MRI and Unilateral NMR Study of Reindeer Skin Tanning Processes

Article pubs.acs.org/ac

MRI and Unilateral NMR Study of Reindeer Skin Tanning Processes Lizheng Zhu,† Eleonora Del Federico,§ Andrew J. Ilott,† Torunn Klokkernes,‡ Cindie Kehlet,§ and Alexej Jerschow*,† †

Department of Chemistry, New York University, New York, New York 10003, United States Museum of Cultural History, University of Oslo, 0130 Oslo, Norway § Department of Mathematics and Science, Pratt Institute, Brooklyn, New York 11205, United States ‡

ABSTRACT: The study of arctic or subarctic indigenous skin clothing material, known for its design and ability to keep the body warm, provides information about the tanning materials and techniques. The study also provides clues about the culture that created it, since tanning processes are often specific to certain indigenous groups. Untreated skin samples and samples treated with willow (Salix sp) bark extract and cod liver oil are compared in this study using both MRI and unilateral NMR techniques. The two types of samples show different proton spatial distributions and different relaxation times, which may also provide information about the tanning technique and aging behavior.

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deterioration patterns that may be characteristic for both the tanning agents and the collagen fibers themselves.23,24 Skin clothing is appreciated for its design and its ability to keep the body warm and dry as well as for the technological processes responsible for these properties. Manually tanned skin is a complex material and includes both depilated skin, skin with attached hair, and a variety of tanning substances or combinations of these tanning substances, depending on the specific use of the skin. Methods for tanning skin consist, among others, of the treatment of the skin with substances such as vegetable tannins, oils, and fats as well as methods in which the skin is smoked. One objective of these treatments is to make the skin water-resistant. Fats and oils can also be used as lubricants. A complicating factor is that in most skin materials used for clothing purposes, a combination of substances and methods are used, making the conservation of these artifacts more difficult.23 Vegetable tannins are obtained from extracts of wood, such as bark, heartwood, and sap wood. Generally, the tannin content is higher in bark than in heartwood and sap wood.25 Tannins can also be extracted from leaves, fruits, galls, and roots of plants, and their specific use is based on their properties as tannins. Tannins are composed mainly of polyphenols and may be divided into two main groups: condensed tannins also called proanthocyanidins and hydrolyzable tannins. The hydrolyzable tannins are further separated into two groups: gallotannins and ellagitannins.26 Another group of tannins is called complex tannins. As the name

he noninvasive analysis and imaging of cultural heritage is of major interest in the field of art conservation because sampling in most cases is strictly limited. Mobile, noninvasive analytical tools, such as in situ micro XRF,1,2 external reflectance FTIR,3−7 and unilateral NMR8−14 among others, have allowed the elucidation of the material’s characteristics, its composition, and stratigraphy as well as its condition and the determination of undocumented conservation treatments. The development of novel nondestructive techniques as well as novel applications continues to be in high demand for the study and conservation of works of art.15 Badea et al.16 used unilateral NMR as well as other noninvasive techniques in their study of historical parchments. They found that gelatinization can be associated with shorter T1 values, whereas hydrolytic processes had the opposite effect. Masic et al.17 studied deterioration of a Dead Sea scroll fragment using solid-state and unilateral NMR. Bardet et al.18 used solid state NMR and ESR to investigate archaeological leather artifacts and their changes with aging. Miu et al. identified tanning agents in the heritage leather items,19 among her extensive research in leather and parchment.20,21 Traditional knowledge, related to the production process of indigenous skin artifacts, is often embedded in the artifact on both tangible and intangible levels. Some of these may be recognized through discussion with tradition-bearers, through observation, and through sensory studies.22 In cases where the tradition-bearer is no longer present, it may be impossible to identify information regarding the leather tanning processes without the use of scientific analysis. The understanding and safeguarding of artifacts such as tanned and decorated skin materials involve complex processes both in production and in alteration through use and repair as well as in their © XXXX American Chemical Society

