1H-Magnetic Resonance Spectroscopy ofRetrobulbar Tissue in Graves Ophthalmopathy

KENJI OHTSUKA, MD, PHD, AND MASATO HASHIMOTO, MD, PHD

● PURPOSE: Accumulation of glycosaminoglycans in theorbit may play an important role in the development ofGraves ophthalmopathy. Therefore, it might be clinicallyuseful to evaluate the concentration of glycosaminogly-cans in the orbit in patients with Graves disease. Weinvestigated the concentration of glycosaminoglycans inretrobulbar tissue using in vivo 1H-magnetic resonancespectroscopy.● METHODS: A model solution of 1% chondroitin sulfate(one of the components of the glycosaminoglycans com-plex) was initially examined using 1H-magnetic reso-nance spectroscopy, and the resonance of chondroitinsulfate was identified. 1H-magnetic resonance spectros-copy spectra of retrobulbar tissue were obtained in 16normal volunteers (28 eyes) and 23 patients with Gravesophthalmopathy (36 eyes). The 1H-magnetic resonancespectroscopy spectrum of chondroitin sulphate in theretrobulbar in vivo tissue was identified by assignmentsthrough lineshape comparisons of spectra of the modelsolutions in vitro and the retrobulbar tissue in vivo.Chondroitin sulphate–peak/H2O-peak ratios were calcu-lated. To verify the results of in vivo 1H-magneticresonance spectroscopy, retrobulbar tissue samples fromfive patients, who underwent orbital decompression sur-gery, were tested for reactivity to chondroitin sulfateproteoglycan by enzyme-linked immunosorbent assay(ELISA).● RESULTS: Multiple peaks, with a large peak at 5.24ppm, were observed in 1H-magnetic resonance spectros-copy spectra of the model solution. A peak at 5.24 ppmwas also observed in spectra of the retrobulbar tissue inall of the normal subjects and the patients. The meanvalue of the 5.24 ppm–peak/H2O-peak ratio was 0.1781(SD 5 0.0498, range, 0.0775 to 0.2282) in the normal

subjects and 0.2874 (SD 5 0.1357, range, 0.1405 to0.7377) in the patients. The 5.24 ppm–peak/H2O-peakratios were significantly increased in the patients withGraves ophthalmopathy (P < .01). The 5.24 ppm–peak/H2O-peak ratio was correlated with the chondroitinsulphate concentration in retrobulbar tissue samples asevaluated by ELISA (r 5 .69).● CONCLUSIONS: This study suggests that 1H-magneticresonance spectroscopy of the retrobulbar tissue allowsus to estimate the concentration of chondroitin sulphateproteoglycan in the retrobulbar tissue. 1H-magnetic res-onance spectroscopy of the retrobulbar tissue may be anew clinical tool for the evaluation of Graves ophthal-mopathy. (Am J Ophthalmol 1999;128:715–719.© 1999 by Elsevier Science Inc. All rights reserved.)

R ECENT STUDIES SUGGEST THAT RETROBULBAR FI-

broblasts are the main targets of the immune processin Graves ophthalmopathy. Histologic examination

of retrobulbar tissue samples shows proliferation of fibro-blasts accompanied by accumulation of glycosaminogly-cans.1 Glycosaminoglycans are hydrophilic molecules thatdirectly increase orbital volume and absorb extracellularfluid, resulting in edematous changes of the orbital con-nective tissue.2,3 Accumulation of glycosaminoglycans inthe orbit may play an important role in the development ofGraves ophthalmopathy. Therefore, it may be clinicallyuseful to evaluate the concentration of glycosaminoglycansin the orbit in patients with Graves disease.

Glycosaminoglycans are compounds showing chemicalreactions characteristic of a carbohydrate containing asmall percentage of protein and include chondroitin sulfateproteoglycan and hyaluronic acid. Glycosaminoglycans inthe retrobulbar tissue consist mainly of chondroitin sulfateproteoglycan.4 In this study, we evaluated the concentra-tion of chondroitin sulfate proteoglycan using in vivo1H-magnetic resonance spectroscopy of the retro-bulbar tissue of patients with Graves ophthalmopathyand normal control subjects. To verify the results of1H-magnetic resonance spectroscopy, retrobulbar tissuesamples from five patients who underwent orbital decom-pression surgery were tested for reactivity to chondroitin

Accepted for publication June 9, 1999.From the Department of Ophthalmology, Sapporo Medical University,

School of Medicine, Sapporo, Japan.This study was supported by Grants-in-Aid for Scientific Research from

the Japanese Ministry of Education, Science and Culture (10770943),Tokyo, Japan.

