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Investigating Water Interactions with Collagen Using 2H Multiple Quantum Filtered NMR Spectroscopy To Provide Insights into the Source of Double Quantum Filtered Signal in Tissue Cassidy R. VanderSchee and Kristopher J. Ooms* Department of Chemistry, The King’s University College, 9125 50th Street, Edmonton, Alberta, T6B 2H3, Canada S Supporting Information *

ABSTRACT: In an effort to provide insight into the molecular origins of the 2H double quantum filtered (DQF) NMR signal observed in connective tissue, specifically spinal disc tissue, 2H multiple quantum filtered (MQF) NMR spectroscopy is used to study the structure and dynamics of D2O in collagen as a function of hydration. Residual quadrupolar coupling constants are measured and decrease from 3500 to 20 Hz while T2 relaxation times increase from 0.65 to 20 ms as hydration increases. Analysis of the data indicates that the quadrupolar coupling and T2 relaxation arises when water molecules spend time in restricted environments. The residual quadrupolar coupling is influenced almost exclusively by the most restricted water sites, the clefts of the triple helices not exposed on the surface of the fibrils, while the T2 relaxation has secondary contributions from less restricted water environments. The magnitudes of the measured values are consistent with results from DQF NMR studies of spinal disc tissue, supporting the assertion that water binding to collagen is a major contributor to the DQF NMR signal observed in spinal disc tissue.

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INTRODUCTION The use of NMR spectroscopy to study specific biomarkers of normal and diseased biological tissue is increasing rapidly. One such technique for studying connective tissues is multiple quantum filtered (MQF) NMR spectroscopy, which is sensitive to subtle changes in the local environment and dynamics of specific biomarkers such as water molecules and sodium ions.1−7 When coupled to modern magnetic resonance imaging techniques, a powerful probe of tissue health is available for both in vitro and in vivo studies that can complement more conventional relaxation based imaging techniques.3,8 While in vivo studies of water rely on 1H double quantum filtered (DQF) signal created by dipolar coupling, in vitro 2H DQF NMR of tissue equilibrated in D2O offers the ability to use the quadrupolar interaction which can be complementary to, and often more sensitive than, 1H dipolar couplings.3 Recent application of quadrupolar DQF to study tissues includes 23Na studies of sodium ions in spinal disc tissue9,10 and 2H studies of cartilage1,6,7 and spinal disc tissues.3 The 2H DQF NMR study of spinal disc tissue revealed potential correlations between DQF parameters and spinal disc degeneration.3 The changes in the 2H DQF spectra were related to the decrease in water content of spinal disc tissue as it degrades, particularly the nucleus pulposus in the soft central part of the tissue. It has been suggested that DQF methods could provide a useful tool for the early diagnosis of degenerative tissue disease.1,3,8 For the study of quadrupolar nuclei such as 2H, DQF techniques offer researchers the ability to measure small residual quadrupolar couplings (CQres) and T2 relaxation times that arise when D2O molecules experience restricted, nonisotropic motion due to interactions with larger macro© 2014 American Chemical Society

molecules or a solid matrix. The experiment begins with a 90x−τ/2−180y−τ/2 pulse sequence. After the first 90x pulse the state of the spin system can be described by the reduced density matrix ρ(0) = −Iy. During the evolution time τ, the spins are allowed to evolve under the influence of the quadrupolar Hamiltonian; the 180° refocusing pulse removes the effect of chemical shift and field inhomogeneities. The effect of this evolution time can be conveniently described using the fictitious spin-1/2 operators of Vega and Pines.11 These include the familiar single quantum operators Ix and Iy and the zero quantum operator Iz as well as the antiphase single-quantum operators Jx = IyIz + IzIy and Jy = IzIx + IxIz and the doublequantum operator Jz = IxIy + IyIx.12 The effect of the evolution time on the spin system can be represented as the precession of the spin vector in Iy−Jy space.12 Therefore, at the end of the evolution period the spin density matrix, ρ(τ), can be represented by two components. If T2 relaxation is also included, the density matrix is given by12 ρ(τ ) = ⌊−Iy cos(Qτ ) + Jy sin(Qτ )⌋ exp( −τ /T2)

