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Quantitative magnetic resonance imaging of fresh and frozen-thawed trout
Magnetic Resonance Imaging, Vol. 17, No. 3, pp. 445– 455, 1999 © 1999 Elsevier Science Inc. All rights reserved. Printed in the USA. 0730-725X/99 $–see front matter
PII S0730-725X(98)00189-1
● Original Contribution
QUANTITATIVE MAGNETIC RESONANCE IMAGING OF FRESH AND FROZEN-THAWED TROUT KEVIN P. NOTT, STEPHEN D. EVANS,
AND
LAURANCE D. HALL
Herchel Smith Laboratory for Medicinal Chemistry, University of Cambridge School of Clinical Medicine, University Forvie Site, Robinson Way, Cambridge, CB2 2PZ, UK Magnetic resonance imaging (MRI) has been used to visualise the major organs and muscular-skeletal framework of fresh rainbow trout (Salmo gairdneri) in two dimensions, and to identify the spatial distribution of lipidand collagen-rich tissues. Quantitative MRI provides the MR parameters (T1, T2, M0, Tsat 1 , Msat/M0, and the Magnetisation Transfer (MT) rate) for the tissue water; variations in those parameters enable distinction to be made between a freshly killed trout and one which has been frozen–thawed. The effects of freezing method, repeat freeze–thawing, and storage time on the MR parameters are discussed. © 1999 Elsevier Science Inc. Keywords: Authentication; Trout; Frozen storage; Freezing.
INTRODUCTION There is ample precedent in the literature for the use of the Nuclear Magnetic Resonance (NMR) parameters of water to study bulk specimens of meat, and it is known that those parameters are sensitive to changes in meat structure such as those due to the development of rigor mortis.1–3 Recently MRI has been used to study changes in those parameters induced by freeze–thawing of red meat, and it has been suggested that such measurements may distinguish fresh from frozen–thawed meat.4 Authentication of fresh fish is of great importance to the consumer for two reasons; as there is a price differential between fresh and frozen produce, retailing of fish as ‘fresh’ when it has been previously frozen is fraudulent; also many fish and shellfish can be hazardous with regards to their microbiological safety if they have been stored carelessly. The main purpose of this project was to evaluate the effects of freeze–thawing on the MRI parameters of water in trout flesh. Specifically, we have measured the values of water concentration (M0%), the spin-lattice relaxation time (T 1 ), the spin-spin relaxation time (T 2 ), the magnetisation transfer parameters Msat / M0, T sat 1 and MT rate for the protons of water. In order to support those measurements we have evaluated dif-
ferent MRI protocols for visualising the anatomical structures of intact trout, and also the spatial distribution of lipid- and collagen-rich tissue. Rainbow trout belongs to the super-order Teleostei, the bony fish with vertebrae; various imaging contrasts have been used previously to show selective enhancement of different tissue types in cartilaginous fish, Elasmobranchii.5 Freezing is an important method for reducing chemical and biological spoilage in fish, but can result in irreversible changes in its quality compared to unfrozen ‘fresh’ fish. The loss of quality as judged by changes in texture, toughening and loss of water-holding capacity,6 is thought to be mainly due to two processes; protein denaturation/aggregation and lipid oxidation. When fish is frozen, the solutes present in the muscles become concentrated as water crystallises during freezing; this increases the ionic strength of the liquid which leads to breaking of electrostatic bonds in proteins and thereby to their denaturation and aggregation. It is believed that myosin and actin, the main contractile proteins, are largely responsible for the functional properties of the muscle and that on frozen-storage, myosin in particular, undergoes denaturation/aggregation reactions.6 –10 Although it is thought that these protein changes upon
RECEIVED 5/22/98; ACCEPTED 10/22/98. Address correspondence to Laurance D. Hall, Herchel Smith Laboratory for Medicinal Chemistry, University of Cam-
bridge School of Clinical Medicine, University Forvie Site, Robinson Way, Cambridge, CB2 2PZ, UK. E-mail: [email protected] 445
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Fig. 1. Series of multi-slice coronal MR images of the head of a rainbow trout, (TR/TE 1000 ms/20 ms, NEX 4), FOV 9 cm, slice 2 mm, 6 mm between slices, 256 3 256 (zero padded from 128) matrix. Anatomical features: (n) nasal cavities; (b) brain; (e) eyes; (o) operculum; (g) gills; (p) pectoral girdle; (c) oesophagus; (h) heart.
freezing are the main causes of quality loss in frozen fish,6 oxidation of lipids can lead to rancid flavours. In addition, lipid oxidation is thought to enhance the deterioration of proteins.11,12 Lipids have been shown to be prevalent in trout by the use of MRI weighted images.13 Changes associated with freeze-storing cod, as well as thermal and high pressure treatment, have been followed14 at 20 MHz (0.47T). It was found that the transverse relaxation time of the water protons showed multicomponent behaviour, with frozen-stored cod having a longer relaxation component that increased with the temperature and duration of frozen-storage. This component was attributed to the water exudate associated with protein denaturation, as well as that from physical damage and enzymatic reaction. A similar MR-relaxation study was undertaken on frozen-storage of post rigor minced cod,15 which also showed bi-exponential relaxation be-
haviour for the water protons. The MR parameters showed variation on frozen-storage between different quality minces, which became more marked in the cooked state. These changes were ascribed to the aggregation of myofibrillar proteins during frozen-storage as opposed to intra/extra-cellular compartmentalisation since the bi-nodal distribution persists into the cooked state. Since the precise history of the fish flesh is of paramount importance to any study of temporal changes,16 attention was focussed in the present study on freshwater rainbow trout which were purchased live from a local trout farm. Initially, a fully automated MRI protocol was used to study a small number of whole trout. However, when those results were analysed it was realised that a much larger number of samples needed to be studied to account for biological variations and so a multiple-sam-
Quantitative MRI of trout ● K.P. NOTT
ple, bulk-MRI approach was used. Given that interanimal variation was likely to be large, it was also decided to investigate the feasibility of a comparative method, which involved repeated freeze–thawing of the sample. The hypothesis is that the change in the MR parameters on re-freezing a sample which had been previously frozen would be smaller than those on freezing a fresh fish, since the major damage had already been inflicted. MATERIALS AND METHODS Intact Trout MRI hardware. All 1H MRI images were acquired using a Bruker BMT (Bruker Medzintechnik Biospec II) imaging console (Karlsruhe, Germany) connected to a 2.35 Tesla, 31 cm horizontal bore super-conducting magnet (Oxford Instruments, Oxford, UK). A 20 cm gradient set built ‘in-house’ with each axis powered by a Techron gradient amplifier (Model 7790, Crown International Inc., Elkart, IN, USA) provided gradient strengths up to 100 mT m21. An ‘in-house’ built, cylindrical, eight strut, bird-cage radio-frequency (RF) probe, internal diameter 9.4 cm, was used in the quadrature mode to transmit and receive the MR signal. Samples were kept at ambient temperature (20°C) by passing a constant stream of air through the probe. Sample preparation. The MRI measurements used 4 trout for each freezing regime, each set was caught by net in November (mean weight, 640 g; mean length, 35 cm) and sacrificed using a Schedule 1 method. Since significant changes occur during at least the first five hours after death,17 the samples were left to equilibrate post mortem in a refrigerator at 2°C. After MRI quantitation of the fresh material, the samples were subjected to one of two freezing regimes: 1) fast freezing in liquid nitrogen, followed by storage in a domestic freezer at 218°C for 4 weeks; 2) slow freezing, and subsequent storage for four weeks in a domestic freezer at 218°C before quantitation of the frozen–thawed samples. Data acquisition and processing. The quantitation of T 2 , T 1 , and MT parameters was achieved by varying the Recovery Delay (TR) the Echo Time (TE), the saturation transfer delay (SP) and the number of echoes in a multiecho Carr Purcell Meiboom Gill (CPMG) MRI sequence developed previously in this laboratory;18 this is based on a sequence devised by Crawley and Henkelman,19 which employs an additional slice-selection gradient to reduce unwanted echoes generated at the slice edge by RF imperfections. This fully automated protocol enables calculation of those parameters from a set of 256 3 64 pixel images (field of view 9 cm, slice thickness 5 mm, 2 transients). The number of phase encoding steps were
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minimised to reduce the acquisition time and increase the signal-to-noise. A set of T 2 -weighted images were obtained through acquisition of 8 echoes for each pixel (7500/20/0 [TR/ TE/SP]). The T 1 -weighted images were obtained by incrementing TR (400, 800, 1600, 3200, 7500/20/0) of a single spin echo sequence to produce images attenuated by T 1 saturation recovery relaxation.20,21 T sat 1 -weighted images were obtained by increasing the duration of the saturation pulse (SP), applied prior to the 90° pulse of a single spin echo sequence (7500/20/0, 100, 325, 750, 1500, 2000). Selective saturation of the macromolecular proton pool was achieved by applying a squared pulse of low amplitude (;10 mT, 10% of the RF power needed to achieve a 90° pulse) at 10 kHz off-resonance.22–24 The raw MRI data sets were transferred from the Bruker Aspect 3000 computer via an ASL data transfer board (designed by A.A. Wilkinson and Dr. N. Dillon) to a network of Unix stations where the subsequent data processing took place. The image-data were zero-padded in the phase direction to produce isometric pixels, baseline corrected, and a Gaussian filter was applied prior to Fourier transformation. The fitting of the data was performed on a curve fitting program (Cmrfit) written-inhouse by Dr. J.J. Attard, based on the Levenberg-Marquandt method.25 All data were visualised using image display software (Cmrview) written by Dr. N.J. Herrod. The MR values were obtained by placing a hand drawn region of interest (ROI) over the muscle in the image. The T 2 - and T 1 -weighted images were fitted assuming mono-exponential relaxation behaviour, to equations (1) and (2) respectively, TE
Mx,y 5 M0e 2 T2
S
(1)
D
TR
Mx,y 5 M0 1 2 e 2 T1
(2)
where Mx,y is the magnetisation at a particular pixel, M0 is the equilibrium magnetisation (proportional to the liquid proton density), T 2 is the transverse relaxation time and T 1 is the longitudinal relaxation time. M0 was quantified from back-projection of the T 2 decay curve and the liquid proton density (M0%) was calculated by comparison of its M0 value with that of a 2% (w/w) agar gel standard which was in place for each experiment. The T sat 1 -weighted images were fitted assuming mono-exponential relaxation behaviour to equation (3). SP
Mx,y 5 Msat 1 (M0 2 Msat)e 2Tsat 1
(3)
to produce the T sat 1 and Msat /M0 images: where M0 is the equilibrium magnetisation (SP 5 0); Mx,y is the magne-
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Fig. 2. Water and fat resolved (from 3 point Dixon TR/TE 6000 ms/20 ms, NEX 2), and T 1 -weighted (TR/TE 1000 ms/20 ms, NEX 4) sagittal MR images of the head of a rainbow trout using FOV 7 cm, slice 2 mm, 256 3 256 matrix (zero padded from 128).
