Comparative 1H NMR studies of saturation transfer in copolymer gels and mouse lenses

Research Article

584

Received: 30 July 2008, Revised: 26 November 2009, Accepted: 2 December 2009, Published online in Wiley InterScience: 15 March 2010

(www.interscience.wiley.com) DOI:10.1002/nbm.1499

Comparative 1H NMR studies of saturationtransfer in copolymer gels and mouse lensesKoji Nakamuraa,e, Masaru Sogamia,c, Seiichi Eraa*, Shigeru Matsushimab,d

and Yasutomi Kinosadab

Saturation transfer in cross-linked copolymer gels an

NMR Biom

d excised intact and perforating trauma-induced cataract mouselenses (4- or 8-week-old) were studied using intermolecular cross-relaxation rates (1/TIS(H2O); 1/TIS), monitored withf2-irradiation at S8.79, S4.00, and 7.13 ppm (gH2/2p� 69Hz). [1] The 1/TIS(7.13 ppm) vs dry weight [W (%)] profilesfor hydrophilic copolymer gels were far steeper than those for hydrophobic copolymer gels, indicating theparticipation of an amount of bound water and a number of copolymer hydroxyl groups in the saturation transferprocess. In contrast, the 1/TIS(S8.79 ppm) vs W (%) profiles exhibited little difference between the hydrophilic andhydrophobic copolymer gels, indicating the major participation of molecular rigidity, i.e. W (%) in the saturationtransfer process. [2] The 1/TIS(7.13 ppm) values for cataractous mouse lenses were larger than those for intact lenses,indicating the formation of large, immobile lens protein associates or aggregates containing a sufficient amount ofbound water for the saturation transfer. [3] The 1/TIS(7.13 ppm) vs W (%) profiles for the hydrophilic copolymer gelsexhibited similar characteristics to the intact and cataractous mouse lenses with regard to the saturation transferprocess. Copyright � 2010 John Wiley & Sons, Ltd.

Keywords: hydrophilic copolymer gels; hydrophobic copolymer gels; intermolecular cross-relaxation rate; intact mouse lens;cataractous mouse lens; spin-lattice relaxation rate

* Correspondence to: S. Era, Department of Physiology and Biophysics, GifuUniversity Graduate School of Medicine, Gifu 501–1194, Japan.E-mail: [email protected]

a K. Nakamura, M. Sogami, S. Era

Department of Physiology and Biophysics, Gifu University Graduate School of

Medicine, Gifu, Japan

b S. Matsushima, Y. Kinosada

Department of Biomedical Informatics, Gifu University Graduate School of

Medicine, Gifu, Japan

c M. Sogami

Department of Physiology, School of Health Sciences, Fujita Health University,

Toyoake, Japan

d S. Matsushima

Department of Diagnostic Radiology, Aichi Cancer Center, Nagoya, Japan

e K. Nakamura

Department of Medical Technology, Gifu University of Medical Technology,

Seki, Japan

�

INTRODUCTION

Intermolecular cross-relaxation times (TIS) (1–4) from irradiatedprotein protons to observed water protons were used toelucidate protein-water and protein-protein interactions (1–9)and have been applied to excised tissues (10–14), living tissues(15–18), and cross-linked copolymer gels (9,12,19–22). Thephysical states of water in excised intact mouse lenses (2-, 4-,or 8-week-old) and hydrophobic copolymer gels (poly(N-VP-stat-MMA/BzMA)s; see Table 1) have been studied using thespin-lattice relaxation rate (1/T1) and the intermolecular cross-relaxation rates (1/TIS) from irradiated protein or polymer protonsto observed water protons (12,13). In 1/TIS vs dry weight (W (%))plots for hydrophobic copolymer gels, the 1/TIS valueswere approximately zero at a W of 21� 27% and increasedsteeply above �50% (12). Those for excised intact mouse lenses(2-, 4-, or 8-week-old) showed an approximately straight line withthe intercept at a W of 23% and with a slope that was almostequal to those of the plots for hydrophobic copolymer gels abovea W of �50% (12), indicating a significant change in the physicalstate of water and/or protein-water interactions above a W of23% (12).In the present study, we investigated the physical states of

water in excised intact and perforating trauma-induced cataractmouse lenses (intact and cataractous mouse lenses) as well as inhydrophilic and hydrophobic copolymer gels, using T1 and TIS.

Abbreviations used: BSA, bovine serum albumin; BSA gel, partially hydro-

lyzed BSA (BSA�) gel; CPMG method, Carr-Purcell-Meiboom-Gill method; CR,

cross-relaxation rate; INV, inversion recovery; MTCI, magnetization transfer

contrast imaging; SAT, saturation transfer; T1, spin-lattice relaxation time;

T1,absc, spin-lattice relaxation time in the absence of exchange between two

pools; T2, spin-spin relaxation time; TIS, cross-relaxation time; HEMA,

2-hydroxyethyl methacrylate; BzMA, benzyl methacrylate; GMA, glycidyl

methacrylate; MMA, methyl methacrylate; N-VP, N-vinyl-2-pyrrolidinone.