(1)

where Q is the quadrupolar half splitting and is related to the CQres by Q (θ , ϕ) =

3π CQ res[3 cos2 θ − 1 − ηQ sin 2 θ cos 2ϕ] 4 (2)

Received: September 24, 2013 Revised: March 10, 2014 Published: March 14, 2014 3491

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where ηQ is the asymmetry parameter of the electric field gradient (EFG) and the angles θ and ϕ define the orientation of the electric field gradient in the magnetic field. After the evolution period, two 90° pulses are used to filter out the signal. The first 90x filter pulse converts the −Iy component to the zero quantum coherence −Iz and the Jy component to double quantum coherence −Jz. If the second filter pulse is a 90x pulse, −Iz is transformed to Iy and −Jz to −Jy. Alternatively if a 90y pulse is used, the resulting coherences are −Ix and Jx, respectively. Thus, implementation of the proper phase cycling on the signal detection allows for the selection of the Jy component of eq 1, which is referred to as the double quantum filtered signal, or the −Iy component that we refer to as the zero quantum filtered (ZQF) signal. Allowing the signal to precess during the acquisition and taking the Fourier transform of the resulting FID leads to the familiar DQF line shape equation3 as well as the analogous ZQF equation.

the observed residual quadrupolar coupling of water in cartilage. However, a full characterization of the DQF signal arising from water−collagen interactions at a wide range of hydrations has not be carried out, a major gap because one of the key macroscopic changes during spinal disc degeneration is a loss of water. In addition to collagen, spinal disc tissue has a large amount of proteoglycan, particularly in the nucleus pulposus.16−19 Initial studies suggest that the interaction of D2O with the proteoglycan is not a major contributor to the 2H DQF signals.5 Other macromolecules, such as elastin, which has been previously studied at high hydration levels using 2H DQF NMR spectroscopy,20 are present in much smaller amounts in the tissue and not expected to be major contributors to the 2H DQF signal.17,21 The hydration structure and dynamics of the collagen triple helix have been studied for many years,15,22−27 and in the past decade, significant advances have been made using magnetic relaxation dispersion (MRD) to determine the different environments and exchange dynamics of the water molecules in collagen and other proteins.26,28−31 For a single triple helix there are generally two types of water environments associated with hydration that need to be considered when modeling exchange dynamics. The first environment is the cleft or water chains. These waters are localized in the clefts of the triple helices and have been identified using both X-ray crystallography and NMR spectroscopy and recently studied using QM studies.23−25,32 On the basis of the structural and NMR titration studies, it has been proposed that approximately 0.26 g of water/g of protein can be accommodated in the cleft sites.33,34 These cleft waters undergo rapid rotational and librational motion, with characteristic correlation times of 6.55 × 10−10 s.33 As well, water exchange between the clefts and more mobile surface and bulk waters also occurs. Different correlation times for this water exchange have been proposed depending on the experiment and details of the model used. Early work on collagen fibrils suggested that the exchange correlation times could be as long as 1 × 10−7 to 5.0 × 10−6 s.25,35 More recent NMR work has indicated that the exchange correlation times are more likely on the nanosecond to subnanosecond time scale.22 In addition, a recent MRD study of a gelatin type B obtained from bovine skin, where the fibril structure was denatured, indicates that the residence times are 3.6 to 39 × 10−9 s,26 suggesting that the longer correlation times do not arise solely from the clefts of the triple helix but may be a consequence of the packing of the triple helices into higher order fibril structures. The formation of fibrils results in water molecules that are hidden from the bulk solvent, leading to longer correlation times.22 According to proton NMR and micro-CT studies, a second water environment becomes important when hydration increases above 0.263 g/g.22,33,34 This layer of water, which has been described as a monolayer34 or multilayer22 of hydration and has ∼1.34 g of water per gram of protein in it, has exchange correlation times of 2.6 × 10−11 s, approximately 4 times that of bulk water.33 This is consistent with other proteins that show water exchange dynamics between surface waters and bulk water on the same order of magnitude.31 It is important to note that in most of these studies the triple helix structure of the protein is assumed to remain relatively constant as hydration is increased and that variations in NMR parameters arise from changes in water mobility and the number of water molecules in different environments.

