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EMBALMING IN THE OLD KINGDOM OF PHARAONIC EGYPT Controversy exists over whether embalming was already being performed in the Old Kingdom in the course of mummifying the deceased. here is controversy as to whether embalming was performed in the course of mummification of the deceased in the Old Kingdom of Pharaonic Egypt (2660-2180 B.C.). Our knowledge of conservation techniques used in the Old Kingdom is limited. In general, mummification was accomplished after dehydrating the body. To improve this mummification process, embalming was progressively used from the time of the Middle Kingdom onward. In an earlier study from the Tubingen laboratory, mummified alkaline phosphatase was successfully isolated from bone samples obtained from a Ptolemaic mummy (1). This mummy had been richly pretreated with phenolic and fungicidal compounds, which suppressed secondary
Ulrich Weser Yoka Kaup Hedwig Etspüler Universität Tübingen (Germany)
Johann Koller Ursula Baumer Doerner Institut München (Germany)
microbial growth. We were very interested in ascertaining the limitations of preserving this zinc-magnesium enzyme in mummified Egyptian bone samples dating back to the Old Kingdom when no conservation, according to general knowledge, was assumed to have been done. Thus, we performed a study on the wellcharacterized, mummified skeleton of IDU II, secretary general of the pine wood trade
office. This high-ranking official lived some 2200 years B.C. and was buried in a solid wood coffin. He was identified by the inscriptions on the outside of his thick, boarded coffin. The tomb of IDU II was unearthed in 1914 at Giza, and its contents were brought to the Pelizaeus Museum (Hildesheim, Germany) (http://mfah.org/ splendor/docs/highlts/hlldemus.html). The mummy (inventory No. 2639) con-
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Figure 1 . Gas chromatogram of the methanol/oxalic acid e x t r a c t . A, methyl dehydroabietate; B, methyl dehydrodehydroabietate; C, dehydroabietic acid; D, dehydrodehydroabietic acid; E, methyl 7-oxo-dehydroabietate; F, methyl 7-oxo-dehydrodehydroabietate; G, 7-oxo-dehydroabietic acid; H, unidentified. Note that the doublet in E is attributed to two isomer forms of the methyl esters.
sisted of an unwrapped skeleton with a ban- tion of Al203-sandblasted clavicle fragment daged head. with organic solvents was used, in which the solvents were geared to the specific Unfortunately, the mummy was soaked solubility of the possible embalming matewith paraffin immediately after recovery (2, 3), which hampered subsequent analyses for rials, that is, natural resins, wood tars, and bitumina. The sandblasting was done to alkaline phosphatase and possible ancient alleviate the severe paraffin contamination embalming components. In this Analytical Approach, we present an improved prepara- that obscured GC analyses. A 1-g piece of clavicle was extracted three times with isotion technique for rsolating aakallne phosphatase that was used on a clavicle fragment octane, each time for 12 h. The pellet of from the mummy of IDUII. The clavicle was remaining material was centrifuged and reextracted two times in 3:1 chloroform/ chosen because it belongs to the group of compact flat bones known to be rich in alka- methanol each time for 70 h. After ration of the solvent the solid residue was line phosphatase. Highly specific monoclonal antibodies directed against the human suspended in methanol and in the last bone used to exclude possible step the resulting extract was acidified with 10% (w/v) oxalic acid microbial contamination The specificity of the reaction was further controlled All extracts were injected into the gas as the same microbial activity can be mimchromatograph onto a DB5-ht fused-silica icked by melanoidins humic substances capillary column with an inner diameter of and lanthanide 0.32 mm and a stationary-phase film thickness of 0.1 um wiihout prior derivatizatton. The temperature-programmed GC run Embalming components started at 80 °C and, after 2 min, the even Two procedures were used for extracting embalming components. In the first proce- temperature was continuously raised at 10 K/min to 320 ° C. For peak identification, dure, a 3:1 (v/v) mixture of chloroform/ the extracts were analyzed by GC/MS. methanol was chosen to dissolve all embalming products for a total extraction. In The hydrocarbons originating from the the second procedure, a stepwise extracparaffin treatment predominate in the gas 512 A