tisation at the particular pixel; Msat is the minimum longitudinal magnetisation observed when saturation sat transfer reaches a steady-state (SP 5 5T sat 1 ) and T 1 is the longitudinal relaxation time measured during saturation transfer. Assuming that the protons of the macromolecular pool are saturated during the application of the irradiating pulse, the apparent MT rate (k) image can be calculated according to equation (4), from the images previ22,23 ously fitted to Msat , M0 and T sat 1 . k5
S
D
1 Msat sat 1 2 M0 T1
(4)
Optimisation of the saturation of the proton pool can only be achieved by time-consuming experiments involving varying the amplitude and offset of the saturation pulse.26 –28 Since full saturation is unlikely to be achieved in practice the value provided by this sequence can only be regarded as an estimate of the true magnetisation transfer rate. Nevertheless the value provides a reliable indication of the relative strength of the MT phenomenon.18,29 Multiple Sample Protocol In order to increase the number of samples studied without increasing the amount of time involved, a multiple-sample, bulk MRI protocol approach was used.30 This involved, extracting 3 tissue samples per trout with a modified cork borer and placing them into an array of wells in a multiple-sample tissue culture plate. Using the MRI protocols summarised above the number of samples that could be analysed simultaneously was only limited30 by the homogeneity of the radio-frequency (B1) and magnetic (B0) fields.31–33 MRI hardware. A 14.5 cm internal diameter gradient was used, with each axis powered by two Techron gradient amplifiers (Models 7570 and 7560, Crown Interna-
tional Inc., Elkart, IN, USA). This gradient set enabled34,35 the use of a short echo time (12 ms) which gave more points on the T 2 decay curve. Sample preparation. Eighteen fresh trout in May were gutted and divided into 5 steaks (thickness 1.5–2 cm) from which all the samples were taken for gravimetric and MR quantitative analysis. All eighteen trout were analysed as ‘fresh’ by MRI on the day the fish was killed and after a suitable equilibration period post mortem in a refrigerator at 2°C. The remaining steaks were frozen at 218°C in a domestic freezer and then subjected to one of three regimes: 1. Six trout were kept frozen for two days and then analysed after thawing. 2. Six trout were kept frozen for two weeks, analysed after thawing and analysed 2 days later after repeated freeze–thawing. 3. Six trout were kept in frozen for 4 weeks prior to further analysis on the thawed samples, and analysed as for (2) above. The trout steaks were thawed to room temperature between 2 aluminum sheets 4 h prior to being placed in the multi-well plate. At each stage they were analysed for moisture content using the protocol detailed by Kirk36 and the pH measured using a pH meter (Model 220, Corning Science Products, USA). Data acquisition. The same fully automated protocol allowed the acquisition of a set of images that enabled the calculation of the T 2 , T 1 and MT parameters for all the samples in each plate. Since the samples were not required for further treatment, the data acquisition protocol was lengthened to increase the accuracy of each parameter.30 A set of 16 T 2 -weighted images were obtained (6000/ 12/0 [TR/TE/SP]). The T 1 -weighted images were ob-
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each well of each of the fitted images, and those values were then used for calculation of each of the quantitation parameters. Bulk NMR Relaxometry Protocols For comparison, smaller scale bulk NMR T 2 relaxometry studies were carried out. Samples were taken from four fresh trout and again after the same trout were frozen-stored for four weeks at 218°C. Bulk CPMG experiments were carried out using echo times of 1 and 12 ms. The resultant decay curves were best fit to a bi-exponential decay. Because imperfections in the RF pulses can cause a large errors,38 the bulk measurements were carried out on a small sample using the large birdcage imaging probe.
Fig. 3. Sagittal images of the head of a rainbow trout under various imaging contrasts; Proton density (TR/TE/SP 7500/ 20/0, NEX 2), T 1 -weighted (TR/TE/SP 400/20/0, NEX 2), T 2 -weighted (TR/TE/SP 7500/60/0, NEX 2), MT-weighted (TR/TE/SP 7500/20/750, NEX 2) using FOV 10 cm, slice 1.5 mm, 256 3 256 matrix (zero padded from 128). Anatomical features: (c) oesophagus; (h) heart.
tained by incrementing TR (120, 375, 500, 750, 1000, 1250, 1500, 1750, 2000, 2500, 3000, 6000/12/0) of a single spin echo sequence to produce images attenuated by T 1 saturation recovery relaxation. T sat 1 -weighted images were obtained by increasing the duration of the saturation pulse (SP), applied prior to the 90° pulse of a single spin-echo sequence (6000/12/0, 50, 150, 300, 750, 1000, 1500). Data processing. All the 128 3 128 pixel images of each plate were measured using a field of view of 7 cm and a 4-mm slice thickness, and the phase direction data zero-padded from 64. This gave the optimal pixel resolution and signal-to-noise ratio of ca 120 required for the subsequent edge detection analysis, with the minimum image acquisition time required for the quantitation of the MR parameters. Estimation of the mean MR parameters for the contents for each well was achieved using fully automated edge detection30 of the first echo image to delineate the edges of each well, followed by location of the well centres using a Hough Transform algorithm for modified circles.37 The local maxima of Hough space identified the coordinates of the centres of the wells, over which a circular mask of a suitable radius was then placed; the mean value of the intensity of all the pixels enclosed within the boundary of that circle was then calculated for
Fig. 4. Sagittal images of the body of a rainbow trout under various imaging contrasts; Proton density (TR/TE/SP 7500/ 20/0, NEX 2), T 1 -weighted (TR/TE/SP 500/20/0, NEX 4), T 2 -weighted (TR/TE/SP 7500/60/0, NEX 2), MT-weighted (TR/TE/SP 7500/20/750, NEX 4), MT difference (between proton density image and MT-weighted image) using FOV 9 cm, slice 2 mm, 256 3 256 matrix (zero padded from 128). Anatomical features: (r) eggs; (l) liver; (s) swim bladder.
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Table 1. MR parameters for water in fresh and frozen–thawed (F/T) trout flesh Freezing Rate Slow Fresh F/T Change Fast Fresh F/T Change
T 1 (s)
T 2 (ms)
M0%
T sat (s) 1
Msat /M0
MT rate (s21)
1.37 6 0.03 1.32 6 0.05 23.6%
41.7 6 0.9 40.5 6 0.7 22.9%
84.6 6 0.4 83.6 6 0.7 21.2%
0.320 6 0.005 0.310 6 0.005 23.1%
0.317 6 0.015 0.257 6 0.010 218.9%
2.15 6 0.05 2.41 6 0.02 112.1%*
1.24 6 0.04 1.25 6 0.04 10.8%
43.3 6 1.0 44.5 6 0.3 12.8%
87.3 6 0.5 84.4 6 0.3 23.3%*
0.304 6 0.007 0.309 6 0.002 11.6%
0.228 6 0.009 0.231 6 0.004 11.3%
2.56 6 0.09 2.49 6 0.03 22.7%
* Significant at 1% level for a paired two-tailed t-test ( p , 0.01). Mean 6 standard error of four samples. Measurements were taken from the transverse section of eight ‘fresh’ rainbow trout caught in November; 4 samples were slow-frozen in a domestic freezer at 218°C, and four were fast-frozen in liquid nitrogen at 2196°C. The same sections were analysed after 4 weeks frozen-storage.