SDQF(τ , ω2) = M 0 sin(Qτ ) exp( −τ /T2) ⎡ ω2 − δ 0 + Q ⎢ ⎣ (ω2 − δ0 + Q )2 + (1/T2*)2 −

⎤ ⎥ (ω2 − δ0 − Q ) + (1/T2*) ⎦ ω2 − δ 0 − Q 2

2

(3)

SZQF(τ , ω2) = M 0 cos(Qτ ) exp( −τ /T2) ⎡ 1/T2* ⎢ ⎣ (ω2 − δ0 + Q )2 + (1/T2*)2 +

⎤ ⎥ (ω2 − δ0 − Q ) + (1/T2*) ⎦ 1/T2* 2

2

(4)

where M0 is the signal intensity immediately after the first 90° pulse, ω2 is the frequency of the spectrum, and δ0 is the frequency offset of the peak in s−1. T∗2 is related to T2 by 1/T∗2 = πW + 1/T2 where W is a line broadening factor resulting from inhomogenous broadening. We have adopted the convention of letting both ZQF and DQF spectra have positive values at their center frequencies. The advantage to performing both the DQF and ZQF experiments is that they respond differently to the quadrupolar coupling, increasing the reliability of the determined CQres values, and they allow for waters with negligible CQres values to be accounted for in the model; the sin(Qτ) and cos(Qτ) terms mean that CQres values near zero contribute to the ZQF spectrum but not the DQF spectrum.13 By fitting of the experimental spectra to the above equations, the CQres and T2 can be determined which can potentially be used to link experimental spectra with molecular level models of hydration. As the application of 2H DQF NMR for studying tissue increases, a better understanding is required of how the hydration of the macromolecular components in tissue leads to the 2H DQF signal. Collagen, one of the main macromolecules in both cartilage and spinal disc tissue, is known to strongly interact with water molecules.14,15 A previous 2H DQF study of water interacting with collagen indicated that the magnitude of the residual quadrupolar coupling for a suspension of collagen in D2O was similar to that observed for a suspension of cartilage powder in D2O, 110 Hz.5 This suggests that the interaction of water with collagen is a dominant contributor to 3492

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A final consideration when modeling exchange dynamics is the importance of proton exchange (or in the case of the current study deuteron exchange) between waters and the COOH groups of the amino acids. This exchange process is typically quite slow; for example, in the study of gelatin type B it was found to be 2.6 × 10−5 s.26 In the same study it was found that the pH did not affect the gel structure or the molecular exchange times; the main effect of pH variations was observed in the MRD relaxation profile at the lowest magnetic fields.26 In this study, 2H DQF and ZQF spectra of D2O are acquired at various levels of collagen hydration to determine the CQres and T2. By determination of these physical parameters, the extent to which collagen is responsible for the changes in the DQF signal observed in spinal disc tissue can be assessed. Comparing the data to previous work on collagen hydration will provide a means of connecting these NMR experiments to current models of collagen hydration. From an application perspective this vital link between experimentally observed spectra of human tissue and molecular level models of hydration is an important step toward using DQF techniques to assess tissue health.

