Brief communication Mass spectroscopic characterization of tetracycline in the skeletal remains of an ancient population from Sudanese Nubia 350Ц550 CE.код для вставкиСкачать
AMERICAN JOURNAL OF PHYSICAL ANTHROPOLOGY 143:151–154 (2010) Brief Communication: Mass Spectroscopic Characterization of Tetracycline in the Skeletal Remains of an Ancient Population From Sudanese Nubia 350–550 CE Mark L. Nelson,1* Andrew Dinardo,2 Jeffery Hochberg,3 and George J. Armelagos3* 1 Paratek Pharmaceuticals, Inc., Boston, MA 02111 Hospital of University of Pennsylvania, Philadelphia, PA 19104 3 Department of Anthropology, Emory University, Atlanta, GA 30322 2 KEY WORDS antibiotic; prehistory; chemical extraction ABSTRACT Histological evidence of tetracycline use has been reported in an ancient X-Group population (350–550 CE) from Sudanese Nubia (Bassett et al., 1980). When bone samples were examined by ﬂuorescent microscopy under UV light at 490 Å yellow–green ﬂuorophore deposition bands, similar to those produced by tetracycline, were observed, suggesting signiﬁcant exposure of the population to the antibiotic. These reports were met skeptically with claims that the ﬂuorescence was the result of postmortem taphonomic inﬁltration of bacteria and fungi. Herein, we report the acid extraction and mass spectroscopic characterization of the antibiotic tetracycline from these samples. The bone samples were demineralized in anhydrous hydrogen ﬂuoride which dissolved the bone-complexed tetracycline, followed by isolation by solid phase extraction on reverse-phase media. Chemical characterization by high pressure liquid chromatography mass-spectroscopic procedures showed that the retention times and mass spectra of the bone extract were identical to tetracycline when treated similarly. These results indicate that a natural product tetracycline was detectable within the sampled bone and was converted to the acidstable form, anhydrotetracycline, with a mass 1 H of 427.1 amu. Our ﬁndings show that the bone sampled is labeled by the antibiotic tetracycline, and that the NAX population ingested and were exposed to tetracycline-containing materials in their dietary regime. Am J Phys Anthropol 143:151–154, 2010. V 2010 Wiley-Liss, Inc. It has been found that human remains from several archaeological sites in Egypt (Cook et al., 1989), Sudan (Bassett et al., 1980; Hummert and VanGerven, 1982), and Jordan (Grauer and Armelagos, 1998) exhibited histological evidence of tetracycline labeling of bone using ﬂuorescence microscopy. Initial reports of ancient tetracycline use were dismissed as postmortem deterioration by soil bacteria and fungi, and it was suggested that tetracycline-like ﬂuorescence in Nubian bone was merely the result of a taphonomic process (Piepenbrink et al., 1983; Piepenbrink, 1986). Tetracyclines are active metal chelators, forming complexes with calcium and proteins (Frost et al., 1961b), and their deposition into bone has been described in treated humans and animals (Milch et al., 1958; Frost et al., 1961a). Here we report the high pressure liquid chromatography mass-spectroscopic (LC/MS) measurement and characterization of tetracycline in human bone from ancient Sudanese Nubia (350–550 CE). The direct determination of the antibiotic tetracycline in the skeletal remains of these ancient people indicates that they were exposed to tetracycline via foods produced through fermentation processes involving grains and cultivated or contaminating antibiotic-producing soil bacteria from the Actinomycetales order or Streptomyces genus. Tetracycline antibiotics are produced industrially via fermentation primarily by different Streptomyces species, where the ﬁrst generation naturally-produced members tetracycline, oxytetracycline, and chlortetracycline are still used today in clinical medicine, and are depicted in Figure 1 (Hunter and Hill, 1997). As little as 5–25 mg/kg given orally is capable of labeling bone in humans, where 0.5–2 g of tetracycline will cause marked microscopic ﬂuorescence in bone, (Milch et al., 1958) producing yellow–green bands of deposition when viewed under UV light at 490 Å. Only the ﬁrst generation tetracyclines, because of their binding afﬁnity to calcium and bone, are used to routinely study bone dynamics and growth patterns under experimental and routine conditions. Fluorescence deposition and banding are observed histologically to determine whether osteon activity and bone resorption and formation followed single or multiple dosing (Sherrod and Maloney, 1989). Fluorophore labeled bone was recovered from an X-Group or Ballana period (350–550 CE) cemetery at the North Argin X-Group (NAX) site on the West bank of the Nile river, opposite the town of Wadi-Halfa. The NAX population was comprised of mainly local individuals who ﬂourished by cultivating the ﬂoodplains of the Nile following the decline of the Meroitic kingdom and preceding the reuniﬁcation of Nubia under Christianity in 550 CE (Adams, 1977). Lake Nubia has since ﬂooded the site after the latest construction that raised the height of the dam at Aswan. C 2010 V WILEY-LISS, INC. C *Correspondence to: Mark L. Nelson, Paratek Pharmaceuticals, Inc., 75 Kneeland St, Boston, MA 02111. E-mail: firstname.lastname@example.org or George J. Armelagos, Department of Anthropology, Emory University, Atlanta, GA 30322. E-mail: email@example.com Received 15 December 2009; accepted 9 April 2010 DOI 10.1002/ajpa.21340 Published online 17 June 2010 in Wiley InterScience (www.interscience.wiley.com). 