MATERIALS AND METHODS

Animal care and treatment were performed according to theinstitutional guidelines for the treatment of experimental

ed. 2010; 23: 584–591 Copyright � 2010

animals. Four- and 8-week-old ddY mice were used in thepresent experiments and were fed standard laboratory food andtap water ad libitum. The mice were sacrificed under 35mg/kgsodium pentobarbital anesthesia. The eye ball was removedimmediately after the animals had been sacrificed. The lens was

John Wiley & Sons, Ltd.

Table 1. Water content (H2O (%)) and percentage weight of monomers of synthetic cross-linked copolymer gels

H2O (%) HEMA GMA N-VP MMA BzMA

Poly(HEMA-stat-GMA)s (gels-1)40.8 10050.6 83.4 16.659.2 65.2 34.867.2 45.4 54.674.2 23.8 76.2

Poly(HEMA-stat-N-VP)s (gels-2)48.7 80.0 20.062.7 60.0 40.076.0 40.0 60.083.1 20.0 80.0

Poly(N-VP-stat-GMA/MMA)s (gels-3)22.6 21.1 26.3 52.745.0 27.8 34.7 37.557.5 32.4 40.6 27.069.7 37.2 46.5 16.480.1 42.0 52.5 5.5

Poly(N-VP-stat-MMA/BzMA)s (gels-4)18.4 33.9 58.1 8.028.3 42.9 49.1 8.040.0 51.9 40.1 8.053.3 60.9 31.1 8.064.7 69.9 22.1 8.073.2 78.9 13.1 8.079.2 87.9 4.1 8.0

BzMA, benzyl methacrylate; HEMA, 2-hydroxyethyl methacrylate; GMA, glycidyl methacrylate; MMA, methyl methacrylate; N-VP,N-vinyl-2-pyrrolidone.Gelation was conducted by adding small amounts of allyl methacrylate (0.3%).

INTERMOLECULAR CROSS-RELAXATION IN COPOLYMER GELS AND MOUSE LENSES

5

carefully excised in Lock’s solution using a posterior approach at�258C and was subjected to 1H NMR measurements within 2 h(12,13). Approximately 10� 15 excised intact lenses werecarefully inserted into a glass capillary of 2.3mm inner diameterwith a funnel-shaped open end. The open end of the glasscapillary was sealed with inert plastic paste (polyvinyl chloride;TERUMO Corp., Tokyo, Japan) and then the glass capillary wasfurther inserted into a 5mm w coaxial NMR tube, containing asmall amount of dimethylsulfoxide-d6 (CEA, Cedex, France) forfield locking (9,12–14,19). After measuring the T1 and TIS valuesfor intact lenses, they were perforated using a set of fine stainlesssteel needles, and then the 1H NMR measurements wereimmediately repeated on these lenses. After 1H NMR measure-ments, the dry weights of the lenses were analyzed as describedpreviously (6,10–13).It is worth noting that the 1H NMR experiments on lenses were

carried out at 258C because of the following reasons: The effect oftemperature on the transparency of lens cytoplasm was reportedin excised rat (23) and calf lenses (24,25). A sharp transition fromthe transparent single phase (above 238C) to the opaquetwo-phase state occurred at approximately 178C in the calf lenscytoplasm (2- to 3-week-old) (24,25). Similar results were alsoreported in young rat lenses (23). Moreover, it is demonstratedthat the opacity associated with cold cataract develops as a resultof changes in the physicochemical state of the lens proteins (23).

NMR Biomed. 2010; 23: 584–591 Copyright � 2010 John Wiley

Although we did not perform the same sort of experiments,1H NMR experiments were carried out at 258C to suppress thebiochemical changes of the lens proteins, that occur in excisedlenses at more normothermic temperatures (12,13).Synthetic cross-linked copolymer gels were supplied by

Menicon Co., Ltd, Nagoya, Japan (see Table 1 in detailedcomposition of each gel). The water contents of each copolymergel, equilibrated with distilled water at 208C, were adjusted to40.8� 74.2, 48.7� 83.1, 22.6� 80.1, and 18.4� 79.2%, respect-ively, by keeping the allyl methacrylate (a cross-linking chemical)content at 0.30% and changing monomer composition for eachcopolymer gel. For the sake of simplicity, the followingabbreviations are used except for in the Table and Figurecaptions: poly(HEMA-stat-GMA)s, gels-1; poly(HEMA-stat-N-VP)s,gels-2; poly(N-VP-stat-GMA/MMA)s, gels-3; poly(N-VP-stat-MMA/BzMA)s, gels-4. Due to their monomer composition, gels-1 andgels-2 show hydrophilic features; whereas, gels-3 and gels-4 showhydrophobic features.Each rod-shaped copolymer gel (1.05� 25.0mm) was inserted

into a glass capillary of 1.1mm inner diameter (9,12,13,19). Theopen end of the capillary was sealed with inert plastic paste, andthen the glass capillary was inserted into a 5mm w co-axial NMRtube, as described previously (9,12,13,19).

