Destructive Sampling of Ivory for Mol by qingyunliuliu


									Sampling of Ivory for Molecular Analysis
Ashley Coutu
Department of Archaeology, University of York

Professor Matthew Collins
BioArch, Department of Archaeology, University of York

Professor Julia Lee-Thorp
Department of Archaeological, Geographical, and Environmental Sciences, University
of Bradford

Sampling Approaches

Taking slices, powders, laser ablation

Ivory is dentine, composed of organic proteins and very poorly crystallised mineral,
all laid down in incremental banded growth structures, which reflect environmental
and dietary conditions at the time of deposition. It is therefore a very useful tissue
to sample for molecular analyses.

In researching ivories, much can be learned by observation without the need for
invasive or chemical analysis. Ivory can be identified to species by an experienced
conservator if the microstructure is visible.

Rapid advances in technology are changing attitudes to invasive sampling, because
for the most part, the level of information which can be obtained is very high
compared to the size of sample required. Sometimes an analytical approach is
unavoidable, to establish, for instance, the cultural attribution or age of an object
where that is in doubt. In all cases sampling for chemical analysis should be carefully
thought through and consideration given to maximizing the information obtained
from any single sample. The rationale for applying a particular method of analysis
must be shown to have a reasonable prospect of success for addressing the question
at hand.

Most chemical analyses require small samples for each analysis (on the order of mg),
which can be obtained by drilling to release powdered ivory, or by shaving small
slivers. Drilling a small hole or across the surface is often the least destructive way
to sample a piece of ivory but there are constraints (see below). Laser ablation is a
minimally destructive, emerging method but applications to mixed organic and
mineral materials are not possible. In ivory all sampling methods have to take
account of the variability across growth increments. For example drilling across
growth increments will produce an averaged sample. This might be desirable but
chemical information is most useful when the tissue can be mapped spatially to
enable analysis of incremental stages within the formation of the ivory, because that
provides sequential and life history information. It requires that a sequence of clearly
defined Schreger lines are sampled, requiring longitudinal or cross-sectional slices, or
cores, to be taken through a tusk. This is obviously a very invasive method. An
alternative used to exploit the life history archive incorporated in the increments and
rather similar to tree-coring, is to remove thin (ca. 0.5cm2) cores at designated
points along a tusk, using a corer. The cores are then sub-sampled by slicing or
drilling and the holes repaired by a professional conservator using ivory powder
mixed with adhesives to allow the tusks to be replaced in their permanent exhibition.
A constraint for all drilling/cutting methods is that since heat is generated, steps
must be taken to avoid over-heating (slow speeds and/or water cooling).

Researchers and museum staff should also be aware that due to CITES regulations
on certain types of ivories (Indian and African elephant ivory), the transport of
samples of (modern and historical) ivory, even if powdered in very small vials, across
national borders is illegal without a permit. You can wait up to 6 months for a CITES
permit to access and transport this material.

Stable and Radiogenic Isotopes

What the different isotopes can tell us

The most common stable light isotopes to measure in either the collagen or mineral
components of ivory are those of nitrogen (15N), carbon (13C), oxygen (18O), and
hydrogen (2H) and more rarely, sulfur (34S). All can be measured from the
collagen (protein) component in the ivory (which is thought to reflect the protein
component of diet), while 13C and 18O can also be measured in the mineral fraction
(representing the bicarbonate isotope values of the blood, linked to energy
metabolism, and in the case of 18O, water). For example, if we consider elephants,
one of the most common sources of ivory, carbon isotope ratios provide a general
indicator of the proportions of browse (trees) vs graze (grasses) in their diet, or
whether the elephants ate foods from densely shaded forests. In environments
typically inhabited by elephants, trees and shrubs are C 3 and isotopically distinct
from C4 grasses, and plants in deep forests have very low, distinct 13C values.
Nitrogen isotopes in ivory come from plant proteins which reflect the nature of the
nitrogen cycle in that habitat, with links, for instance, between enrichment in 15N and
increasing aridity or poor, low-nutrient soils. 18O and 2H values are linked to the
water ingested by the animal and in turn to rainfall and general hydrological
conditions in the area. Terrestrial 34S values in calcified tissues may also be used as
sourcing mechanisms because they are influenced by local geology. Hence these
stable light isotopes provide information on both the animal’s ecology and its
provenience or source.

The most commonly measured radiogenic (or “heavy”) isotopes are strontium
  Sr/86Sr and the many isotopes of lead (206Pb/204 Pb, 207Pb/206 Pb, 208Pb/206 Pb). These
isotopes are calculated from the decay of radiogenic parent material in geological
bedrock. Therefore, the measurement is actually a ratio of the radiogenic isotope to
the stable isotope in that element, and because is based on radioactive decay of the
parent material, measures the age of the bedrock. This in turn can be used to
determine what type of geology the animal is roaming on, and these isotopes are
therefore used primarily for provenancing ivory.