RESULTS AND DISCUSSION Anatomical Images Segmented anatomical images were obtained using T 1 -weighted multi-slice imaging for the head of a fresh rainbow trout in the coronal direction (Fig. 1). Those images revealed numerous anatomical features, including the gill structures, the eyes, nasal passage and vertebrae. They gave high contrast between muscle and fat tissue because the T 1 of fat is approximately half that of muscle. Comparison in Fig. 2 with the fat-resolved im-
Fig. 5. An example of a MT rate image on a transverse section of a ‘fresh’ rainbow trout and the corresponding slice after frozen-storage for 4 weeks. The histograms give the distribution of the pixel values for MT rate taken over the muscle.
age obtained by a three-point Dixon technique39,40 clearly showed how the T 1 -weighted image highlights the lipids. The fat-resolved image was obtained using three-point rather than the two-point41 Dixon method because the latter was found to be too sensitive to B0 field inhomogeneities. There are two main parts to the skull: the chondrocranium (Fig. 1E), which is a set of compartments around the nasal cavities (n), brain (b) and eyes (e); and the branchiocranium, which consists of the bones which support the gill arches, opercular flap, and the jaws or mandibular region. Most of the skull features were observed as high intensity regions. Other visible components of the skeleton includes bone, cartilage and the connective tissue envelope which joins the musculature to the skeleton. The actual distribution of all these tissues were apparent in images which were obtained under different contrasts. The structures of the respiratory gills can be seen in Figs. 1A–D. The gill arches can be clearly seen in D; there are four pairs of gills (g) on each side of the fish, lying in a cavity containing the capillary vessels just in front of the pectoral girdle (p), which are protected on each side by two flaps of skin supported by bones, the operculum (o). The beginning of the oesophagus (c) and the heart (h) can be seen further down the set of images. Figure 1F shows the skeletal framework leading to the brain (b); the brain and spinal cord are enclosed within the skull and vertebral column; the eyeball can be clearly visualised (Fig. 1E) with the pupil appearing as a low intensity region in the centre of the iris. Various other tissues were identified by comparison between a series of sagittal images of the head (Fig. 3) obtained under different image contrast regimes. The T 1 -weighted image highlighted the lipids with respect to
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Table 2. MR parameters for water in fresh and frozen–thawed (F/T) excised flesh of trout, following various storage regimes at 218°C
Fresh Fresh12dayF/T Ratio Fresh12dayF/T /Fresh 2 weeks F/T 2 weeks F/T12dayF/T Ratio 2 weeks F/T12dayF/T /2 weeks F/T 4 weeks F/T 4 weeks F/T12dayF/T Ratio 4 weeks F/T12dayF/T /4 weeks F/T
T 1 (s)
T 2 (ms)
M0%
T sat (s) 1
Msat /M0
MT rate (s21)
1.35 6 0.01 1.09 6 0.01 0.81 6 0.01 1.18 6 0.01 1.08 6 0.01
43.1 6 0.5 42.0 6 0.3 0.97 6 0.01 54.7 6 0.6 50.2 6 0.4
88.3 6 0.8 88.8 6 0.5 1.01 6 0.01 88.1 6 0.6 88.1 6 0.9
0.311 6 0.002 0.206 6 0.002 0.66 6 0.01 0.196 6 0.001 0.203 6 0.001
0.170 6 0.003 0.168 6 0.003 0.99 6 0.03 0.179 6 0.003 0.174 6 0.003
2.70 6 0.02 4.06 6 0.05 1.50 6 0.02 4.19 6 0.04 4.05 6 0.04
0.92 6 0.01 1.12 6 0.02 1.07 6 0.01 0.95 6 0.02
0.92 6 0.01 52.4 6 0.7 49.6 6 0.6 0.95 6 0.02
1.00 6 0.01 84.5 6 0.9 85.7 6 0.6 1.01 6 0.01
1.03 6 0.01 0.196 6 0.002 0.207 6 0.003 1.06 6 0.02
0.97 6 0.02 0.149 6 0.002 0.160 6 0.003 1.07 6 0.02
0.97 6 0.01 4.38 6 0.04 4.10 6 0.05 0.94 6 0.01
Mean 6 standard error (54 fresh, 18 frozen–thawed samples). Measurements were taken on 18 fresh trout caught in May (three samples from each). Equivalent measurements were taken after the trout steaks were subjected to one of three storage regimes: 1) freeze–thawing over two days, 2) 2 weeks frozen-storage and after repeat freeze–thawing over 2 days, 3) 4 weeks frozen-storage and after repeat freeze–thawing over 2 days.
and MT-weighted images) because the connective tissue between the myotomes is visualised. Tissue discrimination due to MT contrast was increased in an MT difference image (Fig. 4), obtained by subtracting two spatially identical images, one with and another without the application of the MT saturation pulse. Although the muscle shows a noticible MT-difference effect, the more intense regions were associated with the backbones and the belly, indicating that those were highly collageneous tissues. Below the swim bladder (s), which has zero intensity, the eggs (r), the liver (l) and, to the right, the spleen and stomach were visible.
the water containing tissues owing to their shorter T 1 values. However, the T 2 - and MT-contrasted images, in particular, showed the greatest distinction between different muscle tissues; one image shows at least three of the 4 heart chambers. The proton density map gave little tissue contrast because all the tissues contain approximately the same concentration of water. Figure 4 shows a sagittal image across the vertebral column of the fish, measured with the 4 types of image contrast. Although slightly masked by partial volume effects associated with limited spatial resolution, the bones in the vertebral column were clearly visible in all four scans because they had low signal intensity. The backbones were masked by a combination of fatty and collageneous tissue, which are indicated in the T 1 - and MT-weighted images respectively. The fish muscle is divided into a series of blocks, called myotomes, by myoseptal connective tissue (which is composed of inextensible collagen fibres). The myotomes form a complex array of segments in a characteristic W-shape along the fish; the patterns of those structures were clearly evident in the different images (particularly in the T 2 -
MRI Relaxometry of Whole Trout Table 1 shows the data obtained from measurements of intact fresh rainbow trout and after subsequent storage for four weeks following either slow freezing in a conventional freezer at 218°C, or fast-freezing in liquid nitrogen at 2196°C. A two-tailed, “paired sample t-test” was used to determine whether freezing had a significant effect on the MR parameters. Some of the MR parameters exhibited larger changes on slow freezing than for
Table 3. Significance at 1% for two-tailed t-test ( p , 0.01), for effects of freeze/thawing (F/T) on the MR parameters of trout flesh given in Table 2 t-test
T1
T2
Fresh v 2 weeks F/T Fresh v 4 weeks F/T 2 weeks F/T v 4 weeks F/T Fresh v Fresh12dayF/T 2 weeks F/T v 2 weeks F/T12dayF/T 4 weeks F/T v 4 weeks F/T12dayF/T
p , 0.01 p , 0.01 NS p , 0.01 p , 0.01 NS
p , 0.01 p , 0.01 NS NS p , 0.01 NS
NS 5 Not significant.
M0% NS NS NS NS NS NS
T sat 1
Msat /M0
MT rate
p , 0.01 p , 0.01 NS p , 0.01 NS NS
NS p , 0.01 p , 0.01 NS NS NS
p , 0.01 p , 0.01 NS p , 0.01 NS p , 0.01
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Table 4. Effect of freeze–thawing (F/T) at 218° on the mean percentage water content (measured by oven drying) and pH of trout steaks
Fresh Fresh12dayF/T Fresh 2 weeks F/T 2 weeks F/T12dayF/T Fresh 4 weeks F/T 4 weeks F/T12dayF/T
pH
% water
6.32 6 0.06 6.23 6 0.03 6.28 6 0.02 6.26 6 0.03 6.29 6 0.01 6.34 6 0.02 6.37 6 0.03 6.38 6 0.03
77.10 6 0.21 77.25 6 0.35 77.86 6 0.31 77.67 6 0.46 77.51 6 0.42 77.71 6 0.41 77.81 6 0.36 77.35 6 0.31
Mean 6 standard error for six trout. The samples were taken at each stage in the experiment outlined on Table 2.
fast freezing. The induction of larger changes by slow freezing can be attributed to the different physical damage to the muscles caused by slower freezing rates.42 The larger ice crystals produced more cellular disruption by dehydration of the intra-cellular space which led to more protein denaturation/aggregation. Fast freezing is thought to rapidly produce smaller ice crystals, therefore, there is no major redistribution of water. However, on frozen storage at 218°C the ice crystals may have had a tendency to grow to larger ones, thereby leading to greater deterioration of the flesh. The magnetisation transfer rate was particularly sensitive to the changes associated with slow freezing and was significantly different at the 1% level ( p , 0.01) for four samples. An example of one set of images, before and after freeze–thawing, is illustrated in Fig. 5 together with the histogram plots for the pixel values of MT rate across the fish muscle. There was a large variation between the MR parameters of the two groups of fresh fish, although not significantly different at the 1% level ( p , 0.01). This variation demonstrates that absolute MR values can only be of use if the inter-animal variations are small, compared to the difference between the fresh and frozen– thawed product.