Received: December 1, 2014 Accepted: February 26, 2015

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Analytical Chemistry indicates, they contain features that are related to both condensed and hydrolyzable tannins, as can be seen in barks of Quercus species.27 Willow bark extract (Salix sp) along with birch bark extract (Betula sp) and alder bark extract (Alnus sp) are all used in the tanning of skin materials in Eurasian arctic and subarctic indigenous cultures.23 The bark is finely cut and boiled in water for approximately 30 min and left to cool down before it is used (lukewarm). These vegetable tannins are all in the condensed tannin group. Various natural and synthetic lipids as well as mixtures of lipids are applied in skin processing technology, either as tanning agents or as lubricants to obtain and maintain certain properties in the skin materials. In nature, lipids appear either as solid fats, such as lard and tallow, or as liquid, such as fish, marine mammals, or vegetable, as well as mineral oils. In these trials, homemade cod liver oil following traditional techniques23 is used. The treatment recipe is based on information revealed by informants from the Sámi culture in northern Norway. Cod liver oil is placed in a glass jar with the lid slightly open and left for approximately 6 months. During this time, it turns into a yellow, clear oil. Only a small amount of residue is filtered off. The color indicates that oxidation of the oil may have occurred, and this may affect the properties of the oil. The fatty acid composition of cod liver oil has a high content of long-chain unsaturated fatty acids. The major saturated fatty acids are palmitic (16:0) and myristic acids (14:0). The major unsaturated fatty acids are oleic acid (18:1 cis-9) and palmitoleic (16:1 cis-9) and eicosenoic acid (20:1 cis-11). In addition, cod liver oil contains proportions of erucic acid (22:1 cis-13) and lower amounts of stearic (18:0), linoleic (18:2 cis9,12), and myristoleic acid (14:1 cis-9). In this article, we describe a study of skin artifacts that have been treated either with cod liver oil or with vegetable tannin and aged for different amounts of time. We discuss the different proton spatial distributions and different relaxation times, as observed via unilateral NMR and MRI.

Figure 1. (a) Sámi winter coat. Photo: Copyright T. Klokkernes, 2004. (b) From left to right, untreated sample, sample treated with cod liver oil, sample treated with willow bark extract. (c) Samples in NMR tubes.

September 2004, treated in August 2013, and measured right afterward. The sample labeled as S0 was treated by logwood extract (logwood extract is a close substitute for willow bark extract). The extract was generously applied to the surface of the sample with a paintbrush, manipulated by hand and then left to dry. The process was repeated the next day. The sample labeled as C0 was treated using the same homemade cod liver oil as described above and was then manipulated by hand after 15 h. The process was repeated the next day, and then the sample was allowed to sit for approximately 36 h to dry. MRI Measurements. All MRI experiments were performed on a Bruker Ultrashield 9.4 T Avance I spectrometer with a microimaging accessory, operating at 400.13 MHz for 1H. Images were collected using a Bruker Micro2.5 gradient assembly with a Bruker Micro2.5 imaging probe and 5 mm resonator. For each sample, a slice-selective, 2D spin echo sequence was employed to acquire images with a 6 × 6 mm field of view, with 96 frequency encoding points to achieve a nominal resolution of 63 μm in the x direction, and 64 phase encoding points for a nominal resolution of 94 μm along y. A 4 mm slice was used. The same sequence was used to collect a series of images for each sample over a range of echo times (TE). The intensity variation at each voxel during this series was then fit to a monoexponential decay to reconstruct the T2 parameter maps for each specimen. Errors on each T2 value were estimated as the square root of the corresponding diagonal component of the covariance matrix for each individual fit. NMR Spectroscopy. 1H chemical shift spectra were collected for the samples using the same spectrometer, probe, and resonator as used for the MRI measurements. A recycle delay of 3 s was used to collect 64 scans with a 120 kHz spectral width, using a 90° pulse with a 10 kHz nutation frequency. The spectra were deconvoluted to obtain the areas of the collagen/ fat (≈1.5 ppm) and water (≈4.5 ppm) peaks: five different models were used with varying constraints on the peak positions, widths, and Gaussian/Lorentzian fraction. The quoted values of the peak areas and error estimates (Table 1) represent the average values from these fits and their standard deviation, respectively. The integrals of the fit peaks were normalized by the mass of each sample to allow for comparison. Unilateral NMR. Unilateral NMR measurements were performed using an ACT (Magritek GmbH, Aachen, Germany) NMR-MOUSE (Mobile Universal Surface Explorer) instru-