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chromatogram of the total extract. Steroids and low-molecular-weight fatty acids were also prevalent. Cyclic alcohols could be detected as marginal components, including cedrol, gujacol,fetf-octylphenol,trimethylcyclohexene methanol, and octahydronaphthalene methanol. These volatile products originate from the liquid fraction of wood tar and are known for their bactericidal and fungicidal properties. In the gas chromatogram, the components of the accompanying solid fraction of this wood tar coelute with the hydrocarbons from the paraffin. Therefore, they could be detected only by their characteristic mass fragments in the corresponding GC/MS analysis. In addition, pyrrol derivatives in the total extract were identified by GC/MS. These rather peculiar compounds are abundant in bone tar, which is produced by smoldering bones. The presence of these compounds indicates that the skeleton of the IDU mummy was possibly flamed with a tar-rich torchlight. Both the observed tar components and the pyrrol derivatives could have been condensed and/or formed in situ on the bone surface Of course, any documentary evidence is missing. The embalmers had to swear an oath not to tell the secret of embalming techniques to anybody. A fragmentary treatise on mummification procedures was compiled some 1700 years later by the Greek historian Herodotus (5th century B.C.), but skeletonization and flaming were not mentioned. Perhaps the technique was abandoned by that time. In the chloroform/methanol extract, neither triterpenoids nor aromatized triterpenoids could be detected by GC or GC/ MS. The lack of triterpenoids in this extraction step eliminates mastic, terebinth (Chios turpentine), frankincense, and/or myrrh as the possible resin source (4), and the lack of aromatized triterpenoids (dehydrogenated, dehydrated, decarboxylated, and dealkylated pentacyclic compounds) excludes bituminous materials (5). As in the total extract volatile wood tar com r)o~ nents were identified again in the chloroform/methanol extract. Analysis of the methanol/oxalic acid extract revealed diterpenoids originating from either natural resins or wood tars Aged diterpenoid resins are composed mainly of triterpenoid
Figure 2. Components of t h e embalming material. A, methyl dehydroabietate; B, methyl dehydrodehydroabietate; C, dehydroabietic acid; D, dehydrodehydroabietic acid; E, methyl 7-oxo-dehydroabietate; F, methyl 7-oxo-dehydrodehydroabietate; G, 7-oxo-dehydroabietic acid. The mass spectrum of dehydrodehydroabietic acid (D) is superimposed on the ions of dehydroabietic acid (C).
acids, whereas aged wood tars contain these resin acids and their methyl esters as well. As a rule, these polar resin acids dissolved in methanol (or chloroform/methanol) cannot be identified by the nonpolar DB5-ht capillary column used in this study unless transformed into their methyl esters. However, this derivatization would obscure the difference between the resin acids and the resin acid esters.
To avoid these undesirable methylation reactions, a different approach was used in which a minimum of 10% (w/v) water-free oxalic acid was added to the methanol solution (6). When these acidified resin solutions are injected, the oxalic acid (unless it decomposes within the injector) is transferred through the column immediately after the solvent and occupies all the active sites in the injector, column, and the detector. This
pretreatment prevents the diterpene resin acids from being absorbed and retained in those places already occupied by the stronger-binding oxalic acid. At the same time, the polarity of the separation column undergoes a short-term shift toward the polar range to the extent that the polar resin acids become capable of interacting with the liquid (stationary) phase and can thus be successfully separated.
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In this extract, high concentrations of diterpenoid resin acids (mainly dehydroabietic acid and its oxidized forms) and methyl esters of these acids were detected (Table 1 and Figure 1)) The high concentration of dehydroabietic acid results from heat conversion and oxidation of abietaneand pimarane-type resin acids found in pine wood resin (7,8). Methyl esters are not abundant in natural resins. They are formed only in the course of smoldering resinous wood (9) but not by the alternative method of heating the resin alone
Thus, oxidized diterpenoid resins as well as their methyl esters exist in this extract and therefore could be identified side by side using this technique. For this reason, methylation was avoided because it would obscure these differences, which reveal the production of the tar from the resinous pine wood. It appears that a process in the presence of excessive air, rather than the more oxygen-deficient smoldering process, was used. Aromatic hydrocarbons normally formed during oxygen-deficient smolder-
ing (e.g., retene) are virtually absent (10). In this case, oxidized diterpenoid resin acids and their esters occur in the extracted embalming material (Figure 2). Therefore, a smoking technique must have been used instead of a smoldering process. Sodium and chloride