the sample, and 22 DQF and 22 ZQF spectra were acquired for each hydration value. ZQF spectra were obtained by signal averaging 128 scans, while DQF spectra were acquired using 256 scans; both experiments used a 1 s recycle delay; T1 values of the highest hydration samples did not exceed 200 ms. The experimental spectra were fit to spectra calculated using eqs 3 and 4 and assumed that ηQ = 0. While it is likely that small deviations from ηQ = 0 exist when water interacts with the collagen, the deviation is expected to be small; other studies typically use values between 0 and 0.1.23,39,40 Test fits using ηQ = 0.1 yielded variations of less than 5%. The fitting was done using a Matlab program written in house and employed a multivariable nonlinear least-squares regression method to minimize the residual for a set of 16−20 calculated and experimental spectra; 8−10 DQF and 8−10 ZQF spectra were selected from the full data set for fitting. The spectra were selected to best map out the build-up and decay of the DQF spectra. The error in the fitting was generally less than 5% for CQres. The greatest challenge when fitting the data was accurate determination of T2. This is reflected both in the fitting error estimates, which ranged from approximately 10% for shorter T2 values up to 20% for the longer T2 values, and in the scatter of the experimental 1/T2 data (see below).

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EXPERIMENTAL SECTION Sample Preparation. Pieces of powdered type 1 collagen (Sigma Aldrich, 9007-34-5) obtained from bovine achillies tendon were used as models for the type 1 collagen found in human spinal disc tissue. Powdered collagen was used in order to reduce the orientation effects caused by preferential alignment of larger fibers in the magnetic field.36,37 Samples weighing between 3 and 20 mg were dehydrated under vacuum. In order to completely dehydrate the collagen, previous studies have shown that the collagen must be placed under vacuum at 90 °C for 7 days.34 Collagen hydration levels in units of grams of D2O per gram of dry collagen (g/g) were calculated based on the mass of the dry collagen and the mass of the D2O (Acros, 99.5% D) added. The hydrated samples were placed in sealed Teflon sample holders and inserted into a 5 mm NMR tube. The collagen and D2O were allowed to equilibrate for various amounts of time depending on the hydration level; the lowest hydrations were left overnight and checked multiple times to ensure that the NMR spectra did not change, while the larger hydrations were left to equilibrate for a few hours. The sample mass was measured before and after the NMR experiment to ensure that no water was lost during the experiment. Signal intensity of a single pulse 2H NMR spectrum was also monitored to ensure that changes in hydration and changes in signal intensity were consistent. No attempts to control pH were made becasue of concerns that the addition of buffer might add further complications to the interpretation of the results; pH in a 10 g/g sample was ∼4.7. Data Acquisition. NMR spectra were collected at 20 °C on a high resolution Bruker Avance 400 MHz NMR spectrometer at The King’s Center for Molecular Structure using a Bruker ATMA multinuclear probe tuned at 61.42 MHz for 2H with a 180° pulse optimized to 40.5 μs. Experiments were referenced to liquid D2O set at 0 Hz. Double and zero quantum filtered experiments were conducted using the 90x−τ/2−180y−τ/2− 90x−t1−90 acquisition pulse sequence where t1 = 10 μs. The four-step phase cycle for the last pulse is x, y, −x, −y for both ZQF and DQF, while the detection phase cycles for ZQF and DQF are x, y, −x, −y and x, −y, −x, y, respectively.5,13,38 The evolution time τ was varied from 0.05 to 300 ms depending on

RESULTS Double and zero quantum filtered 2H NMR spectra were acquired at hydration levels (h) ranging from 0.76 g of D2O per gram of collagen (g/g) to 25.22 g/g. Figure 1 shows a series of

Figure 1. 2H DQF NMR spectra acquired on samples of collagen hydrated with D2O. Blue spectra are experimental, and red spectra are calculated.