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EXPERIMENTAL SECTION Sample Preparation. The samples are labeled by U, S, and C for “untreated”, “salix-treated”, and “cod-liver-oil-treated”, and a number is added for the years of aging. Three sets of reindeer skin material were tested. The samples labeled S6, C6, and U6 (Figure 1b) come from a 4−5-month-old northern Norwegian reindeer calf slaughtered in September 2004 (samples were 6 years old upon measurement). The samples labeled S1.5, C1.5, and U1.5 come from a caribou reindeer (Rangifer tarandus) slaughtered in 1993 and kept at room temperature until samples were made in 2012 (samples were 1.5 years old upon measurement). The six-year-old sample (S6) was manually processed by a Sámi tradition-bearer in Northern Norway by soaking the flesh side of the skin in the tannin extract, manipulating it by hand and then leaving it to dry. The process was repeated the next day. The 1.5-year-old sample (S1.5) was processed in the same way. For the cod-liver-oiltreated sample, the homemade oil (described above) was applied to the flesh side of the skin and folded (flesh side against flesh side) for 30 min before being manipulated by hand. The samples were left for 3 days before the excess oil was scraped off using an unsharpened scraper. Samples labeled U are untreated reindeer skin. The samples labeled S0 and C0 come from the same 4−5 month old northern Norwegian reindeer calf slaughtered in B

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Analytical Chemistry Table 1. 1H NMR Spectral Deconvolution Study of Untreated and Salix- and Cod-Liver-Oil-Treated Samples with 0, 1.5, and 6 Years of Aging sample

peak 1 areaa (1.5 ppm, collagen−fat)

peak 2 areaa (4.5 ppm, water)

collagen−fat/ water ratio

S6 S1.5 S0 U6 C0 C1.5 C6

17 ± 2 22 ± 2 104 ± 6 86 ± 6 530 ± 20 450 ± 20 256 ± 7

90 ± 20 110 ± 10 86 ± 8 70 ± 20 300 ± 40 260 ± 30 140 ± 9

0.18 ± 0.05 0.20 ± 0.03 1.2 ± 0.2 1.2 ± 0.4 1.8 ± 0.3 1.8 ± 0.2 1.8 ± 0.2

a

Normalized by sample weight. Figure 2. (a) 1H NMR spectra of untreated sample (U6) and samples treated with salix under 0 (S0), 1.5 (S1.5), and 6 years (S6) of aging. (b) 1H NMR spectra of untreated sample (U6) and samples treated with cod liver oil under 0 (C0), 1.5 (C1.5), and 6 years (C6) of aging.

ment controlled by a Bruker Minispec (Bruker BioSpin, Rheinstetten, Germany) operating at 18.5 MHz 1H resonance frequency (0.5 T) with a field gradient, G, of 22.6 T m−1 and a 90° pulse of 4.5 μs. The instrument was equipped with a surface rf coil, creating a sensitive volume of ∼10 × 10 mm2 times an adjustable thickness of 2.5 mm away from the rf coil.28 The transverse relaxation decays of 1H were measured using the Carr−Purcell−Meiboom−Gill (CPMG)29 pulse sequence with echo times of 30 μs and acquisition times of 20 μs, which in the presence of the field gradient G define a nominal resolution of ∼50 μm. The magnetic field of the NMR-MOUSE is highly inhomogeneous, leading to an artificial enhancement of the measured T2 relaxation rates as compared to measurements in a homogeneous field.30 The six-year-old reindeer samples were measured using a recycle delay of 3s and 1000 echoes, covering a range of 30 ms. The number of scans varied, depending on tanning technique (16 384 scans for cod liver oil, 24 576 scans for salix and untreated, respectively). A biexponential function with offset was used to fit the T2 relaxation decay curves. The offset was necessary to separate the signal of fluidic components from semisolids, keeping the fitting model robust. The square roots of the diagonal components of the covariance matrix were taken as the standard errors on the fitted parameters. Only even-numbered echoes were included in the fit to minimize systematic errors introduced by pulse imperfections.

Figure 3. Ratios of the visible collagen−fat signals to water signals of untreated and salix- and cod-liver-oil-treated samples with different aging times.

oil-treated samples, the decrease in intensity can be attributed to oil evaporation; however, the interaction between oil and collagen is not yet well understood.31 The spectral deconvolution (Table 1) can be used to separate the signals from the collagen−fat region and the water region, respectively. The ratio between these integrals provides an interesting view into the differences in the two treatment methods (Figure 3). It should be noted that the ratio is between the “visible” signals. An increase in this ratio does not necessarily mean that water is expelled from the tissue, but may also mean that part of the water is immobilized and, hence, gives very broad signals that are undetectable. The same can happen with the collagen−fat signals. Figure 3 shows that the collagen−fat/water signal ratio increases significantly for codliver-oil-treated samples and decreases significantly for the salixtreated samples with respect to the untreated sample. Furthermore, the ratio does not vary much with times of aging, even with respect to the unaged samples, whereas a large decrease is seen for the salix-treated samples upon aging, but not immediately upon treatment. This finding is in line with the general expectation that vegetable-tannin-induced cross-linking with collagen is likely more effective than cod liver cross-linking with the collagen. MRI and T2 Map Study. Figure 4 shows T2 contrast images of an untreated sample using a spin−echo imaging pulse