1H NMR experiments were carried out at 25� 0.058C, using aBruker AM 500 spectrometer (Bruker Analytische Messtechnik

& Sons, Ltd. www.interscience.wiley.com/journal/nbm

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GMBH, Karsruhe, Germany), operating at 500MHz with an inverse5mm w probe. Chemical shifts weremeasured in parts per million(ppm) downfield from the external reference, 3-trimethylsilylpropionate-d4 (CEA, Cedex, France). T1 values were measured bythe inversion recovery method. The intermolecular cross-relaxation times (TIS) from irradiated polymer or protein protonsto observed water protons were measured by Akasaka’s method(2–4), as described previously (2–14,19). The f2-power strength atthe sample position, calibrated using BSA�gel (5–14,19), waschosen to be 69Hz in gH2/2p units.After f2-irradiation of the S-spin system for t (s) in the saturation

transfer (SAT) experiments, the observed time dependence of theI-spin system was given by the following equation(2, 3):

I ¼ I1 þ ðI0 � I1Þ expð�t=T�1 Þ (1)

where I0 and I1 are the longitudinal magnetization before andafter long-time f2-irradiation on the S-spin system, respectively,and 1/T1

�¼ 1/T1þ 1/TIS. In the steady state, (I1/I0) and 1/TIS aregiven by eqns (2) and (3), respectively, which have beenexperimentally confirmed by Akasaka (2,3) and Sogami et al. (19):

I1I0 ¼ T�1=T1 (2)

½ðI0=I1Þ � 1�=T1 ¼ 1=TIS (3)

After long-time f2-irradiation on the S-spin system, thelongitudinal magnetization of the observed I-spin system reachesan equilibrium value (I1). The results of the inversion recovery(INV) experiments, which were performed on the I-spin system inthe presence of f2-irradiation, were given by the followingequation (2,3):

I ¼ I1½1� 2 expð�t=T�1 Þ� (4)

T1 and T�1 in eqns (1) and (4) were calculated using Bruker’snon-linear curve fitting method. In the text, the followingabbreviations are used: [TIS]SAT and [TIS]INV for TIS values obtainedby the SAT and INV methods, respectively.It is worth noting the following evidence. Balaban’s group

(15–17) estimated spin-lattice relaxation time in the absence ofexchange between the two pools using a modification of eqn (2);i.e. T1,absc¼ [T�1 ]INV/(I1/I0)SAT, where the subscripts indicate theINV and SAT methods, respectively, with off-resonancef2-irradiation, and reported that the T1,absc values for tissueswere longer than the corresponding conventional T1 values.Similar results have been reported by several groups. T1,abscvalues, estimated using the procedure given abovewill be written[T1,absc]INV in the coming section. T1,absc values, obtained usingthe original eqn (2) of Akasaka (2–4), i.e. [T1,absc]SAT¼ [T�1 ]SAT/(I1/I0)SAT, with off-resonance f2-irradiation are also given in thecoming section to explain the physical characteristics of the[T1,absc]INV values given by Balaban’s group (15–17).

RESULTS

Copolymer gels

T1,absc: The [T1,absc]INV and [T1,absc]SAT values for hydrophilic andhydrophobic copolymer gels (Table 1) were monitored withf2-irradiation at �8.79, �4.00, 7.13 ppm and gH2/2p� 69Hz. Asshown in Figure 1a, the obtained [T1,absc]INV(�4.00 ppm) valueswere equal to the corresponding conventional T1 values withinexperimental accuracy. The slope of the regression line was[T1,absc]INV¼ 0.97T1, which passes through the origin, or 1.032T1 –

www.interscience.wiley.com/journal/nbm Copyright � 201

0.082. Therefore, a diagonal line is shown in Figure 1a. In thecoming section, [TIS]INV or [TIS]SAT values obtained usingconventional T1 values are given in the text, as previouslyreported (9,12–14,19).1/T1: As shown in Figure 1b, the 1/T1 vs W (%) profiles for

hydrophilic copolymer gels, gels-1 (empty squares) and gels-2(empty triangles), were a little steeper than those for hydrophobiccopolymer gels, gels-3 (empty circles) and gels-4 (empty invertedtriangles) (refer the Materials section and Matsushima et al. (20)on the hydrophilicity and hydrophobicity of each copolymer gel).The 1/T1 vsW (%) profile for gels-4 was similar to that obtained bya 360MHz 1H NMR spectrometer (12). The 1/T1 vs X/(1 - X) profiles,where X is the weight fraction of the polymer, are shown inFigure 1c. Similar results were reported previously for proteinsolution and gel (5).1/TIS: [TIS]INV and [TIS]SAT values were monitored with

f2-irradiation at �8.79, �4.00, and 7.13 ppm (�69Hz).Figure 2a shows [TIS]SAT vs [TIS]INV at �8.79 ppm (filled symbols)and �4.00 ppm (empty symbols). The resulting [TIS]INV valueswere nearly equal to the corresponding [TIS]SAT values withinexperimental accuracy. The regression line was equal to [TIS]SAT¼1.004[TIS]INV, which passes through the origin (Fig. 2a), or0.999[TIS]INVþ 0.172 (data not shown).[1/TIS]INV vs W (%) profiles obtained with f2-irradiation at 7.13,