152 M.L. NELSON ET AL. Fig. 1. Chemical conversion of tetracycline, oxytetracycline, and chlortetracycline to anhydrotetracycline, anhydrooxytetracycline, and anhydrochlortetracycline, respectively during HF acid demineralization of bone. [M 1 H] is the molecular weight of the product plus hydrogen sought after in mass spectral data. Previously, histological samples were prepared using undecalciﬁed bone embedded in Hillquist epoxy, sectioned, ground, and polished (Bassett et al., 1980). The sections were viewed at 490 Å via ﬂuorescence microscopy with appropriate barrier ﬁlters. The ﬂuorophores visible and the patterns that emerged were similar to labeling by tetracycline found in modern clinical settings and agriculture practice. In the NAX sample, 9.2% of the osteons and 6.0% of the bone was labeled in sufﬁcient quantities to inhibit trabecular bone loss (Armelagos et al., 2001). In our studies, the tetracycline-labeled bone chosen was in a state of excellent preservation, possessing no visual evidence of bacterial or fungal surface or embedded contaminants. The bone from a 4-year-old X-Group child was ground to a powder (5 g) and suspended in anhydrous liquid hydrogen ﬂuoride (HF) (15 mL), an acid used to demineralize bone and facilitate dissolution of metal-complexed tetracycline. Three ﬁrst generation tetracyclines, tetracycline, oxytetracycline and chlortetracycline, were each separately dissolved in HF (1 mg/mL) and the solutions incubated overnight at room temperature in sealed polycarbonate tubes. Abbreviations amu LC/MS TIC atomic mass units liquid chromatography mass-spectroscopic total ion chromatogram. American Journal of Physical Anthropology Strong acids such as HF chemically modify tetracyclines possessing a 6-position hydroxyl group to the anhydrotetracyclines, whereby the C ring aromatizes through the loss of water and tautomerization forms a D–C bicyclic aromatic molecular species (Fig. 1). Given the strong acid conditions needed to facilitate the liberation of the tetracycline from the calcium and bone matrix, the modiﬁed anhydrotetracyclines were the molecular species analyzed for in the HF extract. After the HF was removed, the residues were further extracted into methanol saturated with anhydrous HCl gas (50 mL). The methanol extracts were dried in vacuo and the amorphous residues were extracted twice with McIlvaine buffer at pH 3.8 possessing 0.1 M disodium EDTA (2 3 25 mL). The slurries were ﬁltered and further puriﬁed via a solid-phase hydrophobic divinylbenzene chromatography column (20 g) preconditioned with water to retain the analyte tetracyclines. The ﬁltrates were loaded on the column and washed with water (2 3 25 mL). Yellow bands containing anhydrotetracyclines were eluted by methanol containing 20 mM oxalic acid (25 mL) and the eluants were removed in vacuo, producing a bright yellow residue in vacuo for LC/MS analysis. The reference tetracyclines and unlabelled control bone from the NAX population were treated similarly. High pressure liquid chromatography was performed using a binary system of water with 0.1% formic acid (phase A) and 0.1% acetonitrile with 0.1% formic acid TETRACYCLINE IN ANCIENT HUMAN BONE 153 Fig. 2. A: Total ion chromatogram with retention times (minutes) of (R/S)-anhydrotetracycline peaks at 1.404 and 1.498 min and their mass spectra, [M 1 H] 5 427.1 amu., atomic mass units. Red arrows and lines detail peak start and ﬁnish. B: NAX bone extract specimen peaks with retention times at 1.416 and 1.512 min and corresponding mass spectra [M 1 H] 5 427.1 amu. The 427.1 amu is the molecular weight of anhydrotetracycline 1 H where mass/charge is the mass to charge ratio. Red arrows and lines detail peak start and ﬁnish. (phase B) on a linear gradient from 1% B to 60% B over 2.5 min. Separations used a 2 3 20 mm, 3 lm Phenomenex Synergi Hydro-RP column coupled to a Shimadzu LCMS-2010 mass spectrometer for the separation of the eluants, both from the reference tetracyclines and the bone specimen. Mass spectra analyses were acquired in scan mode (100–1,000 MW range), whereas the spectrometer was tuned in positive-ion mode with an ion source voltage of 3.5 keV. The tetracycline, oxytetracycline, and chlortetracycline acid-treated reference eluants showed the total chemical conversion of the compounds to anhydrotetracycline, anhydrooxytetracycline, and