Samples of ancient bone fragments and recent bone were wet-ashed in the presence of HC1 and H202 for 48 h. Diluted allquott were subjected to flame atomic emission spectrometry at 589 nm. In IDU's clavicle, , a2fold-higher sodium content (4100 umol/g dry weight) was found compared with the sodium content of bones from modern autopsies (330 umol/g dry weight). Chloride was assayed using the reaction with mercuric chloranilate to yield HgCl2 and chloranilic acid (11). The concentration of the free violet-colored chloranilic acid is proportional to that of the chloride concentration and was determined from the spectrum taken at 540 nm. Samples of ancient bone fragments and recent bone were wetashed in the presence of H2S04 for 12 h. Aliquots were diluted with water, and 2025 mg of mercuric chloranilate were added. After centrifugation, the absorption of the supematant was recorded at 540 nm The chloride concentration was 3 5-fold higher in the ancient bone fragments (102 umol/g dry weight) compared with recently autoDsied bones (29 umol/g dry weight) .f sodfum chloride were used for desiccation hnrlv tVipn the chloriHp' rnnrpntration
should have been much higher This rates that the skeleton must have been nre, f d .th .. carhnnate/bica h t
together (natronizauon) with a minor poruon of sodium chlonde. Alkaline phosphatase
Figure 3. (a) Protein and (b) activity distribution of gel-filtrated extracts of a clavicle from IDU II and from a modern human rib (dotted line). 514 A
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Fragments of sand-blasted clavicle were finely ground in a porcelain mortar and suspended in Tris acetate at pH 7.4 containing 1 mM magnesium acetate, 0.3% (v/v) Triton X-100, and protease inhibitors (6aminohexanoic acid, phenylmethylsulphonylfluoride, and JV-ethyl-maleimide) under slight agitation for 20 h at 4 °C. After centrifugation the supernatant was concentrated by ultrafiltration through a membrane and gel-filtrated (1). The active fractions were collected concentrated and
Table 1 . Diterpenoid resin components Compound A B C D E (doublet) F G
Methyl dehydroabietate Methyl dehydrodehydroabietate Dehydroabietic acid Dehydrodehydroabietic acid Methyl 7-oxo-dehydroabietate Methyl 7-oxo-dehydrodehydroabietate 7-Oxo-dehydroabietic acid
affinity-chromatographed on a wheat germ lectin agarose column and eluted with Af-acetyl-D-glucoseamine. The enzyme assay in aqueous solution was based on the increase in the formation of ^-nitrophenol as a result of />-nitrophenyl phosphate hydrolysis caused by alkaline phosphatase. The hydrolysis rate was determined by recording at 410 nm for 60 min against a blank containing the solvent buffer and the buffered substrate. Because bone alkaline phosphatase was shown to survive the mummification techniques of the Ptolemaic period (1), we were interested in examining how embalming techniques used in the Old Kingdom affected the integrity of the enzyme. Large aggregates of alkaline phosphatase sometimes associated with cell membranes appeared close to the size-exclusion band near 600 kD (Figure 3a). Six distinctively different protein bands were seen after gel filtration of the modern bone extract. In contrast, distinct protein bands beyond 200 kD disappeared during the ancient protein separation, leaving the major portion in the low relative molecular mass (Afr) region of 20-50 kD. During gel chromatography, enzyme activity was located in the M - 200 ± 30 kD band, which ii virtually identical to the modern sample (Figure 3b). Chromatography of the old enzyme yields an additional activity band that appears at M = 115 5 15 kD. This protein peak is consistent with the polypeptide frame of bone alkaline phosphatase which is composed of two subunits at M = 57.2 2D (12) Unlike in the 600-kD band of fhe recent enzyme no activity was moniiored in the corresponding band of the old en7vmp Further improvement of the enzymic activity was seen after affinity chromatographv when the specific activity rose to 200 mU/rrw
M* (mlz)
Base peak (mlz)
314 312 300 298 328 326 314
239 237 285 197 253 251 253
(One enzyme unit [U] is defined as the amount of enzyme required for 1 umol of substrate to hydrolyze per minute.) Inhibition To exclude the possibility that phosphatase activity may have been simulated by compounds that mimic alkaline phosphatase, several specific inhibition studies were performed on the protein preparation. Denaturation of the enzyme protein was accomplished by heating a gel-filtrated protein sample to 95 °C for 10 mii. A nonheated sample served as the control. Heat denaturation resulted in the complete loss of activity. Aliquots of a gel-filtrated IDUII protein sample were treated with /Hiitrophenyl phosphate in Tris acetate at pH 8.2, and the increase of /Miitrophenol was spectrophotometrically recorded. After 20 min, 1,10phenanthroline was added, and the release of /Miitrophenol was monitored for an additional 40 min. The catalyttc function of the mummified enzyme was inhibited by 80% in the presence of 1,10-phenanthroline. Activity was restored after adding Zn(II). Phenanthroline successfully removes Zn(II) by chelation or at teass firmly binds it at the substrate Zn(II) coordination site within the protein. For inhibition with L-homoarginine, an allosteric-acting specific inhibitor of bone alkaline phosphatase regulation, an ancient and a recent protein preparation were incubated in Tris buffer and L-homoarginine at 4 °C for 1 h. The rate of inhibition was expressed as a percentage of the inhibited control in which L-homoarginine was omitted. Enzyme activity diminished by 77%. Zn(II) displacement studies in the active center were performed with Cd (II). About 0.4 uM protein was incubated in Tris buffer and cadmium sulfate for 1 h. The rate of inhibition was expressed as a per-
