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Brief communication Mass spectroscopic characterization of tetracycline in the skeletal remains of an ancient population from Sudanese Nubia 350Ц550 CE.

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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: mnelson@paratekpharm.com
or George J. Armelagos, Department of Anthropology, Emory University,
Atlanta, GA 30322. E-mail: antga@learnlink.emory.edu
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
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Bassett EJ, Keith MS, Armelagos GJ, Martin DL, Villanueva A.
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Dekker. p 659–682.
McCormick JRD, Sjolander NO, Johnson S, Doerschuk AP.
1959. Biosynthesis of tetracyclines. II. Simple defined media
for growth of Streptomyces aureofaciens and elaboration of 7chlortetracycline. J Bacteriol 77:475–477.
Milch R, Koll DP, Tobie JE. 1958. Flourescence of tetracycline
antibiotics in bone. J Bone Joint Surg Am 40:897–909.
Novotny K, Herold M. 1960. The production of chlortetracycline
containing feed supplement by direct enrichment of wheat
bran. Antibiot Khimioter 5:42–46.
Piepenbrink H. 1986. Two examples of biogenous dead bone
decomposition and their consequences for taphonomic interpretation. J Archaeol Sci 13:417–430.
Piepenbrink H, Herrmann B, Hoffmann P. 1983. Tetracyclintypische Fluoreszenzen an bodengelagerten skeletteilen. Z
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