�4.00, and �8.79 ppm (�69Hz) are shown in Figures 3a, 3band 3c, respectively. As shown in Figures 3a and 3b, the [1/TIS]INVvalues for hydrophobic gels (gels-4 (empty inverted triangles))were small, i.e. approximately zero at a W of 20.8 or 26.8% andincreased gradually from 35.3% and then steeply above�50%, asreported previously (12). However, the profiles for the hydrophilicgels (gels-1 (empty squares) and gels-2 (empty triangles)) weresteeper than those for the hydrophobic gels (gels-3 (emptycircles) and gels-4 (empty inverted triangles)). In contrast, no suchdifferences between hydrophilic and hydrophobic gels wereobserved in the profiles obtained at �8.79 ppm (Fig. 3c). Thisresult was similar to the reported [1/TIS]SAT vs W (%) profiles,monitored with f2-irradiation, below�10 and above 14 ppm (19).

Mouse lenses

T1,absc: [T1,absc]INV and [T1,absc]SAT value for intact and perforatingtrauma-induced cataract mouse lenses (8-week-old) weremeasured with f2-irradiation at �8.79, �4.00, and 7.13 ppm(�69Hz). As shown in Figure 1a, the [T1,absc]INV values forcataractous lenses (filled triangles) were nearly equal to thecorresponding conventional T1 values within experimentalaccuracy, but were a little larger for the intact lenses (emptyrhombuses). However, the [T1,absc]SAT values for intact lenses werenearly equal to the corresponding conventional T1 values withinexperimental accuracy (data not shown), as reported previously(12,13,19). Therefore, the [TIS]INV and [TIS]SAT values for mouselenses, obtained using the conventional T1 values, are giventhroughout this paper, as reported previously (9,12–13,19). The[T1,absc]INV values for 4-week-old mouse lenses are not shown inFigure 1a, because we did not have (I1/I0)SAT data.1/T1: 1/T1 vs W (%) profiles for intact (empty rhombuses) and

cataractous (filled triangles) lenses (4- and 8-week-old) are shownin Figure 1b. There was a significant difference in the 1/T1 valuesbetween paired intact and cataractous lenses (n¼ 14, 0.53� 0.06vs 0.57� 0.06 s�1, p< 0.005, paired Student’s t-test; not signifi-cant, unpaired Student’s t-test), as reported previously (13). The 1/T1 vs X/(1 - X) plots are shown in Figure 1c.

0 John Wiley & Sons, Ltd. NMR Biomed. 2010; 23: 584–591



1/TIS: [TIS]SAT vs [TIS]INV plots for intact (rhombuses) andcataractous (triangles) mouse lenses (8-week-old), obtained withf2-irradiation at �8.79 (filled symbols) and �4.00 ppm (emptysymbols) and gH2/2p� 69Hz, are shown in Figure 2b. The [TIS]INVvalues for cataractous lenses were nearly equal to the corres-ponding [TIS]SAT values (regression line: [TIS]SAT¼ 0.98[TIS]INV).However, those for intact lenses, especially those obtained at�8.79 ppm, were significantly longer than the corresponding[TIS]SAT values, as shown in Figure 2b (filled rhombuses).As shown in Figure 3a, there was a significant difference in the

[1/TIS]INV values obtained at 7.13 ppm (�69Hz) between pairedintact (empty rhombuses) and cataractous (filled triangles) lenses(n¼ 14, 0.61� 0.33 vs 1.38� 0.33 s�1, p< 0.001, paired Student’st-test). Those obtained at �4.00 ppm (Fig. 3b) exhibited asignificant difference between paired intact and cataractouslenses (n¼ 14, 0.20� 0.10 vs 0.46� 0.18 s�1, p< 0.001, pairedStudent’s t-test). Those obtained at �8.79 ppm (Fig. 3c) alsoexhibited a significant difference between paired intact andcataractous lenses (8-week-old) (n¼ 5, 0.20� 0.05 vs0.30� 0.04 s�1, p< 0.001, Student’s t-test).