centage of the inhibited control in which Cd(II) was omitted. Replacement of coordinated Zn(II) in the active center by Cd(II) decreases the activity to 48% compared with that of the native zinc enzyme. For inhibition with ortho-vanadate as a phosphate analogue, ortho-vanadate in concentrations of 1,10,100, and 1000 uM was added to an ancient protein preparation. The rate of inhibition was expressed as a percentage of the inhibited control in which ortho-vanadate was omitted. As the concentration of ortho-vanadate increased, activity decreased. From the controls it is clear that the observed alkaline phosphatase activity is unequivocally attributable to the ancient enzyme and not to possible mimetic compounds. Immunoassay
Binding of the ancient protein to the monoclonal antihuman bone alkaline phosphatase antibody (BAP A) was examined in an enzyme-antigen immunoassay based on the activity of the bound enzyme (13). Wells of a microtiter plate were coated with goat antimouse IgG, and the specific BAP A was bound to the IgG, incubated, and washed with buffer. ^-Nitrophenyl phosphate buffer was added, and the formation of ^-nitrophenol was allowed to develop for 60 min and monitored at 405 nm on a mii croplate reader. Reproducibility was better than 3%. There was an unequivocal immunoreactivitv of 23% compared with that of obtained from modern autopsy Furthermore no microbial contamination of the bones was detectable usincr t h e prnrpHnrpQ dpcrrihpH e a r l i e r (1) TTQino1
established cultivatinc techniques neither bacterial nor fun pal prowth was seen revealing essentiallv stprile conditions as a result of the ancient embalming. Conclusion
The rich abundance of pine wood tar components and sodium in the bones supports the earlier proposal that IDU II had been skeletonized or defleshed, at least in part. As the bone appeared natronized and soaked with these embalming compounds, the earlier debated skeletonization becomes increasingly plausible (2). Embalming proved to be beneficial for keeping bone Zn2Mg-alkaline phosphatase intact.
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Analytical Approach (5) Roller, J.; Baumer, U. Acta Praehistorica et search focuses on structure-function Archaeologica 1995,25,129-31. correlation of metal proteins and metal com(6) Roller, J.; Baumer, U.; Grosser, D.; Walch, plexes. An important sideline of his research R. In Baroque and Rococo Lacquers, Arbeitsheft 81 des Bayerischen Landesamtes fur includes chemical and biochemical aspects of molecular archaeology, artworkk and conserDenkmalpflegeg Lipp Verlag: Munchen, 1997,359-78. vation problems. Yoka Kaup is a postdoc(7) Sandermann, W. Fette und Seifen n942, toral fellow in the same laboratory and is 49, 578-85. interested in general enzymology and ancient (8) Sandermann, W. In Analyse der Fette und Parts of thiistudy were supported by DFG-grant metalloenzymes. Hedwig Etspuler, a recent Fettprodukte; Raufmann, H. P. Ed.; We 401-39./1 and by yhe Fonds der Chemischen Ph.D. graduate, is interested in general imSpringer Verlag: Berlin, 1958,1, Industrie. Thanks go to Dres. J. P. Luzio (Univer1049-1104. munology and in the immunological aspects sity of Cambridge), Bettina Schmitz and A. Egge- (9) Beck, C.W.;Borromeo,L. MASC4 Reof archaeology. Johann Roller, ,enior rebrecht tPelizaeus Museum), P. Eiring ang H-J. search Papers in Science and Archaeology search scientist at the Doerner Institut Hartmann (University of Tubingen), and J. Tay1990, 7, 551-58. lor (British Museum, London) for support and (10) Evershed, R. P.; Jerman, R; Eglinton, G. Munchen focuses his research on elucidathelpful lrititism. ing ancient organic materials including Nature 1985,314, 528-30. (11) Barney, J. E.; Bertolacini, R. J. Anal. Chem. dried oils lipids waxes resins and proteins 1957,1187-88. References in paintings varnishes artwork and ar(12) Weiss, M. J.; Henthorn, P. S.; Lafferty, (1) Kaup,Y. etal. Z.f.Naturforsch. .194, chaeological artifacts Ursula Baumer iis M. A.; Slaughter, C; Raducha, M.; Harris, 49 C, 489-500. research assistant whose interests focus so H. Proc. Natl. Acad. Sci. USU 1986,838 (2) Schmitz, B. In HildesheimerAgyptologische GC GC/MS and contemporary and ancient 7182-86. Beitrage 38; Schmitz., B., Ed.; Gerstenberg (13) Etspuler, H.; Raup, Y.; Bailyes, E. M.; Lupitches and tars Address correspondence Verlag: Hildesheim, 1996; pp 7-42. zio, J. P.; Weser, U. Immunol. Lett. 1995, about this article to Weser at Anoreanische (3) Roller, J.; Baumer, U.; Kaup, Y.; Etspuler, 48,187-91. H.; Weser, U. Nature 1998,391,343—33. Binrhemip I Jniversitiit TiJhiftffpn Untotop(4) Proefke, M.; Rinehart, K. L; Raheel, ML; < spvlpr\tras