DQF spectra acquired at three representative hydrations. At low hydrations a broad peak is observed, ∼2 kHz in breadth, which narrows as the hydration increases indicating a decrease in the residual quadrupolar coupling arising from a smaller fraction of the total water residing in restricted environments. The DQF signal intensity for all samples shows the expected build-up and decay pattern, Figure 2. The signal intensity for each curve in Figure 2 is standardized by normalizing the signal to the intensity of the single-pulse 2H NMR spectrum acquired for each sample. The signal build-up follows a sine function that depends on the magnitude of the CQres and τ, eq 2. At the lowest hydration, 0.76 g/g, the build-up of the signal occurs rapidly with an initial build-up rate of 3.4 ms−1 and decreases as hydration increases, Table 1. This normalized initial build-up 3493

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Figure 2. Plots of the normalized DQF intensity versus the experimental evolution time τ. The DQF intensities were normalized by dividing by the intensity of a NMR spectrum obtained using a single 90° pulse.

Table 1. Select Data Obtained from the ZQF and DQF Experiments for the Large CQres Sitea

a

hydration (g/g)

τmax (ms)

initial build-up rate (ms−1)

0.76 1.43 1.76 3.29 5.29 9.72 18.12

0.2 0.4 0.4 0.7 0.8 1.6 3

3.4 2.1 1.7 0.65 0.10 0.17 0.071

CQres (Hz)

1/T2 (ms−1)

× × × × × × ×

1.4 0.83 0.90 0.50 0.47 0.29 0.18

3.5 1.5 1.2 8.0 5.0 3.3 1.8

103 103 103 102 102 102 102

Figure 3. Plots of (a) CQres and (b) 1/T2 versus the hydration of the collagen. Black squares are data for the high CQres site, while red circles are for the low CQres water sites. Blue × symbols are values calculated using eqs 5 and 6 and a model for hydration based on trapped cleft and monolayer water environments; see text.

A full table of data is available in the Supporting Information.

concentration of the two sites approaches 50% each. At this point the T2 and CQres values also converge, suggesting that at higher hydrations a single site fit might be more appropriate. With no clear evidence in the spectra for returning to a single site fit, we have chosen to maintain the two-site fitting at all hydrations above 2.05 g/g. The appearance of this low CQres site in the fitting procedure indicates a different dynamic water regime where the D2O molecules do not have easy access to the sites that cause restricted motion; regions of water became visually apparent in the NMR tube at around 6 g/g. Consistent with the changes in build-up rate and τmax, the CQres and the relaxation rate, 1/T2, for the large CQres site both show similar changes as the hydration is increased, Figure 3. The CQres values decrease from 3.5 × 103 Hz at the lowest hydrations to values of around 97 Hz, while the 2H relaxation rate is rapid, ∼1.7 ms−1 at low hydration, and decreases to around 0.2 ms−1 at the highest hydrations. All the parameters showed scatter of the data points. The CQres and 1/T2 parameters show scatter that is consistent with the approximate error noted above for the fitting procedure. The scatter in the build-up rate, Supporting Information Table S1, matches that of the CQres. While the scatter in τmax is low at low hydrations, it increases at higher hydrations, mostly because of the broad, flat shape of the DQF signal intensity curve, Figure 2. From a characterization perspective, this suggests that CQres and the initial build-up rate are the most diagnostic of collagen hydration while τmax is useful at low hydrations but becomes less reliable at the highest hydrations. 1/T2 shows the most scatter, suggesting that relaxation may be the most sensitive to factors other than just the sample hydration, such as sample preparation, packing, sample quality, equilibration time, etc.