Effect of Storage and Repeat Freeze–Thawing The data obtained from excised samples of trout flesh using the multiple sample protocol are shown in Table 2. The results of statistical analyses on those data are given in Table 3. Table 4 shows the equivalent data for moisture content and pH at each stage of the experiment; there was a general decrease in the moisture content, though not statistically significant at the 1% level ( p , 0.01). There are 2 main differences between the data in Table 1 and Table 2. Firstly, the data for these 2 sets of fresh fish may indicate seasonal variations;43 the multiple sample experiments were conducted 6 months after the initial study. Secondly, the differences on freeze–thawing trout steaks was much greater than that observed for freeze–thawing the whole trout; this may reflect some additional tissue disruption associated with the preparation of the steaks. It was found that the magnetisation transfer parameand MT rate were particularly sensitive to ters T sat 1 freeze–thawing; thus there was a significant difference at the 1% level ( p , 0.01) between the fresh sample and that after freeze–thaw treatment. Since there was no significant difference between storage over 2 and 4 weeks, it is clear that most of the damage must occur during the initial freezing process; furthermore, a repeated freeze–thaw cycle on a previously frozen–thawed sample shows less change than for the first freeze–thaw cycle of a fresh product (Table 2). The 2 day freeze–thaw cycle on the fresh fish had no significant effect on the T 2 parameter; however, a statistically significant increase was observed after frozen storage. The observed increase in the mono-exponential transverse relaxation time could be a result of a redistribution of the water within the muscle, which increases the population of the longer relaxation component.14,44 This rationale is based on the ‘bulk’ NMR T 2 data given in Table 5. M0 and T 2 of the observed relaxation components can be dependent on the echo time (TE).45,46 No significant change was observed in the mono-exponential T 2 due to freeze–thawing. However, a significant increase was observed in the longer T 2 component of a bi-exponential decay analysis of data obtained with an
Table 5. ‘Bulk’ mono-exponential and bi-exponential T 2 parameters for water in fresh and frozen–thawed (F/T) trout flesh Sample Fresh F/T
TE (ms)
Bulk T 2 (ms)
T A2 (ms)
PA (%)
T B2 (ms)
PB (%)
1 12 1 12
52.2 6 0.7 50.0 6 0.7 57.4 6 0.4 55.4 6 0.6
45.3 6 0.7 43.8 6 1.1 46.4 6 0.9 43.9 6 0.4
82.4 6 3.2 87.1 6 4.1 81.0 6 2.0 84.9 6 0.9
98.3 6 6.9 121.2 6 20.6 130.0 6 8.5 128.6 6 11.3
17.6 6 3.2 12.9 6 4.1 19.0 6 2.0 15.1 6 0.9
Mean 6 standard error for four samples. The trout were frozen for a period of 4 weeks in a domestic freezer at 218°C. PA and PB are the populations of the bi-exponential components T A2 and T B2 respectively.
Quantitative MRI of trout ● K.P. NOTT
echo time of 1 ms; this was not observed when an echo time of 12 ms was used. The mono-exponential T 2 ’s from bulk and imaging were similar. The liquid proton density, which was extrapolated from the mono-exponential fit of a T 2 decay curve, showed a general, though not statistically significant, decrease with frozen storage; this was most likely due to water loss through ‘drip’ on thawing the muscle. Water exudes not from the breakage of the sarcolemma (cell wall) as was first thought, but through changes in the protein properties on frozen-storage.42 The statistically significant decrease in T 1 could be a result of a reduced water content through drip loss; however, the large size of the changes could indicate that the relaxation rate is enhanced47 by the reduction in protein mobility through protein aggregation. The changes in the MT rate could be the result of a combination of factors.48 However, a significant contribution could stem from the freeze denaturation of proteins, which subsequently leads to protein aggregation involving increased rigidity by intermolecular cross-links. Magnetisation transfer rates are known to increase with the increasing weight of protein,47 owing to the enhanced cross-relaxation mechanism. Given that Tsat 1 and MT rate appeared to be more sensitive to the effects of freeze–thawing, future studies should concentrate on the magnetisation transfer phenomenon in relation to the mechanism of the protein deterioration during freeze–thawing. CONCLUSIONS The original aims of this work were 2-fold; first, to demonstrate the type of anatomical resolution that MRI can achieve between individual organs of intact trout. Second, to explore the potential use of MRI as a basis for the authentication of fresh trout from that which had been frozen–thawed. It is clear that MRI enabled the anatomical details of rainbow trout to be visualised by highlighting various soft tissues using a combination of protocols that gave different contrasts. This has implications regarding fish quality since the distribution of fat and the content of connective tissue are also important factors affecting quality. In the future it will be feasible to achieve a time course study on living fish.49 Quantitative MRI provided parameters that were sensitive to the effects of freeze–thawing and to the method of freezing and duration of frozen-storage. Thus, there were no significant changes in the MR parameters on freezing on intact trout in liquid nitrogen, whereas there was a significant change in the MT rate as a result of slow freezing. For the trout steaks T 1 , T sat 1 and MT rate exhibited the most prominent effects. For fish which had been previously frozen, the change in the MR parameters
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after repeat freeze–thawing were smaller than those observed for the initial 2 day freeze–thaw of fresh fish, indicating a method which could be used to allow for the variability observed for fresh trout. The same methodology could be applied to sea-water fish, crustacea and molluscs, which are prone to loss of quality through mistreatment but which are less readily available in the ‘fresh’ state. Acknowledgments—The authors thank the UK Ministry of Agriculture Fisheries and Food (MAFF) and the Herchel Smith Endowment for collaborative funding of this work; Miss A. Kshirsagar for providing the edge detection software; Dr. J.J. Tessier for the MRI quantitation protocols; Dr. T.A. Carpenter, Mr. C. Bunch, Mr. S. Smith and Mr. C. Harbird for supplying and maintaining the MRI facilities.
REFERENCES 1. Chang, D.C.; Hazlewood, C.F.; Woessner, D.E. The spinlattice relaxation times of water associated with early postmortem changes in skeletal muscle. Biochim. Biophys. Acta. 437:253–258; 1976. 2. Currie, R.W.; Jordan, R.; Wolfe, F.H. Changes in water structure in postmortem muscle, as determined by NMR T 1 values. J. Food Sci. 46:822– 823; 1981. 3. Pearson, R.T.; Duff, I.D.; Derbyshire, W.; Blanshard, J.M.V. An NMR investigation of rigor in porcine muscle. Biochim. Biophys. Acta. 362:188 –200; 1974. 4. Guiheneuf, T.M.; Parker, A.D.; Tessier, J.J.; Hall, L.D. Authentication of the effect of freezing/thawing of pork by quantitative magnetic resonance imaging. Mag. Reson. Chem. 35:S112–S118; 1997. 5. Waller, G.N.H.; Williams, S.C.R.; Cookson, M.J.; Kalkoudi, E. Preliminary analysis of elasmobranch tissue using magnetic resonance imaging. Magn. Reson. Imaging 12: 535–539; 1994. 6. Mackie, I.M. The effects of freezing on flesh proteins. Food. Rev. Int. 9:575– 610; 1993. 7. Matsumoto, J.J. Denaturation of fish muscle proteins during frozen storage. Adv. Chem. Ser. 180:205–224; 1980. 8. Matsumoto, J.J. Chemical deterioration of muscle proteins during frozen storage. A.C.S. Symp. Ser. 123:95–124; 1980. 9. Shenouda, S.Y.K. Theories of protein denaturation during frozen storage of fish flesh. Adv. Food Res. 26:275–311; 1980. 10. Sikorski, Z.; Olley, J.; Kostuch, S. Protein changes in frozen fish. C.R.C. Crit. Rev. Food Sci. Nutr. 8:97–129; 1976. 11. Takama, K. Insolubilisation of rainbow trout actomyosin during storage at 220°C. I. Properties of insolubilised proteins formed by reactions of propanal and caproic acid with actomyosin. Bull. Jap. Soc. Sci. Fish 40:585–588; 1974. 12. Takama, K.; Zama, K.; Igarashi, H. Changes in the flesh lipids of fish during frozen storage. III. Relation between rancidity in fish flesh and protein extractability. Bull. Jap. Soc. Sci. Fish 38:607– 612; 1972.