DISCUSSION

Copolymer gels

1/T1: As shown in Figure 1a ([T1,absc]INV(�4.00 ppm)), the[T1,absc]INV values for copolymer gels were nearly equal to thecorresponding conventional T1 values within experimentalaccuracy. Those obtained at �8.79 and 7.13 ppm were alsosimilar to those shown in Figure 1a. As written in the Methodssection, Balaban’s group (15–17) estimated [T1,absc]INV using thefollowing relationship [T1

�]INV/(I1/I0)SAT [modified eqn (2)] withoff-resonance f2-irradiation and reported that the [T1,absc]INVvalues for tissues are longer than the corresponding conventionalT1 values. However, in the present study, the [T1,absc]INV and[T1,absc]SAT values were nearly equal to the correspondingconventional T1 values (Fig. 1a). One possible reason for thefinding by Balaban’s group (15–17), i.e. [T1,absc]INV� T1 could beaccounted for by [T�1 ]INV� [T�1 ]SAT, i.e. [TIS]INV� [TIS]SAT, asdiscussed in the section on mouse lenses. Indeed, the [TIS]SATvalues obtained with f2-irradiation at 7.13, �4.00 (emptysymbols), and �8.79 ppm (filled symbols) were nearly equal tothe corresponding [TIS]INV values, as shown in Figure 2a.

3———————————————————Figure 1. (a) Spin-lattice relaxation times in the absence of exchangebetween the two pools ([T1,absc(H2O)]INV; i.e. [T�1(H2O)]INV/(I1/I0)SAT),

obtained with f2-irradiation at �4.00 ppm and gH2/2p � 69Hz, as a

function of the corresponding conventional T1(H2O) values for poly(HE-

MA-stat-GMA)s, poly(HEMA-stat-N-VP)s, poly(N-VP-stat-GMA/MMA)s,poly(N-VP-stat-MMA/BzMA)s and excised intact and cataractous mouse

lenses (8-week-old) at 258C. See the text on [T1�(H2O)]INV and (I1/I0)SAT.

(b) 1/T1 values as a function of dry weight [W (%)] for poly(HE-MA-stat-GMA)s, poly(HEMA-stat-N-VP)s, poly- (N-VP- stat-GMA/MMA)s,

poly(N-VP-stat-MMA/BzMA)s and excised intact and cataractous mouse

lenses (4- and 8-week-old) at 258C. All of the excised intact and perforat-

ing trauma-induced cataract mouse lenses were paired samples.(c) 1/T1 values as a function of X/(1 - X), where X is the weight fraction

for poly(HEMA-stat-GMA)s, poly(HEMA-stat-N-VP)s, poly(N-VP-stat-GMA/

MMA)s, poly(N-VP-stat-MMA/BzMA)s and excised intact and cataractous

mouse lenses (4- and 8-week-old) at 258C. All of the excised intactand perforating trauma-induced cataract mouse lenses were paired

samples.


587

Figure 2. (a) [TIS]SAT values, obtained by the SATmethod with f2-irradiationat �8.79 (filled symbols) or �4.00 ppm (empty symbols) and gH2/2p �69Hz, as a function of the corresponding [TIS]INV values, obtained by the

INV method, for poly(HEMA-stat-GMA)s, poly(HEMA-stat-N-VP)s, poly(N-

VP-stat-GMA/MMA)s, poly(N-VP-stat-MMA/BzMA)s at 258C. (b) [TIS]SATvalues, obtained by the SAT method with f2-irradiation at �8.79 (filled

symbols) or �4.00 ppm (empty symbols) and gH2/2p � 69Hz, as a

function of the corresponding [TIS]INV values, obtained by the INVmethod, for excised intact and cataractous mouse lenses (8-week-old)

at 258C.

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The 1/T1 vsW (%) plots were re-analyzed using 1/T1 vs X/(1 – X)plots, as shown in Figure 1c. It has been shown thatcross-relaxation between water protons and macromolecularprotons provides the most effective relaxation pathway for thewater molecules in the immediate vicinity of the macromolecularsurface (26–29). If the two-phase model of water molecules (freeand bound) exchanging rapidly is assumed, the observed 1/T1parameters can be successfully expressed as follows: 1/T1 vs X/(1– X) plots, i.e. 1/T1¼C[1/T1,b - 1/T1,f ]X/(1 – X)þ 1/T1,f, where X isthe weight fraction of macromolecules, T1,f and T1,b are thespin-lattice relaxation times for free and bound water, respect-


ively, and C is the amount of bound water (g/g macromolecule)(5,13,27–29). It can be seen from Figure 1c that plots forcopolymer gels exhibited increases of slope in the followingorder: gels-4< gels-3< gels-2� gels-1. Assuming the 1/T1,bvalues for copolymer gels to be similar, then the increases inbound water seem to be in agreement with the above writtenorder, indicating that hydrophobic copolymer gels are less hydratedthan hydrophilic copolymer gels. The obtained results weresimilar to the reported plots for frog muscle (27), biologicalmembranes (28,29), bovine mercaptalbumin (BMA) solutions,and BSA�gels (5).It should be noted that Pope’s group (30,31) found a similar