rate is a rough measure of the magnitude of the residual quadrupolar coupling and is a useful way of monitoring changes in collagen hydration. It is most useful at low hydrations where signal build-up occurs rapidly. Another defining feature of the DQF intensity curve is the evolution time at which maximum DQF signal intensity is observed, τmax, Table 1. τmax increases as the collagen is hydrated, from 0.2 ms at the lowest hydration to values around 3 ms at the highest. Since the τmax value is affected by both CQres and T2 values, it can be a useful composite parameter for monitoring changes in hydration. In order to obtain values for CQres and the T2, sets of DQF and ZQF spectra were fit using eqs 3 and 4 as a function of hydration. Representative values of CQres and the T2 are presented in Table 1 while the full set of data, plotted in Figure 3, is available in the Supporting Information. Both the DQF and ZQF spectra were fit simultaneously, allowing for sites with large CQres and negligible CQres values to be included in the fits. While fitting the spectra, it became clear that the spectra at longer τ values were no longer being fully fit using a single site. Therefore, at hydration values above 2 g/g, a second site with a much smaller CQres value was included. Between hydration values of 2.05 and 3.29 g/g, the fitting program could not automatically identify this small CQres site, a consequence of the method used to perform the least-squares fitting. In an attempt to overcome this limitation the CQres of the second site was locked at 100 Hz between hydrations of 2.05 and 3.29 g/g; 100 Hz was chosen as an extrapolation of the last three low CQres data points fit by the program. The relative amounts of water in the two sites vary with hydration; the fraction of water in the low CQres site increases with increasing hydration until the 3494

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component that can be determined from the splitting23 and is most closely related to the apparent order parameter used here. This close agreement suggests that the rapid exchange model and corresponding assumptions made above are adequate for interpreting the NMR data. The observed CQres values are a result of water spending time in the trapped cleft sites in the collagen fibrils and no observable contribution is observed from the trapped monolayer and exposed surface sites. Interestingly, the calculated data also shows scatter between h = 3 and 8 g/g, the same region where the experimental data showed the most significant scatter. This suggests that some of the experimental scatter is due to inaccuracies in the fraction of total water assigned negligible CQres values in the fitting procedure. To confirm that the sites responsible for the observed CQres are connected to the fibril structure of the collagen, experiments were performed on a sample treated with NaCl and warmed until the collagen sample denatured and formed a gel phase.42,43 CQres dropped dramatically, and the T2 relaxation time increased; a gel sample at a hydration of 10.92 g/g had a CQres of 14 Hz compared to 3.3 × 102 Hz for the fibrous sample at a hydration of 9.72 g/g. This result also suggests that deuteron exchange with the protein is not a major contributor to the larger CQres values observed for the more fibrous collagen. Quadrupolar T2 relaxation is influenced by the magnitude of the quadrupolar coupling experienced by the 2H nucleus and the motion of the water molecules, both the exchange of water molecules and their motion within individual environments. In many protein systems NMR relaxation is dominated by the interaction experienced by water when it is in the most restricted environment. This restricted environment acts as a relaxation sink, dramatically increasing the relaxation rate of the quadrupolar nuclei in water. The observed relaxation rate can be modeled in a way similar to that used for CQres, where the observed 1/T2 is the weighted sum of the rates in the various environments the water visits during the experiment.29,35

DISCUSSION Compared to other hydrated systems studied using 2H DQF NMR spectroscopy such as hydrated proton exchange membranes where CQres values up to 250 Hz were observed, the CQres values measured here for D2O on collagen are large, 3.5 kHz at h = 0.76 g/g.13,38 In a simple model, the CQres value for water undergoing rapid, random exchange between different sites is determined by the weighted average of quadrupolar coupling of the water molecules in each site. The assumption that water is rapidly exchanging between the sites during the NMR experiment is supported by the correlation times discussed in the Introduction. On the basis of previous studies of collagen hydration, initial hydration occurs in the cleft sites of the collagen triple helix. When the helices form fibrils, many of these water molecules experience restricted exchange, a proposed explanation for the longer water correlation times observed when studying fibrils;22 exchange is still fast enough to be considered rapid on the time scale of the NMR experiment. It is expected that the occupation of these restricted cleft sites will play a more important role in determining the average CQres than when the water is in the more mobile trapped monolayer sites in the fibrils or when it is exposed on the surface of the fibrils. If we use a three-site approximation for water hydration, cleft waters and monolayer waters trapped between helices, and exposed waters, which represent all exposed surface waters as well as the bulk water near the fibril, CQres can be expressed as CQ res = fcleft ⟨CQ ⟩cleft + fmono ⟨CQ ⟩mono