anhydrochlortetracycline, respectively (Fig. 1). The total ion chromatogram (TIC) of the mass spectra of anhydrotetracycline, shown in Figure 2, shows two peaks over the time of the run at retention times (Rt) of 1.404 and 1.498 min (Fig. 2A), corresponding to the R and S (natural) epimers, respectively. Epimers occur because of the C4 dimethylamino group isomerization located within the tetracycline A ring. Their mass spectra showed molecular ions for both peaks corresponding to anhydrotetracycline for both epimers, with a [M 1 H] 5 427.10, consistent with acid cat- alyzed dehydration and aromatization of the C ring. Anhydrotetracycline also formed a fragmentation ion at [M 1 H] 5 410.0, consistent with fragmentation patterns typical of the 2 position carboxamide group and loss of NH2. Anhydrooxytetracycline possessed a TIC with peaks detected at a Rt of 1.450 min, no epimer pair because of the 5-position hydroxyl group in ring B, and a molecular ion of [M 1 H] 5 443.10 and a fragmentation ion of [M 1 H] 5 426.05. The ﬁnal tetracycline standard, chlortetracycline, possessed TIC peak retention times of 1.602 and 1.739 min, with a mass spectra consistent with the conversion of chlortetracycline to anhydrochlortetracycline [M 1 H] 5 461.05 (data not shown). The chromatogram derived from the extracted bone of the NAX child, shown in Figure 2B, showed the presence of anhydrotetracycline with TIC peak retention times of 1.416 and 1.512 min, also corresponding to the C4 dimethylamino group (R/S) isomer pair formed during acid extraction. The mass spectra of both peaks (Fig. 2B) were identical to that produced by anhydrotetracycline, with a molecular ion of [M 1 H] 5 427.10 and fragmentation ion at [M 1 H] 5 410.5. Comparisons of the NAX extract with an authentic sample of anhydrotetracycline American Journal of Physical Anthropology 154 M.L. NELSON ET AL. also conﬁrmed similarity of both the TIC retention times and mass spectra (data not shown). Given the identical retention times of the chromatograph peaks derived from the NAX specimen and anhydrotetracycline, and their identical mass spectra, the data indicates that the NAX skeletal remains contained readily detectable levels of the antibiotic tetracycline. The NAX specimen also had one of the highest levels of tetracycline in our study by direct visualization, with 34.2% of the osteons and 11.3% of the bone tetracycline labeled. Mass spectroscopic characterization conﬁrms that the labeled bone and eluting extraction bands are the chemically modiﬁed derivative anhydrotetracycline, and that members of the NAX population were frequently exposed to tetracycline via regularly consumed grains or broths to afford bone labeling. In past studies of NAX bone labeling, there were no correlations between either age or gender and osteon labeling, but 95% of the individuals examined had labeled osteons whereas 56% had over 5% of their osteons labeled (Bassett et al., 1980). The extent of the labeling suggests that the population received tetracycline during osteon mineralization, which occurs during periods of 80 days. This ﬁnding contradicts the notion that the osteons were labeled by a one time event of bacterial contamination of grains or foodstuffs. Single exposure would lead to low numbers of labeled osteons, lowering the ﬂuorescent band deposition and limiting visual or instrumental detection. If the NAX population did produce gruels or beer fermentations using Actinomycete bacteria, they would also have needed to inoculate the media used with greater than 10% of an active culture or previous fermentation broth to achieve the growth needed to produce sufﬁcient quantities of tetracycline in a liquid fermentation medium (McCormick et al., 1959). In contrast, surface inoculation of cracked and water-treated grains would produce tetracycline, but in low yields compared with liquid fermentation (Novotny and Herold, 1960). The NAX population was skilled in the science of fermentation, and now it is evident that they could have produced fermentation mixtures containing Streptomyces or other species that imparted nutritive and pharmacological effects. The mass spectroscopic detection of tetracycline in the bone samples of the NAX population and the extent of osteon labeling demonstrates that the ability to produce the antibiotic tetracycline through American Journal of Physical Anthropology fermentation processes was occurring almost 2,000 years ago. LITERATURE CITED Adams WY. 1977. Nubia: corridor to Africa. Princeton: Princeton University Press. Armelagos GJ, Kolbacher K, Collins K, Cook J, KrafeldDaugherty M. 2001. Tetracycline consumption in pre-history. In: Nelson M, Hillen W, Greenwald R, editors. Tetracyclines in biology, chemistry and medicine. Switzerland: Birkahauser Verlag. p 219–236. Bassett EJ, Keith MS, Armelagos GJ, Martin DL, Villanueva A. 1980. 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