid and lower concentrations of oleic acid, the skin as the exposed of the Tyrolean Iceman be from samples of the other glacier corpses (Fig. sample All samples from both the glacier corpses and the corpse recovered from the lake were by the presence of 10-hydroxystearic acid, which was likely from the of a to the in oleic acid. This was the samples originating from the frozen Scythian corpses, which were found buried in the same The specimen of the Scythian enclosed in ice was dominated by the presence of 10-hydroxystearic acid and was within the glacier on the score plot (Fig. sample whereas the sample of the Scythian skeleton was by saturated fatty acids completely 10-hydroxystearic acid (Fig. sample F). In contrast to the Scythian the corpse of the Scythian has been immersed in water (e.g., for several a which then into ice. These the formation of 10-hydroxystearic acid to be associated with storage conditions. This is by the presence of of this fatty acid in the body permanently immersed in the mountain lake as well as in the glacier corpses, which were exposed to to the storage temperatures of these corpses, the in T. Investigations on the mechanism of adipocere formation and its relation to other biochemical reactions.Forensic Sci. Int. 1996; 80: 49-61Google is to a in the formation of 10-hydroxystearic acid. The mechanism of the post-mortem fatty acid however, to be In of fatty acids with an number of carbon atoms is Thus, the high concentrations of a fatty acid with carbon atoms in samples of the freeze-dried mummy from the Peruvian Andes was an However, the a the origin of this fatty acid. As from the of 10-hydroxystearic acid as well as the high concentrations of unsaturated fatty acids, the score plot the samples of this mummy in to the fresh specimens (Fig. samples The preserved however, was the one excavated in the Peruvian The sample obtained from this mummy was with fresh tissue on the score plot (Fig. sample even in this well-preserved specimen acid was not which may be due to the nature of this fatty acid. that multivariate of fatty acid profiles of anthropologic samples from different epochs and the respective preservation and about the post-mortem storage conditions. The Tyrolean Iceman was found to be better preserved than corpses buried in glaciers for much shorter This can be by rapid desiccation of the Tyrolean Iceman by mountain winds after death, as by (1Seidler H. Bernhard W. Teschler-Nicola M. Platzer W. zur Nedden D. Henn R. Oberhauser A. Sjøvold T. Some anthropological aspects of the prehistoric Tyrolean Ice Man.Science. 1992; 258: 455-457Google Scholar). The corpse was probably then enclosed in ice, including periods of in water as indicated by the presence of 10-hydroxystearic acid, which is a for storage of anthropologic specimens in