(5)

where f k is the fraction of water in site k, fcleft + f mono + fexposed = 1, and ⟨CQ⟩k is the averaged site specific quadrupolar coupling. ⟨CQ⟩k is often written as SkC0Q where C0Q is the quadrupolar coupling of static water (∼220 kHz)40 and Sk is an apparent order parameter reflecting both a reduction due to rapid librational motion and any deviation of the actual instantaneous coupling constant from C0Q; Sk can range from 0, total isotropic averaging, to 1, no rotational averaging.41 The fexposed⟨CQ⟩exposed term is not included in eq 5 because it will be nearly zero for these rapidly exchanging and tumbling waters. In order to determine approximate values for f k, the numbers obtained by Fullerton, using NMR titration and mirco-CT cross-sectional diameter measurements, for the amount of water accommodated in each site were used: 0.26 g/g in the cleft and an additional 1.34 g/g in the monolayers. Using these values assumes that the number of water molecules in the sites that are trapped due to the fibril structure is much larger than the number that is exposed on the surface of the fibrils. It also assumes that rapid, random exchange allows each water molecule to sample many sites during the NMR experiment, averaging the site specific quadrupolar couplings in a way that is proportional to the number of sites available to the water. By use of Fullerton’s values, the experimental hydration level, and the fraction of water assigned significant CQres values (black data points in Figure 3a), eq 5 was fit to the experimental CQres values. Figure 3a shows the close agreement (R2 = 0.95) between experimental and calculated values (blue ×). The fit yielded ⟨CQ⟩cleft = 8.4 kHz and ⟨CQ⟩mono = 0 Hz. By use of a value for C0Q of 220 kHz, the average order parameter for water in the trapped cleft sites is 0.038. This order parameter compares favorably with the magnitude of the S22 component of the Saupe’s orientation matrix previously determined for the cleft waters, |S22| = 0.036;23,27 the S22 component is the

1 1 1 1 = fcleft cleft + fmono mono + fexposed exposed T2 T T2 T2 2

(6)

As above, the exposed environment includes waters on the exposed surface of the fibrils as well as bulk waters near the fibril that are in exchange with the surface waters while the cleft and mono layer sites are those trapped between helices in the fibrils. By use of the same values discussed above for the faction of water in each site, eq 6 was fit to the experimental values, blues × in Figure 3b (R2 = 0.93). The fit yields values of Tcleft = 2 0.3 ms, Tmono = 4.5 ms, and Texposed = 25 ms. This analysis 2 2 indicates that the most restricted waters are indeed acting as the dominant relaxation sink. However, sites that do not contribute to CQres, in this model the trapped monolayer and the exposed surface water sites, are still contributing to relaxation that is faster than that of pure water; T1 of pure water was 387 ms, which under ideal conditions should be close to T2. This is an important finding, as it means that T2 relaxation could be affected by changes in the structure and dynamics of the trapped monolayer and exposed surface water, making it sensitive to physical changes that do not affect CQres. This finding may partly explain why the T2 parameters measured in this study show higher errors and more scatter than the CQres data. With the CQres and T2 parameters as a function of collagen hydration determined, connection to the DQF study of spinal 3495

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CONCLUSIONS The DQF and ZQF spectra of D2O interacting with collagen reveal that the residual quadrupolar coupling is created when rapidly exchanging water molecules sample sites on the collagen that restrict the motion and exchange of the water. On the basis of current models of hydration, we associate these sites with the clefts of the triple helix that are trapped when the helices form fibrils. There is very little contribution to CQres from the trapped monolayer or exposed waters. The short T2 relaxation times are also dominated by these trapped cleft water sites but also show a significant T2 attenuation from the trapped monolayer and exposed surface water sites. This new insight could have implications for using 2H NMR to study tissue. CQres is likely a more robust measure of collagen hydration and fibril structure, while T2 will be affected by more subtle biochemical changes that could interfere with the highly mobile monolayer and exposed waters near the surface of the collagen fibrils. When compared to the 2H DQF spectra acquired of human spinal disc samples, these results show that the water binding to collagen is a major contributor to the DQF signal observed in spinal disc tissue and that changes in the NMR signal due to tissue degeneration are strongly connected to the changing hydration of collagen.