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RESULTS AND DISCUSSION NMR Spectroscopy Study. Figure 2 shows the static 1H NMR spectra of reindeer skin samples treated with salix and cod liver oil with 0, 1.5, and 6 years of natural aging. The spectra are normalized by sample weight. The biggest difference between the two treated samples is that although an increase in intensity upon treatment is seen in both types of samples, the cod liver oil treatment provides a much larger enhancement, by 14 times. This effect most probably relates to the fact that the addition of the oil increases the overall proton density. The peak at 1.5 ppm can be assigned primarily to mobile collagen components.17 Subsequent effects are similar in both samples: with increasing aging times, the overall proton intensity decreases, along with the integrals (see Table 1 and Figure 3), although for the samples treated with cod liver oil, even at 6 years of aging, the signal remains larger than from the original (untreated) one. The signals also become broader with increasing aging time. In the salix-treated samples, this effect can be explained by an increased degree of immobilization due to cross-linking between the collagen and the tannin,31 and repulsion of water provides additional effects. In the cod-liverC

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Analytical Chemistry sequence at different echo times. By fitting the images voxelwise to relaxation decays, the T2 maps (Figure 5) are obtained for the 6-year-old untreated, salix-treated, and codliver-oil-treated samples. Compared with the untreated sample,

Figure 4. T2 Contrast images of the untreated sample (U6) with increasing echo times.

Figure 6. (a) NMR MOUSE and MRI depth profiles (dotted lines, extracted from MRI T2 maps, with shaded regions representing the fitting error) of the untreated sample (U6, black triangle), salix-treated sample (S6, blue dot), and cod-liver-oil-treated sample (C6, red square). (b) Representative biexponential fits of the relaxation decays for each of the series in part a at a depth of 400 μm, showing the first 15 ms of the 30 ms acquisition.

Figure 5. T2 maps of 6-years-aged untreated (U6), salix-treated (S6), and cod-liver-oil-treated (C6) samples. The red lines on each plot indicate the position of the slice used for the 1D depth profile shown in Figure 6a.

profiles obtained by acquiring CPMG decays in steps of 50 μm into the samples starting from the flesh side, which is the treated side, and progressing to the hair side to a final depth of 800 μm. The relaxation decays were modeled by a biexponential decay function, which fit the data very well, as shown in Figure 6b. It can be seen that there is some deviation from the fitted line at short delay times (around 1 ms), which we ascribe to the complicated relaxation behavior in strongly dipolar-coupled systems. This precludes the use of the fast T2 components (relaxation times vary between 0.2 and 0.4 ms) as a differentiating measure; therefore, we concentrate on the interpretation of the slowly relaxing components, which do allow a distinction among the untreated and the two treated samples. The untreated sample has an intermediate relaxation time that is well distinguished from the cod-liver-oil-treated skin, which is the slowest relaxing, and the salix-treated sample exhibiting the fastest relaxation behavior. Moreover, it can be noticed that whereas the relaxation profile for the untreated sample remains fairly constant throughout the first 500 μm, both treated samples show variations. These variations are most pronounced for the salix-treated sample, which has significantly shorter relaxation times in the region from 200 to 400 μm depth. The depth-dependence of the MRI profiles is slightly different from the MOUSE profiles, although overall, they do show excellent agreement in the relaxation times of the samples and in the trends between them. The development of a layered structure in the treated samples is also apparent in both sets of results. The relaxation times are measured at different magnetic fields, and their similarity indicates that residual dipolar couplings are the dominant T2 relaxation mechanism. The remaining differences between the MOUSE and MRI data are likely due to sample variations as it was difficult to compare the