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13. McCarthy, M.J.; Kauten, R.J. Magnetic resonance imaging applications in food research. Trends Food Sci. Technol. 1:134 –139; 1990. 14. Lambelet, P.; Renevey, F.; Kaabi, C.; Raemy, A. Lowfield nuclear magnetic resonance relaxation study of stored or processed cod. J. Agric. Food Chem. 43:1462– 1466; 1995. 15. Lillford, P.J.; Jones, D.V.; Rodger, G.W. Water in fish tissue—a proton relaxation study of post rigor minced cod. In Adv. Fish Sci. Technol. 495– 497; 1979. 16. Love, R.M. The Technology of Fish Utilization. London: Fishing News (Books) Ltd., 1965. 17. Foucat, L.; Bielicki, G.; Peyronnet, N.; Blachere, L.; Troquier, C.; Renou, J.P. Post mortem energy catabolism and water dynamic changes in trout muscle. Poster at the Third International Conference on Applications of Magnetic Resonance in Food Science, Nantes, France; 1996. 18. Guiheneuf, T.M.; Tessier, J.J.; Herrod, N.J.; Hall, L.D. Magnetic resonance imaging (MRI) of meat products: 1—Automated quantitation of the NMR relaxation parameters of cured pork, by ‘bulk’ and MRI methods. J. Sci. Food Agric. 71:163–173; 1996. 19. Crawley, A.P.; Henkelman, R.M. Errors in T 2 estimation using multislice multiple-echo imaging. Mag. Reson. Med. 4:34 – 47; 1987. 20. Kaldoudi, E.; Williams, S.C.R. Relaxation time measurements in NMR imaging, Part I: Longitudinal relaxation time. Conc. Magn. Reson. 5:217–242; 1993. 21. Pykett, I.L.; Rosen, B.R.; Buonanno, F.S.; Brady, T.J. Measurement of spin-lattice relaxation times in nuclear magnetic resonance imaging. Phys. Med. Biol. 28:723– 729; 1983. 22. Wolff, S.D.; Balaban, R.S. Magnetisation transfer contrast (MTC) and tissue water proton relaxation in vivo. Mag. Reson. Med. 10:135–144; 1989. 23. Eng, J.; Ceckler, T.L.; Balaban, R.S. Quantitative 1H magnetisation transfer imaging ex-vivo. Mag. Reson. Med. 17:304 –314; 1991. 24. Hajnal, J.V.; Baudouin, C.J.; Oatridge, A.; Young, I.R.; Bydder, G.M. Design and implementation of magnetisation transfer pulse sequences for clinical use. J. Comp. Assist. Tomog. 16:7–18; 1992. 25. Marquardt, D.W. An algorithm for least-squares estimation of non linear parameters. J. Soc. Ind. Appl. Math 11:431– 441; 1963. 26. Henkelman, R.M.; Huang, X.; Xiang, Q.X.; Stanisz, G.J.; Swanson, S.D.; Bronskill, M.J. Quantitative interpretation of magnetisation transfer. Mag. Reson. Med. 29:759 –766; 1993. 27. Morrison, C.; Henkelman, R.M. A model for magnetization transfer in tissues. Mag. Reson. Med. 33:475– 482; 1995. 28. Tessier, J.J.; Dillon, N.; Carpenter, T.A.; Hall, L.D. Interpretation of magnetisation transfer and proton cross-relaxation spectra of biological tissues. J. Magn. Reson. Series. B 107:138 –144; 1995. 29. Tessier, J.J. Magnetisation Transfer Imaging and Proton
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Cross-relaxation Spectroscopy. PhD thesis, University of Cambridge, U.K., 1995. Evans, S.D.; Nott, K.P.; Kshirsagar, A.; Hall, L.D. Efficient magnetic resonance imaging methods for automated quantitation of MR parameters from multiple samples. Mag. Reson. Chem. 35:S76 –S80; 1997. Majumdar, S.; Orphanoudakis, S.C.; Gmitro, A.; O’Donnell, M.; Gore, J.C. Errors in the measurements of T 2 using multiple-echo MRI techniques I. Effects of radiofrequency pulse imperfections. Mag. Reson. Med. 3:397– 417; 1986. Majumdar, S.; Orphanoudakis, S.C.; Gmitro, A.; O’Donnell, M.; Gore, J.C. Errors in the measurements of T 2 using multiple-echo MRI techniques II. Effects of static field inhomogeneity. Mag. Reson. Med. 3:562–574; 1986. Poon, C.S.; Henkelman, R.M. Practical T 2 quantitation for clinical applications. J. Mag. Reson. Imag. 2:541–553; 1992. Fisher, B.J.; Dillon, N.; Carpenter, T.A.; Hall, L.D. Design by genetic algorithm of a z gradient set for magnetic resonance imaging of the human brain. Meas. Sci. Technol. 6:904 –909; 1995. Fisher, B.J.; Dillon, N.; Wilkinson, A.A.; Carpenter, T.A.; Hall, L.D. Design and evaluation of a transverse gradient set for magnetic resonance imaging of the human brain. Meas. Sci. Technol. 7:1– 6; 1996. Kirk, R.S. Pearson’s composition and analysis of foods. Harlow: Longman, 9th Edition, 1991. Chan, R.; Siu, W.C. New parallel hough transform for circles. IEEE Proceedings, Computers and Digital Techniques 138(5):335–344; 1991. Vold, R.L.; Vold, R.R.; Simon, H.E. Errors in measurements of transverse relaxation rates. J. Magn. Reson. 11: 283–298; 1973. Lodes, C.C.; Felmlee, J.P.; Ehman, R.L.; Sehgal, C.M.; Greenleaf, J.F.; Glover, G.H.; Gray, J.E. Proton MR chemical shift imaging using double and triple phase contrast acquisition methods. J. Comp. Assist. Tomog. 13:855– 861; 1989. Glover, G.H.; Schneider, E. Three-point Dixon technique for true water/fat decomposition with B0 inhomogeneity correction. Mag. Reson. Med. 18:371–383; 1991. Dixon, W.T. Simple proton spectroscopic imaging. Radiology 153:189 –194; 1984. Love, R.M. Ice formation in frozen muscle. In: J. Hawthorn and E.J. Rolfe (Eds). Low temperature biology in foodstuffs. Pergamon Press, Oxford, 1968: pp. 105–123. Love, R.M. The Chemical Biology of Fishes, Vol 2. Academic Press, New York, 1980. Cole, W.C.; LeBlanc, A.D.; Jhingran, S.G. The origin of biexponential T 2 relaxation in muscle water. Mag. Reson. Med. 29:19 –24; 1993. Belton, P.S. Can nuclear magnetic resonance give useful information about the state of water in foodstuffs? Comments Agric. Food Chem. 2:179 –209; 1990. Grucker, D.; Mauss, Y.; Steibel, J.; Poulet, P.; Chambron, J. Effect of interpulse delay on NMR transverse relaxation rate of tissues. Magn. Reson. Imaging 4:441– 443; 1986.
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47. Zhong, J.; Gore, J.C.; Armitage, I.M. Quantitative studies of hydrodynamic effects and cross-relaxation in protein solutions and tissues with proton and deuteron longitudinal relaxation times. Mag. Reson. Med. 13:192–203; 1990. 48. Virta, A.; Komu, M.; Kormano, M.; Lundbom, N. Magnetisation transfer in protein solutions at 0.1T: Dependence
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on concentration, molecular weight, and structure. Acad. Radiol. 2:792–798; 1995. 49. Van Den Berg, C.; Van Dusschoten, D.; Van As, H.; Terlouw, A.; Schaafsma, T.J.; Osse, J.W.M. Visualising the water flow in a breathing carp using NMRI. Neth. J. Zool. 45:338 –346; 1995.
PII S0730-725X(98)00189-1
● Original Contribution
QUANTITATIVE MAGNETIC RESONANCE IMAGING OF FRESH AND FROZEN-THAWED TROUT KEVIN P. NOTT, STEPHEN D. EVANS,
AND
LAURANCE D. HALL
Herchel Smith Laboratory for Medicinal Chemistry, University of Cambridge School of Clinical Medicine, University Forvie Site, Robinson Way, Cambridge, CB2 2PZ, UK Magnetic resonance imaging (MRI) has been used to visualise the major organs and muscular-skeletal framework of fresh rainbow trout (Salmo gairdneri) in two dimensions, and to identify the spatial distribution of lipidand collagen-rich tissues. Quantitative MRI provides the MR parameters (T1, T2, M0, Tsat 1 , Msat/M0, and the Magnetisation Transfer (MT) rate) for the tissue water; variations in those parameters enable distinction to be made between a freshly killed trout and one which has been frozen–thawed. The effects of freezing method, repeat freeze–thawing, and storage time on the MR parameters are discussed. © 1999 Elsevier Science Inc. Keywords: Authentication; Trout; Frozen storage; Freezing.
INTRODUCTION There is ample precedent in the literature for the use of the Nuclear Magnetic Resonance (NMR) parameters of water to study bulk specimens of meat, and it is known that those parameters are sensitive to changes in meat structure such as those due to the development of rigor mortis.1–3 Recently MRI has been used to study changes in those parameters induced by freeze–thawing of red meat, and it has been suggested that such measurements may distinguish fresh from frozen–thawed meat.4 Authentication of fresh fish is of great importance to the consumer for two reasons; as there is a price differential between fresh and frozen produce, retailing of fish as ‘fresh’ when it has been previously frozen is fraudulent; also many fish and shellfish can be hazardous with regards to their microbiological safety if they have been stored carelessly. The main purpose of this project was to evaluate the effects of freeze–thawing on the MRI parameters of water in trout flesh. Specifically, we have measured the values of water concentration (M0%), the spin-lattice relaxation time (T 1 ), the spin-spin relaxation time (T 2 ), the magnetisation transfer parameters Msat / M0, T sat 1 and MT rate for the protons of water. In order to support those measurements we have evaluated dif-
ferent MRI protocols for visualising the anatomical structures of intact trout, and also the spatial distribution of lipid- and collagen-rich tissue. Rainbow trout belongs to the super-order Teleostei, the bony fish with vertebrae; various imaging contrasts have been used previously to show selective enhancement of different tissue types in cartilaginous fish, Elasmobranchii.5 Freezing is an important method for reducing chemical and biological spoilage in fish, but can result in irreversible changes in its quality compared to unfrozen ‘fresh’ fish. The loss of quality as judged by changes in texture, toughening and loss of water-holding capacity,6 is thought to be mainly due to two processes; protein denaturation/aggregation and lipid oxidation. When fish is frozen, the solutes present in the muscles become concentrated as water crystallises during freezing; this increases the ionic strength of the liquid which leads to breaking of electrostatic bonds in proteins and thereby to their denaturation and aggregation. It is believed that myosin and actin, the main contractile proteins, are largely responsible for the functional properties of the muscle and that on frozen-storage, myosin in particular, undergoes denaturation/aggregation reactions.6 –10 Although it is thought that these protein changes upon
RECEIVED 5/22/98; ACCEPTED 10/22/98. Address correspondence to Laurance D. Hall, Herchel Smith Laboratory for Medicinal Chemistry, University of Cam-
bridge School of Clinical Medicine, University Forvie Site, Robinson Way, Cambridge, CB2 2PZ, UK. E-mail: [email protected] 445
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Fig. 1. Series of multi-slice coronal MR images of the head of a rainbow trout, (TR/TE 1000 ms/20 ms, NEX 4), FOV 9 cm, slice 2 mm, 6 mm between slices, 256 3 256 (zero padded from 128) matrix. Anatomical features: (n) nasal cavities; (b) brain; (e) eyes; (o) operculum; (g) gills; (p) pectoral girdle; (c) oesophagus; (h) heart.