disc tissue can be made. In the study of spinal disc tissue, spectra acquired for samples of human nucleus pulposus showed correlations between the age of the tissue sample and both the initial build-up rate and the τmax value of the DQF signal.3 In healthy nucleus pulposus tissue 70−80% of the mass is water and 4% is collagen,17 leading to a ratio of water to collagen of around 20 g/g. The normalized initial build-up rates for the DQF signal ranged from approximately 0.009 ms−1 for the more hydrated 15 year old tissue sample to 0.08 ms−1 for a highly degraded tissue sample.3 The τmax values near 3 ms were measured for degenerated nucleus pulposus samples, increasing to 20 ms for healthy tissue samples. In both cases these values place the degenerated nucleus polposus at the very highest collagen hydration levels, with the healthy tissue samples having slower signal build-ups and longer τmax values than anything observed in the collagen hydration study. The tissue τmax values are also longer than those previously observed for suspended collagen and bovine nasal cartilage immersed in D2O which showed values between 1 and 4 ms.2,5,8 On the basis of these comparisons, it can be concluded that collagen is a major contributor to the DQF signal observed in the study of spinal disc tissue. The signal intensity and the magnitude of CQres and T2 parameters of highly hydrated collagen approach the order of magnitude needed to explain the initial build-up rates and τmax values observed for the tissues. However, at similar hydration levels the build-up rate for pure collagen is about 3 times larger than for the tissue sample while the τmax is about 7 times shorter, indicating that other components in the tissue are attenuating the effect of the collagen. One species that is present in spinal disc tissue in concentrations up to 0.3 M is sodium ions. To investigate the effect of elevated Na+ levels on the parameters, 2H DQF and ZQF NMR spectra were acquired on fibrous collagen samples immersed in 0.3 M NaCl, Supporting Information Table S2. The CQres and T2 parameters did not change significantly; values were within the scatter of the neat D2O data, indicating that the presence of Na+ does not significantly affect the value of ⟨CQ⟩cleft or the distribution of water in the different environments. The most likely explanation then for the difference between the pure collagen and the tissue is that the other components give rise to uneven distributions of water, leading to localized variations in the apparent distribution of water. On the basis of the model used here, the change to fcleft would not have to be large, ∼0.008, to achieve a reduction in CQres of 70 Hz which would explain the tissue build-up rate and τmax. The different sensitivities of CQres and T2 relaxation to the water interacting with collagen fibrils have implications for the study of spinal disc health. One of the proposed reasons for turning to DQF methods as a tool for studying tissue was that T2 and T1 relaxation based imaging methods are not always diagnostic of tissue health, particularly in the early stages of disc degeneration.3,18 Our findings suggest that the utility of using DQF lies in the sensitivity of CQres to changes in the hydration of collagen fibrils, i.e., the presence and fraction of time water spends in the tightest binding sites, while T2 relaxation has more substantial contributions from secondary binding sites and changes to the exposed surface water environments. Further work to fit spectra obtained from disc tissue to determine CQres is required to further untangle the connection between observed DQF spectra, collagen structure, and tissue health.

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AUTHOR INFORMATION

S Supporting Information *

CQres and T2 values obtained from the fitting of the DQF and ZQF spectra. This material is available free of charge via the Internet at http://pubs.acs.org. Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank colleagues at The King’s University College, Canada, for helpful comments. The Natural Sciences and Engineering Research Council of Canada and TKUC are thanked for financial support. Dr. Alex Vega is thanked for help with the DQF and ZQF theory.

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REFERENCES

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