relationship for 1/T1 and W(%) in commercially available contactlens hydrogels, such as IGEL (N-VP/MMA) (W of 36, 50 and 62%;Capricornia Contact Lens, Brisbane, Australia), Benz (HEMA)(65%), and Benz G (HEMA/GMA) (26, 44, 56, and 65%; BenzResearch and Development Corp., FL, USA).1/TIS: While conducting the research on [1/TIS]SAT or [1/TIS]INV

values, obtained with f2-irradiation at 7.13, �4.00 or �8.79 ppm,we have studied action spectra ([1 – (I1/I0)] vs f2(ppm))(4,8,9,13,14,19) and intermolecular cross-relaxation rate (CR)spectra (CR spectra; [1/TIS]SAT vs f2(ppm)) (8,9,13,14,19) for BSA(BMA) solution, BSA�gel (8,9), intact or cataractous mouse lenses(13), packed human red blood cells (RBCs) with normal orunstable hemoglobin (14), and hydrophilic or hydrophobiccopolymer gels (19) (gels in Table 1), obtained with f2-irradiationfor 5 x T1 from �100 to 100 ppm except chemical shift of waterprotons� 1.5 ppm at gH2/2p of 69� 250Hz at 258C. We alsoevaluated CR ratio spectra (13,14,19) to estimate the effects ofbound water, number of polymer hydroxyl group and associationof macromolecules [see Fig. 1 in (13), Fig. 5 in (14) and Fig. 4 in(19)]. It is worth noting that z-spectrum, i.e. water proton spectralintensity ratio offset frequencydispersion curve (32) is amirror profileof action spectrum, i.e. [1 – (I1/I0)] vs f2(ppm) [refer Table 1 in (19)].As reported previously, the [1/TIS]SAT vs W (%) profiles for

copolymer gels obtained with f2-irradiation from �10 to 14 ppm(�250Hz) indicate the participation of an amount of boundwater, monomer composition (number of hydroxyl groups) andthe rigidity of copolymer gels (� W (%)) in the saturation transferprocess [9,19, refer Fig. 3c in (19)]. Indeed, the [1/TIS]INV vs W (%)profiles obtained with f2-irradiation at 7.13 ppm (Fig. 3a) and�4.00 ppm (Fig. 3b) exhibited significant differences betweenhydrophilic (gels-1 and gels-2) and hydrophobic (gels-3 andgels-4) copolymer gels. On the other hand, it was also reportedthat those monitored with f2-irradiation below �10 and above14 ppm (�250Hz) seem to be independent of monomercomposition and mainly indicate the participation of W (%)(rigidity) in the saturation transfer process (9,19). The [1/TIS]SATvs W (%) plots obtained with off-resonance f2-irradiation (above16.0 and below �12.0 ppm at �250Hz) showed approximatelystraight lines with the intercept at a W of �27% (19) (seeFig. 2c therein). As shown in Figure 3c, the [1/TIS]INV(�8.79 ppm)vs W (%) profiles exhibited little difference among each othercompared with those at 7.13ppm (Fig. 3a) and�4.00ppm (Fig. 3b).

Mouse lenses

1/T1: The intact mouse lenses kept in a glass capillary of 2.3mminner diameter were found to be transparent by visual inspectionduring 1H NMR measurement at 258C, indicating no aqueousphase separation in the lens cytoplasm (23–25). These lenses,which were perforated with a set of fine stainless needles,




immediately formed a homogeneous viscous opaque gel,indicating perforating trauma-induced cataract formation (13).Thus, there might be no change in water content before and aftercataractous lens formation in the present paired experiments.To study whether there is a significant difference in 1/T1 values

between the paired experiments or not, we carried out 1/T1 vs X/(1 – X) analyses (5,13,27–29). As shown in Figure 1c, the obtainedregression lines for intact and cataractous lenses were 1/T1¼ 0.39X/(1 – X)þ 0.33 and 1/T1¼ 0.48X/(1 – X)þ 0.34 (linearitytest; n¼ 14; p< 0.005, Student’s t-test), respectively, exhibitingsome indication of increases in the fraction of bound waterduring cataract formation, as increased hydration (water content)in the localized cataracts of excised intact rabbit lenses (33) and inhereditary cataract mouse lenses detected by laser Ramanspectroscopy (34,35). However, a significant difference betweenthe two linear regression coefficients of 0.39 (intact) and 0.48(cataractous lenses) was not observed (Fig. 1c).T1,absc and TIS: As shown in Figures 1a and 2b, the [T1,absc]INV

and [TIS]INV values for cataractous lenses were nearly equal to thecorresponding conventional T1 and [TIS]SAT values, respectively,within experimental accuracy (9,12–14,19). However, the[T1,absc]INV and [TIS]INV values for intact lenses were longer thanthe corresponding conventional T1 and [TIS]SAT values, respect-ively, as shown in Figure 1a (empty rhombuses) andFigure 2b (empty and filled rhombuses). On the other hand, aswe previously described (12–14,19), the [T1,absc]SAT values forintact lenses were nearly equal to the corresponding T1 valueswithin experimental accuracy.Before discussing the above evidence, it is worth noting that