freezing are the main causes of quality loss in frozen fish,6 oxidation of lipids can lead to rancid flavours. In addition, lipid oxidation is thought to enhance the deterioration of proteins.11,12 Lipids have been shown to be prevalent in trout by the use of MRI weighted images.13 Changes associated with freeze-storing cod, as well as thermal and high pressure treatment, have been followed14 at 20 MHz (0.47T). It was found that the transverse relaxation time of the water protons showed multicomponent behaviour, with frozen-stored cod having a longer relaxation component that increased with the temperature and duration of frozen-storage. This component was attributed to the water exudate associated with protein denaturation, as well as that from physical damage and enzymatic reaction. A similar MR-relaxation study was undertaken on frozen-storage of post rigor minced cod,15 which also showed bi-exponential relaxation be-
haviour for the water protons. The MR parameters showed variation on frozen-storage between different quality minces, which became more marked in the cooked state. These changes were ascribed to the aggregation of myofibrillar proteins during frozen-storage as opposed to intra/extra-cellular compartmentalisation since the bi-nodal distribution persists into the cooked state. Since the precise history of the fish flesh is of paramount importance to any study of temporal changes,16 attention was focussed in the present study on freshwater rainbow trout which were purchased live from a local trout farm. Initially, a fully automated MRI protocol was used to study a small number of whole trout. However, when those results were analysed it was realised that a much larger number of samples needed to be studied to account for biological variations and so a multiple-sam-
Quantitative MRI of trout ● K.P. NOTT
ple, bulk-MRI approach was used. Given that interanimal variation was likely to be large, it was also decided to investigate the feasibility of a comparative method, which involved repeated freeze–thawing of the sample. The hypothesis is that the change in the MR parameters on re-freezing a sample which had been previously frozen would be smaller than those on freezing a fresh fish, since the major damage had already been inflicted. MATERIALS AND METHODS Intact Trout MRI hardware. All 1H MRI images were acquired using a Bruker BMT (Bruker Medzintechnik Biospec II) imaging console (Karlsruhe, Germany) connected to a 2.35 Tesla, 31 cm horizontal bore super-conducting magnet (Oxford Instruments, Oxford, UK). A 20 cm gradient set built ‘in-house’ with each axis powered by a Techron gradient amplifier (Model 7790, Crown International Inc., Elkart, IN, USA) provided gradient strengths up to 100 mT m21. An ‘in-house’ built, cylindrical, eight strut, bird-cage radio-frequency (RF) probe, internal diameter 9.4 cm, was used in the quadrature mode to transmit and receive the MR signal. Samples were kept at ambient temperature (20°C) by passing a constant stream of air through the probe. Sample preparation. The MRI measurements used 4 trout for each freezing regime, each set was caught by net in November (mean weight, 640 g; mean length, 35 cm) and sacrificed using a Schedule 1 method. Since significant changes occur during at least the first five hours after death,17 the samples were left to equilibrate post mortem in a refrigerator at 2°C. After MRI quantitation of the fresh material, the samples were subjected to one of two freezing regimes: 1) fast freezing in liquid nitrogen, followed by storage in a domestic freezer at 218°C for 4 weeks; 2) slow freezing, and subsequent storage for four weeks in a domestic freezer at 218°C before quantitation of the frozen–thawed samples. Data acquisition and processing. The quantitation of T 2 , T 1 , and MT parameters was achieved by varying the Recovery Delay (TR) the Echo Time (TE), the saturation transfer delay (SP) and the number of echoes in a multiecho Carr Purcell Meiboom Gill (CPMG) MRI sequence developed previously in this laboratory;18 this is based on a sequence devised by Crawley and Henkelman,19 which employs an additional slice-selection gradient to reduce unwanted echoes generated at the slice edge by RF imperfections. This fully automated protocol enables calculation of those parameters from a set of 256 3 64 pixel images (field of view 9 cm, slice thickness 5 mm, 2 transients). The number of phase encoding steps were
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minimised to reduce the acquisition time and increase the signal-to-noise. A set of T 2 -weighted images were obtained through acquisition of 8 echoes for each pixel (7500/20/0 [TR/ TE/SP]). The T 1 -weighted images were obtained by incrementing TR (400, 800, 1600, 3200, 7500/20/0) of a single spin echo sequence to produce images attenuated by T 1 saturation recovery relaxation.20,21 T sat 1 -weighted images were obtained by increasing the duration of the saturation pulse (SP), applied prior to the 90° pulse of a single spin echo sequence (7500/20/0, 100, 325, 750, 1500, 2000). Selective saturation of the macromolecular proton pool was achieved by applying a squared pulse of low amplitude (;10 mT, 10% of the RF power needed to achieve a 90° pulse) at 10 kHz off-resonance.22–24 The raw MRI data sets were transferred from the Bruker Aspect 3000 computer via an ASL data transfer board (designed by A.A. Wilkinson and Dr. N. Dillon) to a network of Unix stations where the subsequent data processing took place. The image-data were zero-padded in the phase direction to produce isometric pixels, baseline corrected, and a Gaussian filter was applied prior to Fourier transformation. The fitting of the data was performed on a curve fitting program (Cmrfit) written-inhouse by Dr. J.J. Attard, based on the Levenberg-Marquandt method.25 All data were visualised using image display software (Cmrview) written by Dr. N.J. Herrod. The MR values were obtained by placing a hand drawn region of interest (ROI) over the muscle in the image. The T 2 - and T 1 -weighted images were fitted assuming mono-exponential relaxation behaviour, to equations (1) and (2) respectively, TE
Mx,y 5 M0e 2 T2
S
(1)
D
TR
Mx,y 5 M0 1 2 e 2 T1
(2)
where Mx,y is the magnetisation at a particular pixel, M0 is the equilibrium magnetisation (proportional to the liquid proton density), T 2 is the transverse relaxation time and T 1 is the longitudinal relaxation time. M0 was quantified from back-projection of the T 2 decay curve and the liquid proton density (M0%) was calculated by comparison of its M0 value with that of a 2% (w/w) agar gel standard which was in place for each experiment. The T sat 1 -weighted images were fitted assuming mono-exponential relaxation behaviour to equation (3). SP
Mx,y 5 Msat 1 (M0 2 Msat)e 2Tsat 1
(3)
to produce the T sat 1 and Msat /M0 images: where M0 is the equilibrium magnetisation (SP 5 0); Mx,y is the magne-
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Fig. 2. Water and fat resolved (from 3 point Dixon TR/TE 6000 ms/20 ms, NEX 2), and T 1 -weighted (TR/TE 1000 ms/20 ms, NEX 4) sagittal MR images of the head of a rainbow trout using FOV 7 cm, slice 2 mm, 256 3 256 matrix (zero padded from 128).
tisation at the particular pixel; Msat is the minimum longitudinal magnetisation observed when saturation sat transfer reaches a steady-state (SP 5 5T sat 1 ) and T 1 is the longitudinal relaxation time measured during saturation transfer. Assuming that the protons of the macromolecular pool are saturated during the application of the irradiating pulse, the apparent MT rate (k) image can be calculated according to equation (4), from the images previ22,23 ously fitted to Msat , M0 and T sat 1 . k5
S
D
1 Msat sat 1 2 M0 T1
(4)
Optimisation of the saturation of the proton pool can only be achieved by time-consuming experiments involving varying the amplitude and offset of the saturation pulse.26 –28 Since full saturation is unlikely to be achieved in practice the value provided by this sequence can only be regarded as an estimate of the true magnetisation transfer rate. Nevertheless the value provides a reliable indication of the relative strength of the MT phenomenon.18,29 Multiple Sample Protocol In order to increase the number of samples studied without increasing the amount of time involved, a multiple-sample, bulk MRI protocol approach was used.30 This involved, extracting 3 tissue samples per trout with a modified cork borer and placing them into an array of wells in a multiple-sample tissue culture plate. Using the MRI protocols summarised above the number of samples that could be analysed simultaneously was only limited30 by the homogeneity of the radio-frequency (B1) and magnetic (B0) fields.31–33 MRI hardware. A 14.5 cm internal diameter gradient was used, with each axis powered by two Techron gradient amplifiers (Models 7570 and 7560, Crown Interna-
tional Inc., Elkart, IN, USA). This gradient set enabled34,35 the use of a short echo time (12 ms) which gave more points on the T 2 decay curve. Sample preparation. Eighteen fresh trout in May were gutted and divided into 5 steaks (thickness 1.5–2 cm) from which all the samples were taken for gravimetric and MR quantitative analysis. All eighteen trout were analysed as ‘fresh’ by MRI on the day the fish was killed and after a suitable equilibration period post mortem in a refrigerator at 2°C. The remaining steaks were frozen at 218°C in a domestic freezer and then subjected to one of three regimes: 1. Six trout were kept frozen for two days and then analysed after thawing. 2. Six trout were kept frozen for two weeks, analysed after thawing and analysed 2 days later after repeated freeze–thawing. 3. Six trout were kept in frozen for 4 weeks prior to further analysis on the thawed samples, and analysed as for (2) above. The trout steaks were thawed to room temperature between 2 aluminum sheets 4 h prior to being placed in the multi-well plate. At each stage they were analysed for moisture content using the protocol detailed by Kirk36 and the pH measured using a pH meter (Model 220, Corning Science Products, USA). Data acquisition. The same fully automated protocol allowed the acquisition of a set of images that enabled the calculation of the T 2 , T 1 and MT parameters for all the samples in each plate. Since the samples were not required for further treatment, the data acquisition protocol was lengthened to increase the accuracy of each parameter.30 A set of 16 T 2 -weighted images were obtained (6000/ 12/0 [TR/TE/SP]). The T 1 -weighted images were ob-
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each well of each of the fitted images, and those values were then used for calculation of each of the quantitation parameters. Bulk NMR Relaxometry Protocols For comparison, smaller scale bulk NMR T 2 relaxometry studies were carried out. Samples were taken from four fresh trout and again after the same trout were frozen-stored for four weeks at 218°C. Bulk CPMG experiments were carried out using echo times of 1 and 12 ms. The resultant decay curves were best fit to a bi-exponential decay. Because imperfections in the RF pulses can cause a large errors,38 the bulk measurements were carried out on a small sample using the large birdcage imaging probe.