which has an intermediate relaxation time (∼8 ms), the salixtreated sample has a shorter T2 (∼2 ms), indicating that the skin becomes more rigid, and the molecules become less mobile as a result of the tanning process. A number of mechanisms have been proposed in vegetable tannage.32 Among these theories, the explanation of tannin fixation into the collagen lattice with participation of ionic and nonionic groups of collagen appears to be the most plausible one.33 The cod-liver-oil-treated sample has longer relaxation times (∼12 ms), which indicates a greater mobility. A more subtle effect is the cross-linking of cod liver oil itself,34 which does not lead to the same degree of rigidity as when tannins are used. This is in line with the relatively longer T2 relaxation times, and larger collagen−fat/water ratios found for cod-liver-oil-treated samples. The images of one salix-treated sample also showed that a layered structure formed. The outside layer (flesh side and the treated side) had a shorter T2 (1 ms) than the inside layer (hair side, 4 ms). This finding would indicate that cross-linking in such samples slowly propagates through the skin, whereas a more uniform process is found in cod-liver-oil-treated samples. Depth Profiling Studies by Unilateral NMR. Although one could, in principle, use MRI noninvasively on a whole skin sample, this approach is expensive, and sensitivity will be limited in large scanners. Therefore, the capabilities of unilateral NMR measurements are also explored here, particularly with respect to their ability to reproduce the MRI findings and differentiate between depth-dependent T2 relaxation properties of samples treated using different methods. No sampling is needed to study works of art by unilateral NMR, and therefore, this technique would be preferable because of the invasiveness of the MRI technique. The NMR MOUSE can provide relaxation data at various depths of an artwork noninvasively. Figure 6a shows depth D

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Analytical Chemistry Notes

analysis methods at identical points on the reindeer skin in practice. However, some differences can also be attributed to the different methods used to acquire the data (a CPMG for the MOUSE and a spin echo for the MRI), as well as the fitting models employed. A monoexponential fitting function is used for the MRI results, where the minimum echo time in the sequence is 1.4 ms, precluding the resolution of the quickly relaxing components (T2 of 0.2−0.4 ms) that are adequately fitted with the biexponential model used for the MOUSE data. However, the quickly relaxing components may still impact the fitted T2 values in the MRI results and appear to do so, especially in the first 400 μm of the salix-treated sample. Despite these differences between the results from each technique, the excellent overall agreement supports the classification of the samples by their T2 relaxation times. For this particular set of samples, the studies suggest that the salix-induced cross-linking process with the collagen is at a maximum between 200 and 400 μm, whereas noticeable effects are seen throughout the whole sample. The slower relaxation shown in the first 100 μm of the cod-liver-oil-treated sample in the MRI depth profile is most likely due to some evaporation of the oil and drying at the surface of the skin. The overall increase in relaxation behavior throughout the skin profile when compared with the untreated sample clearly indicates that the oil has penetrated the whole skin sample.

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Alexej Jerschow acknowledges funding from the U.S. National Science Foundation, Award No. CHE 1412064. Cindie Kehlet and Eleonora Del Federico acknowledge funding from the Alfred P. Sloan Foundation and the Henry and Camille Dreyfus Foundation and the assistance of Pratt Institute alumni Megan Welchel, Amelia Catalano, and Hiba Schahbaz.

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CONCLUSIONS It is found that proton NMR spectra and relaxation times change significantly as a function of treatment of reindeer skin samples and aging time. Cod-liver-oil-treated samples generally show stronger signals in the collagen−fat region, and tannintreated samples lead to a strong decrease of those signals. Part of this effect is likely due to the strong cross-linking propensity of tannin−collagen interactions. Although cod liver oil can cross-link, as well, these effects were found to contribute much less to a reduction in mobility in the samples. The spectroscopic results were further supported by spatially localized MRI T2 maps and by unilateral NMR measurements. Similar spatial variations in the samples are observed using both methods, with the development of a layered structure evident in the treated samples. Given the fact that unilateral NMR is a noninvasive technique and can be applied directly on the work of an art or artifact of interest, we suggest that it could be an important technique for the characterization and analysis of indigenous skin clothing and skin treatment processes. In particular, it appears that differences between oil-treated and vegetabletannin-treated samples can be discerned on the basis of large differences in their T2 relaxation behavior. The utility of spectroscopic NMR studies and MRI as complementary tools is also apparent: they provide a more complete picture of the variation of properties across the samples and give critical insight into the mechanisms of the preservation techniques. In this manner, these methods provide a chemical basis for the unilateral NMR results and a justification for the classification of preservation techniques and samples. This analysis could be further enhanced by feature extraction and pattern recognition techniques, such as principal component analysis.

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