saturation transfer from irradiated protein (macromolecule)protons to water protons occurs as a result of the followingsequential events (36): Saturation of f2-irradiated proto-ns¼¼> Subsequent intramolecular saturation transfer to thewhole proton spin system of the protein except protons in themobile regions by cross-relaxation¼¼> Saturation transfer fromprotein to bound water by intermolecular cross-relaxation¼¼>Transfer of saturation from the saturated bound water to theunsaturated water in the bulk solution by chemical exchan-ge¼¼> Recovery of the magnetization of the protons of freewater by the spin-lattice relaxation process. A schematic diagramof Akasaka’s model (36) was reported by Kuwata et al. (12) (see

3———————————————————Figure 3. (a) [1/TIS]INV values, obtained by the INV method withf2-irradiation at 7.13 ppm and gH2/2p� 69Hz, as a function of dry weight

[W (%)] for poly(HEMA-stat-GMA)s, poly(HEMA-stat-N-VP)s, poly(N-VP-

stat-GMA/MMA)s, poly(N-VP-stat-MMA/BzMA)s and excised intact and

cataractous mouse lenses (4- and 8-week-old) at 258C. All of the excisedintact and cataractous mouse lenses were paired samples. (b) [1/TIS]INVvalues, obtained by the INV method with f2-irradiation at �4.00 ppm and

gH2/2p � 69Hz, as a function of dry weight [W (%)] for poly(HE-MA-stat-GMA)s, poly(HEMA-stat-N-VP)s, poly(N-VP- stat-GMA/MMA)s,

poly(N-VP-stat-MMA/BzMA)s and excised intact and cataractous mouse

lenses (4- and 8-week-old) at 258C. All of the excised intact and catar-

actous mouse lenses were paired samples. (c) [1/TIS]INV values, obtainedby the INV method with f2-irradiation at �8.79 ppm and gH2/2p � 69Hz,

as a function of dry weight [W (%)] for poly(HEMA-stat-GMA)s, poly(-

HEMA-stat-N-VP)s, poly(N-VP-stat-GMA/MMA)s, poly(N-VP-stat-MMA/

BzMA)s and excised intact and cataractous mouse lenses (4- and8-week-old) at 258C. All of the excised intact and cataractous mouse

lenses were paired samples.


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K. NAKAMURA ET AL.

590

Fig. 3 therein), and Akasaka’s interpretation (36) could beapplicable to intact and cataractous lenses.[T1,absc]INV> T1 (Fig. 1a, empty rhombuses), i.e. [TIS]INV> [TIS]SAT

(Fig. 2b, empty and filled rhombuses) for intact lenses may beattributed to the following mechanism, which we havedemonstrated in BSA�gel (5–9), where BSA� represents partiallyhydrolyzed BSA (37). The intramolecular cross-relaxation times(TIS) from irradiated protein protons to observed side-chainprotons were studied in BMA solution (mono-dispersed state)and BSA�gel (poly-dispersed state of BSA�) (5–8). The [TIS]SAT and[TIS]INV values for the side-chains of BMA solution showed asimilar single kind of TIS for each side-chain (7). However, the SATmeasurement on BSA�gel exhibited the presence of at least twokinds of intramolecular saturation transfer processes given in eqn(1), i.e. Ia¼ Ia,1þ (Ia,0 - Ia,1)exp(-t/T�a;1 ) and Ib¼ Ib,1þ (Ib,0 –Ib,1)exp(-t/T�b;1 ), where T

�b;1 � 0.05 T�a;1, as described previously (7)

(see schematic Fig. 3 therein). The Ib process of BSA�gel seems to

be attributed to large, immobile protein associates and/oraggregates (2–8), resulting in a short T�b;1 value. On the otherhand, the Ia process seems to be attributed to smaller proteinassociates and/or monomers, resulting in a long T�a;1 value. Theobserved [TIS]SAT values seem to be affected by the short T�b;1 andlong T�a;1 processes. On the other hand, the corresponding [TIS]INVvalues seem to be weakly affected by the short T�b;1 processesbecause of the technical restriction of the INVmethod resulting in[TIS]INV> [TIS]SAT (7) and [T1,absc]INV> [T1,absc]SAT� T1.