Fig. 3. Sagittal images of the head of a rainbow trout under various imaging contrasts; Proton density (TR/TE/SP 7500/ 20/0, NEX 2), T 1 -weighted (TR/TE/SP 400/20/0, NEX 2), T 2 -weighted (TR/TE/SP 7500/60/0, NEX 2), MT-weighted (TR/TE/SP 7500/20/750, NEX 2) using FOV 10 cm, slice 1.5 mm, 256 3 256 matrix (zero padded from 128). Anatomical features: (c) oesophagus; (h) heart.
tained by incrementing TR (120, 375, 500, 750, 1000, 1250, 1500, 1750, 2000, 2500, 3000, 6000/12/0) of a single spin echo sequence to produce images attenuated by T 1 saturation recovery relaxation. T sat 1 -weighted images were obtained by increasing the duration of the saturation pulse (SP), applied prior to the 90° pulse of a single spin-echo sequence (6000/12/0, 50, 150, 300, 750, 1000, 1500). Data processing. All the 128 3 128 pixel images of each plate were measured using a field of view of 7 cm and a 4-mm slice thickness, and the phase direction data zero-padded from 64. This gave the optimal pixel resolution and signal-to-noise ratio of ca 120 required for the subsequent edge detection analysis, with the minimum image acquisition time required for the quantitation of the MR parameters. Estimation of the mean MR parameters for the contents for each well was achieved using fully automated edge detection30 of the first echo image to delineate the edges of each well, followed by location of the well centres using a Hough Transform algorithm for modified circles.37 The local maxima of Hough space identified the coordinates of the centres of the wells, over which a circular mask of a suitable radius was then placed; the mean value of the intensity of all the pixels enclosed within the boundary of that circle was then calculated for
Fig. 4. Sagittal images of the body of a rainbow trout under various imaging contrasts; Proton density (TR/TE/SP 7500/ 20/0, NEX 2), T 1 -weighted (TR/TE/SP 500/20/0, NEX 4), T 2 -weighted (TR/TE/SP 7500/60/0, NEX 2), MT-weighted (TR/TE/SP 7500/20/750, NEX 4), MT difference (between proton density image and MT-weighted image) using FOV 9 cm, slice 2 mm, 256 3 256 matrix (zero padded from 128). Anatomical features: (r) eggs; (l) liver; (s) swim bladder.
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Table 1. MR parameters for water in fresh and frozen–thawed (F/T) trout flesh Freezing Rate Slow Fresh F/T Change Fast Fresh F/T Change
T 1 (s)
T 2 (ms)
M0%
T sat (s) 1
Msat /M0
MT rate (s21)
1.37 6 0.03 1.32 6 0.05 23.6%
41.7 6 0.9 40.5 6 0.7 22.9%
84.6 6 0.4 83.6 6 0.7 21.2%
0.320 6 0.005 0.310 6 0.005 23.1%
0.317 6 0.015 0.257 6 0.010 218.9%
2.15 6 0.05 2.41 6 0.02 112.1%*
1.24 6 0.04 1.25 6 0.04 10.8%
43.3 6 1.0 44.5 6 0.3 12.8%
87.3 6 0.5 84.4 6 0.3 23.3%*
0.304 6 0.007 0.309 6 0.002 11.6%
0.228 6 0.009 0.231 6 0.004 11.3%
2.56 6 0.09 2.49 6 0.03 22.7%
* Significant at 1% level for a paired two-tailed t-test ( p , 0.01). Mean 6 standard error of four samples. Measurements were taken from the transverse section of eight ‘fresh’ rainbow trout caught in November; 4 samples were slow-frozen in a domestic freezer at 218°C, and four were fast-frozen in liquid nitrogen at 2196°C. The same sections were analysed after 4 weeks frozen-storage.
RESULTS AND DISCUSSION Anatomical Images Segmented anatomical images were obtained using T 1 -weighted multi-slice imaging for the head of a fresh rainbow trout in the coronal direction (Fig. 1). Those images revealed numerous anatomical features, including the gill structures, the eyes, nasal passage and vertebrae. They gave high contrast between muscle and fat tissue because the T 1 of fat is approximately half that of muscle. Comparison in Fig. 2 with the fat-resolved im-
Fig. 5. An example of a MT rate image on a transverse section of a ‘fresh’ rainbow trout and the corresponding slice after frozen-storage for 4 weeks. The histograms give the distribution of the pixel values for MT rate taken over the muscle.
age obtained by a three-point Dixon technique39,40 clearly showed how the T 1 -weighted image highlights the lipids. The fat-resolved image was obtained using three-point rather than the two-point41 Dixon method because the latter was found to be too sensitive to B0 field inhomogeneities. There are two main parts to the skull: the chondrocranium (Fig. 1E), which is a set of compartments around the nasal cavities (n), brain (b) and eyes (e); and the branchiocranium, which consists of the bones which support the gill arches, opercular flap, and the jaws or mandibular region. Most of the skull features were observed as high intensity regions. Other visible components of the skeleton includes bone, cartilage and the connective tissue envelope which joins the musculature to the skeleton. The actual distribution of all these tissues were apparent in images which were obtained under different contrasts. The structures of the respiratory gills can be seen in Figs. 1A–D. The gill arches can be clearly seen in D; there are four pairs of gills (g) on each side of the fish, lying in a cavity containing the capillary vessels just in front of the pectoral girdle (p), which are protected on each side by two flaps of skin supported by bones, the operculum (o). The beginning of the oesophagus (c) and the heart (h) can be seen further down the set of images. Figure 1F shows the skeletal framework leading to the brain (b); the brain and spinal cord are enclosed within the skull and vertebral column; the eyeball can be clearly visualised (Fig. 1E) with the pupil appearing as a low intensity region in the centre of the iris. Various other tissues were identified by comparison between a series of sagittal images of the head (Fig. 3) obtained under different image contrast regimes. The T 1 -weighted image highlighted the lipids with respect to
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Table 2. MR parameters for water in fresh and frozen–thawed (F/T) excised flesh of trout, following various storage regimes at 218°C
Fresh Fresh12dayF/T Ratio Fresh12dayF/T /Fresh 2 weeks F/T 2 weeks F/T12dayF/T Ratio 2 weeks F/T12dayF/T /2 weeks F/T 4 weeks F/T 4 weeks F/T12dayF/T Ratio 4 weeks F/T12dayF/T /4 weeks F/T
T 1 (s)
T 2 (ms)
M0%
T sat (s) 1
Msat /M0
MT rate (s21)
1.35 6 0.01 1.09 6 0.01 0.81 6 0.01 1.18 6 0.01 1.08 6 0.01
43.1 6 0.5 42.0 6 0.3 0.97 6 0.01 54.7 6 0.6 50.2 6 0.4
88.3 6 0.8 88.8 6 0.5 1.01 6 0.01 88.1 6 0.6 88.1 6 0.9
0.311 6 0.002 0.206 6 0.002 0.66 6 0.01 0.196 6 0.001 0.203 6 0.001
0.170 6 0.003 0.168 6 0.003 0.99 6 0.03 0.179 6 0.003 0.174 6 0.003
2.70 6 0.02 4.06 6 0.05 1.50 6 0.02 4.19 6 0.04 4.05 6 0.04
0.92 6 0.01 1.12 6 0.02 1.07 6 0.01 0.95 6 0.02
0.92 6 0.01 52.4 6 0.7 49.6 6 0.6 0.95 6 0.02
1.00 6 0.01 84.5 6 0.9 85.7 6 0.6 1.01 6 0.01
1.03 6 0.01 0.196 6 0.002 0.207 6 0.003 1.06 6 0.02
0.97 6 0.02 0.149 6 0.002 0.160 6 0.003 1.07 6 0.02
0.97 6 0.01 4.38 6 0.04 4.10 6 0.05 0.94 6 0.01
Mean 6 standard error (54 fresh, 18 frozen–thawed samples). Measurements were taken on 18 fresh trout caught in May (three samples from each). Equivalent measurements were taken after the trout steaks were subjected to one of three storage regimes: 1) freeze–thawing over two days, 2) 2 weeks frozen-storage and after repeat freeze–thawing over 2 days, 3) 4 weeks frozen-storage and after repeat freeze–thawing over 2 days.
and MT-weighted images) because the connective tissue between the myotomes is visualised. Tissue discrimination due to MT contrast was increased in an MT difference image (Fig. 4), obtained by subtracting two spatially identical images, one with and another without the application of the MT saturation pulse. Although the muscle shows a noticible MT-difference effect, the more intense regions were associated with the backbones and the belly, indicating that those were highly collageneous tissues. Below the swim bladder (s), which has zero intensity, the eggs (r), the liver (l) and, to the right, the spleen and stomach were visible.