Reprint requests to Kenji Ohtsuka, MD, PhD, Department ofOphthalmology, School of Medicine, Sapporo Medical University, S-1,W-16, Chuo-ku, Sapporo 060, Japan; fax: 81-11-613-6575; e-mail:kohtsuka@sapmed.ac.jp

© 1999 BY ELSEVIER SCIENCE INC. ALL RIGHTS RESERVED.0002-9394/99/$20.00 715PII S0002-9394(99)00229-9

sulfate proteoglycan by enzyme-linked immunosorbent as-say (ELISA).

METHODS

THE SUBJECTS OF OUR STUDY WERE 23 PATIENTS (36 EYES)

with Graves ophthalmopathy (age range, 15 to 66 years;mean, 42 years; 17 women, six men) examined at SapporoMedical University Hospital between September 1996 andJuly 1997 and 16 normal subjects (28 eyes) (age range, 24to 56 years; mean, 38 years; 12 women, four men). Allthe subjects were volunteers. Informed consent wasobtained from all the patients and normal subjects afterthe nature of the examination had been explained.Tenets of the Declaration of Helsinki were followed,and Institutional Human Experimentation Committeeapproval was obtained.

The diagnosis of Graves disease was based on history,conventional symptoms of thyrotoxicosis associated with adiffusely enlarged goiter, elevated levels of serum free T3and free T4, and increased thyroidal 131I uptake; elevatedanti–thyroid stimulating hormone (TSH) receptor anti-body, antithyroglobulin antibody or antithyroidperoxidaseantibody titers, and the presence of ophthalmopathy pro-vided supporting evidence for the diagnosis. All 23 pa-tients exhibited a diffusely enlarged goiter, elevated levelsof serum free T3 and free T4, and elevated titers ofanti–thyroid stimulating hormone (TSH) receptor anti-body. Ophthalmologic examination of each patient wasperformed by the same ophthalmologist. The mean valueof the exophthalmometry measurements was 19.85 mm(range, 10 to 25 mm). The classification of the EyeChanges of Graves’ Disease of the American ThyroidAssociation (NOSPECS) was adopted for evaluation ofophthalmopathy.5 The NOSPECS class in all patients wascompared with the results of 1H-magnetic resonance spec-troscopy of the orbit.

Localized 1H-magnetic resonance spectroscopy spectraof the orbit were obtained using a whole-body 1.5-Tmagnetic resonance system (General Electric MedicalSystem, Signa, Milwaukee, Wisconsin) with a standardhead coil. The localization of the volume of interest wasguided by T1 magnetic resonance images using three-dimensional spoiled gradient recalled acquisition in thesteady state (repetition time 5 40 ms, echo time 5 5 ms,2-mm thick, contiguous, and interleaved slices; number ofexcitation 5 1). The size and the location of the volumeof interest was set 6 3 6 3 6 mm at the retrobulbar spacecircumscribed by the posterior pole of the globe and thefour rectus muscles. To avoid including the optic nervewithin the volume of interest, the volume of interest wasactually displaced laterally (Figure 1). Localized 1H-mag-netic resonance spectroscopy spectra were acquired with ashort echo-time-stimulated echo-acquisition mode se-quence with short tau inversion recovery methods for fat

suppression accumulating 256 scans for signal averaging(repetition time 5 1500 ms, echo time 5 30 ms, mixingtime 5 13.7 ms, inversion time 5 170 ms). Total exami-nation time was less than 30 minutes.

Before the in vivo study, 1H-magnetic resonance spec-troscopy spectra of a 1% solution of chondroitin sulfateproteoglycan were obtained. The pH of the model solutionwas adjusted to 7.1 to 7.3 using a phosphate buffer.1H-magnetic resonance spectroscopy spectra of the modelsolution were compared with spectra of the orbit. Fornumerical analysis, the raw data of the 1H-magneticresonance spectroscopy spectra were transferred toSA/GE software (General Electric Medical System,Milwaukee, Wisconsin). For each quantitative 1H-mag-netic resonance spectroscopy spectrum, the raw free-induction decay was zero filled, Fourier transformed, andphase corrected. Baseline correction was performed forthe purpose of presentation.