#1 Thephysical characteristics of the crystallin proteins in the lenscytoplasm in intact lenses may be similar to those for BSA� inBSA�gel (5–8), resulting in [TIS]INV> [TIS]SAT [Fig. 2b, filled(�8.79ppm) and empty (�4.00ppm) rhombuses), and [T1,absc]INV> T1� [T1,absc]SAT (Fig. 1a, empty rhombuses (�4.00 ppm)]. It is worthnoting that the [T1,absc]INV> T1 relationship reported by Balaban’sgroup (15–17) seems to be attributed to the above long and veryshort saturation transfer processes.1

Although we have no knowledge of the exact initiationmechanism of this type of cataract formation, it seems likely thatthe strengthening of the secondary bonding may cause theformation of large, immobile association or aggregation of lensproteins (5,13), resulting in a single saturation transfer process.Therefore, the strong and nearly homogeneous association and/or aggregation of lens proteins in perforating trauma-inducedcataract formation seems to exhibit an increase in [1/TIS]INV(Figs 3a and 3b) and the following relationship [1/TIS]INV� [1/TIS]SAT (Fig. 2b, empty and filled triangles), as observed incopolymer gels (Fig. 2a) and excised tissues, such as packed RBCs(14), rat liver, hepatocellular tumors, and brain tissues (unpub-lished data), which are packed with various kinds of macromol-ecules and show macromolecular crowding-induced organiz-ation of cytoskeletal elements (38–40).

Correlation of our findings with other reports

Moffat and Pope (41) demonstrated differences in T2 relaxationdecays between excised intact lenses and lens homogenates ofhuman and porcine lenses. They found that the T2 relaxation ofintact lens is bi- or multi-exponential when observed by theCPMG method and that the breakdown of the lens cellularstructure by homogenization reduces the T2 relaxation decaysfrommulti-exponential to mono-exponential (41) (see Tables 1, 2,and 3 therein). Moreover, bi- or multi-exponential T2 relaxationdecay in intact lenses reflects spatial variations in water-solublecrystallin contents throughout the lens rather than the presence


of distinct bound and free water pools. In the present study, the T2values for mouse lenses were not measured, thus we can notcomment on whether T2-relaxation decays are single or multi-exponential in intact and cataractous mouse lenses. As discussedin the last section, our findings that [1/TIS]INV< [1/TIS]SAT and [1/TIS]INV� [1/TIS]SAT for paired intact and cataractous mouse lenses(5–7,13), respectively, seem to be qualitatively similar to thestatements by Moffat and Pope (41).With regard to MR imaging, Lizak et al. (42) studied the

preliminary magnetization transfer contrast imaging (MTCI) ofnormal and cataractous human lenses using a Signa 1.5 T clinicalscanner with an off-resonance saturation pulse at 24 ppm(off-water resonance frequency). They reported that for theprotein-dense lens nucleus, cataractous changes could be readilyobserved by MTCI. They also reported that in the nuclear cataract,the observed changes in MTC signal are probably due to alteredprotein hydration associated with protein cross-linking (42). Inrelation to the MTCI of Lizak et al. (42), it is worth noting ourfollowing findings. While conducting studies on [1/TIS]SAT vs f2(ppm) profiles (CR spectra) (8,9,13,14,19) and [(I0/I1) – 1]vs f2(ppm) profiles (equivalent CR spectra) (19) for copolymergels, we have developed equivalent CR imaging (equivalent CRI)with an off-resonance saturation pulse at 7 ppm (off-waterresonance frequency) and applied it to copolymer gels (21,22)and breast cancer (22,43–46). As reported by Eng et al. (16),Balaban and Ceckler (17), and Ueshima et al. (47), CRI is usefulfor quantitative tissue characterization. However, CRI is quitetime- consuming compared with MTCI (16,17). In contrast,equivalent CRI is not time-consuming compared with MTCI, andmoreover, it exhibits interesting tissue characterization(22,43–46). Thus, equivalent CRI in human cataractous lensesmay be more useful for quantitative tissue characterization thanMTCI (42).

Comparison of mouse lenses with copolymer gels

As demonstrated in Figure 3a, [1/TIS]INV(7.13 ppm) vs W (%)profiles for hydrophilic copolymer gels showed similar charac-teristics in cross-relaxation processes to intact and/or cataractousmouse lenses. However, the profiles for hydrophobic copolymergels showed quite different characteristics compared with mouselenses. Therefore, results from our approach indicated that [1/TIS]INV vsW (%) profiles are useful for searching the physical stateof water for cross-linked copolymer gels as well as tissuecharacterization for the excised and/or living tissues.

Acknowledgements

We are grateful to the Late Professor K. Fujita (Dean of FujitaHealth University) for his gracious support for the NMR exper-iments. We wish to thank Drs H. Amano (Amano Eye Clinic, Gifu,Japan), K. Takahashi (Takahashi Eye Clinic, Gifu, Japan), K. Kato(Department of Pathology, Fujita Health University, Toyoake,Japan), and T. Hirabayashi (Department of Environmental Tech-nology and Urban Planning, Graduate School of Engineering,Nagoya Institute of Technology, Nagoya, Japan) for their kinddiscussion and helpful comments. We also wish to thank Drs K.Nakada, K. Takahashi, and S. Kanome (Menicon Co. Ltd, NagoyaJapan) for their helpful comments and for their supplies ofsynthetic cross-linked copolymer gels, and Ms K. Hayashi of thislaboratory for her kind help in preparing the manuscript.



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Comparative 1H NMR studies of saturation transfer in copolymer gels and mouse lenses