It is also possible to use a combination of 15N, 13C and 87Sr/86Sr to definitively
distinguish marine mammal ivories from terrestrial animal ivories. This is especially
useful when material is fragmentary and cannot be distinguished on the basis of the
microstructure of the ivory.

Sample sizes

Extraction of collagen for C, N, H and O requires enough ivory to produce around 1
mg of collagen, which is the amount required for each simultaneous 15N and 13C
analysis, and similar amounts for 18O and 2H analysis, and the two sets must be
done separately. Collagen quality can be conveniently be assessed using the C:N
ratio (the theoretical atomic ratio is 3.1, but values of C/N are typically in the range
3.1-3.5) Approximately 20% by weight of ivory is collagen, but to ensure that
sufficient collagen for analysis and replication, sample sizes are typically in the order
of 30-150 mg. Extraction of mineral for 13C, 18O 87Sr/86Sr and Pb requires 20-50
mg of material, dependent on how many elemental analyses are run and duplicates if
necessary. The sample taken for the collagen extraction is separate from the
mineral extraction, so if measuring all light isotopes on one ivory sample, it is
possible that 50-200 mg of material will be needed.

Combining Analyses

Collagen extracted for stable isotope analysis is suitable for protein sequencing. The
use of acid to extract collagen will cause severe hydrolysis of any residual DNA, but if
EDTA extraction was used in order to isolate collagen, any surviving insoluble
collagen could form the basis of a subsequent DNA analysis. However, it would
seem more logical to extract the DNA first (see section below).

Trace Elements
Another method for determining provenance in biological tissues such as ivory is
trace element analysis. This method measures the amount, in parts per million
(ppm) in ivory and is therefore used often to measure heavy elements that collect in
the mineral portion of the bone, such as strontium (Sr) and zinc (Zn). This method
distinguishes populations based on concentrations of specific elements in the ivory.
The most common methods used are through neutron activation, atomic absorption
spectroscopy, and inductively coupled mass spectrometry (ICP-MS), all of which
require small sample sizes of between 1-200 mg for analysis.

Trace element (and light element isotope) analysis should be avoided in
archaeological samples due to the risk of diagenetic exchange with the burial

Dating ivory
It is often beneficial to date ivory, particularly when dating artefacts or pieces that
are pre-CITES ban, or are antique (over 100 years old according to CITES
regulations) and therefore do not require a CITES permit for transport or analysis.
The age range for radiocarbon dating is between 400 and 50,000 years. Dating is
most routinely analysed on collagen. Therefore, it is also possible to extract collagen
for combining radiocarbon dating with isotope analyses. When using conventional
radiocarbon dating, samples of 1-10 grams are required. If using accelerator mass
spectrometry (AMS) samples of 0.1 to 1 mg of carbon are required (approximately
50% of the mass of collagen is carbon), meaning that typical sample sizes are
usually between 50 – 200mg.

Protein Sequence Analyses (ZooMS)

What proteins can tell us

The dominant protein in ivory is collagen, which is very slowly evolving and in order
to discriminate between ivories, the species should have an evolutionary divergence
time of at least 5 million years. As yet, no other proteins have been routinely
extracted from ivories.

Sample size

In principle it is not necessary to sub-sample the ivory with this method as enough
collagen can be extracted using warm buffered water (65 degrees C) PH 8 for 2
hours. It does not result in any measurable deterioration of the ivory. The minimum
sample size this analysis can be conducted on is yet to be determined, but appears
to be less than 1 milligram.

DNA Analysis

What DNA can tell us

The advent of next generation sequencing technologies means that in the future, the
potential of DNA analysis on ivory is almost limitless. So far, mitochondrial DNA
(mtDNA) and single-nucleotide polymorphism (SNP) data have been used to source
ivory and explore population genetics, and we envisage that such analyses will
increase in number and resolution. As a consequence of these new technologies, we
would strongly recommend that researchers (working with museums) no longer
isolate DNA for targeted PCR amplification. Instead, we envisage the production and
immortalisation of DNA libraries for each sample. The libraries would ideally be
curated by the museum and if not, by another national facility or failing that the
researcher themselves. These libraries are DNA copies of the original genome and
are not subject to CITES regulations, thereby enabling them to be transported across
national boundaries.

Sample size

Approximately 20 mg of ivory. Sample size may vary slightly if the ivory is poorly

Combining with other analyses

In order to prepare a DNA library the DNA is typically extracted using EDTA, which
damages the mineral phase and prevents the residue from being analysed for
inorganic isotope analysis, although this has never been verified. The protein is
destroyed enzymatically in order to liberate the DNA, again, preventing organic
isotope analysis or protein sequence analysis. If the enzymatic digestion is omitted
this typically halves the total DNA yields but would allow further analyses of the
residual collagen. However the EDTA (C/N = 5) is difficult to fully remove and will
interfere with radiocarbon and isotopic analysis.

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