Retrobulbar fat tissue was sampled from five patientswho underwent orbital decompression and stored at 280 Cfor later analysis. Extraction of proteins from orbital fattissues was performed as follows. Approximately 50 mg oforbital fat tissue was homogenized in 5 ml of phosphate-buffered saline containing 2% Triton X-100, with a glass/glass homogenizer, on ice. An aliquot (0.1 ml) of thesample diluted with 0.8 ml of phosphate-buffered salinewas successively mixed with 3 ml of chloroform/methanol(1:2); 0.9 ml of 0.7% sodium chloride solution; and 1 ml ofchloroform. The mixture was centrifuged for 5 minutes at2,000 revolutions per minute, resulting in clear separationof chloroform (bottom layer) and methanol/water (upperlayer). The interface between the two layers, containingprotein components, was carefully collected, lyophilized,dissolved in methanol, and subjected to ELISA and pro-tein concentration assay.

For ELISA, samples containing the protein components

FIGURE 1. The localization of volume of interest (a square inthe right orbit) in an axial T1-weighted magnetic resonanceimage of the orbit using three-dimensional spoiled-gradientrecalled acquisition in the steady state. The size and location ofthe volume of interest was set at 6 3 6 3 6 mm in theretrobulbar space.

AMERICAN JOURNAL OF OPHTHALMOLOGY716 DECEMBER 1999

(diluted 1:200) were placed in individual slide wells, andrabbit antichondroitin sulphate proteoglycan antibody(Bioline; diluted 1:200) was added. Peroxidase-conjugatedrabbit anti-rabbit immunoglobulin antibodies were used asthe second antibodies. The absorbance values at 492 nmwere calculated by subtraction of the optical density(OD492) obtained for bovine serum albumin. The proteinconcentration of each sample was also analyzed accordingto the methods reported previously.6 Finally, the chon-droitin sulphate proteoglycan concentration index wascalculated in the five patients as a measure of the chon-droitin sulphate proteoglycan concentration in total pro-tein components of the orbital fat tissue.

RESULTS

FIGURE 2 SHOWS EXAMPLES OF IN VITRO SPECTRA OF A 1%

solution of chondroitin sulfate proteoglycan and in vivospectra of the retrobulbar tissue in a normal subject and apatient with Graves ophthalmopathy. Multiple peaks, witha large peak at 5.24 ppm, were observed in 1H-magneticresonance spectroscopy spectra of the model solution. Apeak at 5.24 ppm was also observed in spectra of theretrobulbar tissue in all the normal subjects and thepatients. The mean value of the peak at 5.24 ppm/H2O-peak ratio was 0.1781 (SD 5 0.0498, range, 0.0775 to0.2282) in normal subjects and 0.2874 (SD 5 0.1357,range, 0.1405 to 0.7377) in patients. Figure 3 shows thedistribution of chondroitin sulphate–peak (5.24 ppm–peak)/H2O-peak ratios in normal subjects and patientswith Graves ophthalmopathy. Chondroitin sulphate–peak/H2O-peak ratios were significantly increased in the patientgroup (P , .01).

In five patients, retrobulbar fat tissue was sampled duringorbital decompression and the chondroitin sulphate pro-teoglycan concentration was evaluated by ELISA. Figure 4shows the relationship between the chondroitin sulphate–peak/H2O-peak ratio and the chondroitin sulphate proteo-glycan concentration index in these five patients. Thechondroitin sulphate–peak/H2O-peak ratio correlated withthe chondroitin sulphate proteoglycan concentration in-dex (r 5 .69).

The classification of the Eye Changes of Graves’ Diseaseof the American Thyroid Association (NOSPECS) in allthe patients was compared with the results of 1H-magneticresonance spectroscopy of the retrobulbar tissue. Figure 5shows the relationship between the chondroitin sulphate–peak/H2O-peak ratio and the classification of NOSPECSin each patient. In classes 1 and 2, the chondroitinsulphate–peak/H2O-peak ratios were within normal range(0.1405 to 0.1771), whereas the ratios in classes 3 and 4were significantly larger than normal (P , .01). The meanvalue of the chondroitin sulphate–peak/H2O-peak ratiowas 0.1640 (SD 5 0.0164, n 5 4) in class 1, 0.1755 (SD 50.0233, n 5 2) in class 2, 0.2796 (SD 5 0.1012, n 5 17)

in class 3, and 0.3992 (SD 5 0.1574, n 5 13) in class 4.The ratio was significantly larger in class 4 than in class 3(P , .01). The ratio was not correlated with the results ofexophthalmometric measurements.