the water containing tissues owing to their shorter T 1 values. However, the T 2 - and MT-contrasted images, in particular, showed the greatest distinction between different muscle tissues; one image shows at least three of the 4 heart chambers. The proton density map gave little tissue contrast because all the tissues contain approximately the same concentration of water. Figure 4 shows a sagittal image across the vertebral column of the fish, measured with the 4 types of image contrast. Although slightly masked by partial volume effects associated with limited spatial resolution, the bones in the vertebral column were clearly visible in all four scans because they had low signal intensity. The backbones were masked by a combination of fatty and collageneous tissue, which are indicated in the T 1 - and MT-weighted images respectively. The fish muscle is divided into a series of blocks, called myotomes, by myoseptal connective tissue (which is composed of inextensible collagen fibres). The myotomes form a complex array of segments in a characteristic W-shape along the fish; the patterns of those structures were clearly evident in the different images (particularly in the T 2 -
MRI Relaxometry of Whole Trout Table 1 shows the data obtained from measurements of intact fresh rainbow trout and after subsequent storage for four weeks following either slow freezing in a conventional freezer at 218°C, or fast-freezing in liquid nitrogen at 2196°C. A two-tailed, “paired sample t-test” was used to determine whether freezing had a significant effect on the MR parameters. Some of the MR parameters exhibited larger changes on slow freezing than for
Table 3. Significance at 1% for two-tailed t-test ( p , 0.01), for effects of freeze/thawing (F/T) on the MR parameters of trout flesh given in Table 2 t-test
T1
T2
Fresh v 2 weeks F/T Fresh v 4 weeks F/T 2 weeks F/T v 4 weeks F/T Fresh v Fresh12dayF/T 2 weeks F/T v 2 weeks F/T12dayF/T 4 weeks F/T v 4 weeks F/T12dayF/T
p , 0.01 p , 0.01 NS p , 0.01 p , 0.01 NS
p , 0.01 p , 0.01 NS NS p , 0.01 NS
NS 5 Not significant.
M0% NS NS NS NS NS NS
T sat 1
Msat /M0
MT rate
p , 0.01 p , 0.01 NS p , 0.01 NS NS
NS p , 0.01 p , 0.01 NS NS NS
p , 0.01 p , 0.01 NS p , 0.01 NS p , 0.01
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Table 4. Effect of freeze–thawing (F/T) at 218° on the mean percentage water content (measured by oven drying) and pH of trout steaks
Fresh Fresh12dayF/T Fresh 2 weeks F/T 2 weeks F/T12dayF/T Fresh 4 weeks F/T 4 weeks F/T12dayF/T
pH
% water
6.32 6 0.06 6.23 6 0.03 6.28 6 0.02 6.26 6 0.03 6.29 6 0.01 6.34 6 0.02 6.37 6 0.03 6.38 6 0.03
77.10 6 0.21 77.25 6 0.35 77.86 6 0.31 77.67 6 0.46 77.51 6 0.42 77.71 6 0.41 77.81 6 0.36 77.35 6 0.31
Mean 6 standard error for six trout. The samples were taken at each stage in the experiment outlined on Table 2.
fast freezing. The induction of larger changes by slow freezing can be attributed to the different physical damage to the muscles caused by slower freezing rates.42 The larger ice crystals produced more cellular disruption by dehydration of the intra-cellular space which led to more protein denaturation/aggregation. Fast freezing is thought to rapidly produce smaller ice crystals, therefore, there is no major redistribution of water. However, on frozen storage at 218°C the ice crystals may have had a tendency to grow to larger ones, thereby leading to greater deterioration of the flesh. The magnetisation transfer rate was particularly sensitive to the changes associated with slow freezing and was significantly different at the 1% level ( p , 0.01) for four samples. An example of one set of images, before and after freeze–thawing, is illustrated in Fig. 5 together with the histogram plots for the pixel values of MT rate across the fish muscle. There was a large variation between the MR parameters of the two groups of fresh fish, although not significantly different at the 1% level ( p , 0.01). This variation demonstrates that absolute MR values can only be of use if the inter-animal variations are small, compared to the difference between the fresh and frozen– thawed product.
Effect of Storage and Repeat Freeze–Thawing The data obtained from excised samples of trout flesh using the multiple sample protocol are shown in Table 2. The results of statistical analyses on those data are given in Table 3. Table 4 shows the equivalent data for moisture content and pH at each stage of the experiment; there was a general decrease in the moisture content, though not statistically significant at the 1% level ( p , 0.01). There are 2 main differences between the data in Table 1 and Table 2. Firstly, the data for these 2 sets of fresh fish may indicate seasonal variations;43 the multiple sample experiments were conducted 6 months after the initial study. Secondly, the differences on freeze–thawing trout steaks was much greater than that observed for freeze–thawing the whole trout; this may reflect some additional tissue disruption associated with the preparation of the steaks. It was found that the magnetisation transfer parameand MT rate were particularly sensitive to ters T sat 1 freeze–thawing; thus there was a significant difference at the 1% level ( p , 0.01) between the fresh sample and that after freeze–thaw treatment. Since there was no significant difference between storage over 2 and 4 weeks, it is clear that most of the damage must occur during the initial freezing process; furthermore, a repeated freeze–thaw cycle on a previously frozen–thawed sample shows less change than for the first freeze–thaw cycle of a fresh product (Table 2). The 2 day freeze–thaw cycle on the fresh fish had no significant effect on the T 2 parameter; however, a statistically significant increase was observed after frozen storage. The observed increase in the mono-exponential transverse relaxation time could be a result of a redistribution of the water within the muscle, which increases the population of the longer relaxation component.14,44 This rationale is based on the ‘bulk’ NMR T 2 data given in Table 5. M0 and T 2 of the observed relaxation components can be dependent on the echo time (TE).45,46 No significant change was observed in the mono-exponential T 2 due to freeze–thawing. However, a significant increase was observed in the longer T 2 component of a bi-exponential decay analysis of data obtained with an
Table 5. ‘Bulk’ mono-exponential and bi-exponential T 2 parameters for water in fresh and frozen–thawed (F/T) trout flesh Sample Fresh F/T
TE (ms)
Bulk T 2 (ms)
T A2 (ms)
PA (%)
T B2 (ms)
PB (%)
1 12 1 12
52.2 6 0.7 50.0 6 0.7 57.4 6 0.4 55.4 6 0.6
45.3 6 0.7 43.8 6 1.1 46.4 6 0.9 43.9 6 0.4
82.4 6 3.2 87.1 6 4.1 81.0 6 2.0 84.9 6 0.9
98.3 6 6.9 121.2 6 20.6 130.0 6 8.5 128.6 6 11.3
17.6 6 3.2 12.9 6 4.1 19.0 6 2.0 15.1 6 0.9
Mean 6 standard error for four samples. The trout were frozen for a period of 4 weeks in a domestic freezer at 218°C. PA and PB are the populations of the bi-exponential components T A2 and T B2 respectively.
Quantitative MRI of trout ● K.P. NOTT
echo time of 1 ms; this was not observed when an echo time of 12 ms was used. The mono-exponential T 2 ’s from bulk and imaging were similar. The liquid proton density, which was extrapolated from the mono-exponential fit of a T 2 decay curve, showed a general, though not statistically significant, decrease with frozen storage; this was most likely due to water loss through ‘drip’ on thawing the muscle. Water exudes not from the breakage of the sarcolemma (cell wall) as was first thought, but through changes in the protein properties on frozen-storage.42 The statistically significant decrease in T 1 could be a result of a reduced water content through drip loss; however, the large size of the changes could indicate that the relaxation rate is enhanced47 by the reduction in protein mobility through protein aggregation. The changes in the MT rate could be the result of a combination of factors.48 However, a significant contribution could stem from the freeze denaturation of proteins, which subsequently leads to protein aggregation involving increased rigidity by intermolecular cross-links. Magnetisation transfer rates are known to increase with the increasing weight of protein,47 owing to the enhanced cross-relaxation mechanism. Given that Tsat 1 and MT rate appeared to be more sensitive to the effects of freeze–thawing, future studies should concentrate on the magnetisation transfer phenomenon in relation to the mechanism of the protein deterioration during freeze–thawing. CONCLUSIONS The original aims of this work were 2-fold; first, to demonstrate the type of anatomical resolution that MRI can achieve between individual organs of intact trout. Second, to explore the potential use of MRI as a basis for the authentication of fresh trout from that which had been frozen–thawed. It is clear that MRI enabled the anatomical details of rainbow trout to be visualised by highlighting various soft tissues using a combination of protocols that gave different contrasts. This has implications regarding fish quality since the distribution of fat and the content of connective tissue are also important factors affecting quality. In the future it will be feasible to achieve a time course study on living fish.49 Quantitative MRI provided parameters that were sensitive to the effects of freeze–thawing and to the method of freezing and duration of frozen-storage. Thus, there were no significant changes in the MR parameters on freezing on intact trout in liquid nitrogen, whereas there was a significant change in the MT rate as a result of slow freezing. For the trout steaks T 1 , T sat 1 and MT rate exhibited the most prominent effects. For fish which had been previously frozen, the change in the MR parameters
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after repeat freeze–thawing were smaller than those observed for the initial 2 day freeze–thaw of fresh fish, indicating a method which could be used to allow for the variability observed for fresh trout. The same methodology could be applied to sea-water fish, crustacea and molluscs, which are prone to loss of quality through mistreatment but which are less readily available in the ‘fresh’ state. Acknowledgments—The authors thank the UK Ministry of Agriculture Fisheries and Food (MAFF) and the Herchel Smith Endowment for collaborative funding of this work; Miss A. Kshirsagar for providing the edge detection software; Dr. J.J. Tessier for the MRI quantitation protocols; Dr. T.A. Carpenter, Mr. C. Bunch, Mr. S. Smith and Mr. C. Harbird for supplying and maintaining the MRI facilities.
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