DISCUSSION

THIS STUDY SUGGESTS THAT THE PEAK AT 5.24 PPM IN1H-magnetic resonance spectroscopy spectra can be aparameter of the concentration of chondroitin sulphateproteoglycan in the retrobulbar tissue. Michaelis andassociates7 suggested that qualitative assignments throughlineshape comparisons of spectra of metabolite solutions invitro and human tissue in vivo are helpful for the identi-fication of the spectrum of a given metabolite. In the

FIGURE 2. 1H-magnetic resonance spectroscopy spectra of a1% solution of chondroitin sulphate (A), retrobulbar tissue ina normal subject (B), and retrobulbar tissue in a patient withGraves ophthalmopathy (C). CS 5 chondroitin sulphate.

1H-MAGNETIC RESONANCE SPECTROSCOPYVOL. 128, NO. 6 717

present study, a peak at 5.24 ppm was observed in1H-magnetic resonance spectroscopy spectra of both themodel solution and of the retrobulbar tissue, suggestingthat the peak at 5.24 ppm in 1H-magnetic resonancespectroscopy spectra of the retrobulbar represents spectrumof chondroitin sulphate proteoglycan.

In a study using high-field in vitro 1H-magnetic reso-nance spectroscopy, resonances for C2HOC protons werereported to appear at 5.20 to 5.28 ppm.8 Lipid content of

cell membranes, such as phosphatidylcholine, phosph-atidylethanolamine, phosphatidylinositol, phosphatidyl-serine, phosphatidylglycerol, phosphatidic acid, and tri-glyceride, also contain C2HOC. Therefore, the elevationof the peak at 5.24 ppm may reflect one or more of thepathologic changes within the retrobulbar tissue inGraves ophthalmopathy. In addition, the acyl chains(–CH5CH–) of all lipid classes appear at 5.12 to 5.45ppm.8–10 Even minor variations of the field strength maycause spectral changes.7 Therefore, spectra of all thesecomponents will change in 1.5-T magnetic resonancespectroscopy. It is equally possible for any of these compo-nents to participate as well as chondroitin sulphate pro-teoglycan in the formation of the peak at 5.24 ppm in1H-magnetic resonance spectroscopy of retrobulbar tissue.

The 5.24 ppm–peak/H2O-peak ratio was significantlyincreased in the patient group. Therefore, the elevation ofthe peak at 5.24 ppm may represent some pathologicchanges of the retrobulbar tissue in Graves ophthalmopa-thy. Histologic examination of retrobulbar tissue samplesof patients with Graves ophthalmopathy shows prolifera-tion of fibroblasts accompanied by accumulation of chon-droitin sulfate proteoglycan and T-cell infiltration.1,11,12 Itis possible that the peak at 5.24 ppm represents cellularityin retrobulbar tissue as well as the concentration ofchondroitin sulphate proteoglycan, because 1H-magneticresonance spectroscopy spectra of the lipid content of cellmembranes appear at around 5.24 ppm. We thereforeverified the results of 1H-magnetic resonance spectroscopyby ELISA of retrobulbar tissue samples. The 5.24 ppm–peak/H2O-peak ratio in 1H-magnetic resonance spectros-copy spectra was correlated with the results of chondroitinsulphate proteoglycan assay by ELISA. These findingssuggest that the peak at 5.24 ppm of 1H-magnetic reso-nance spectroscopy spectra of the orbit can be a usefulparameter of the concentration of chondroitin sulphateproteoglycan in retrobulbar tissue.

FIGURE 3. Distribution of chondroitin sulphate–peak (a peakat 5.24 ppm)/H2O-peak ratios in normal subjects (A) andpatients with Graves ophthalmopathy (B).

FIGURE 4. Relationship between chondroitin sulphate–peak (apeak at 5.24 ppm)/H2O-peak ratio and chondroitin sulphateproteoglycan concentration index (CSPCI) of retrobulbar tissuesamples in five patients who underwent orbital decompression.

FIGURE 5. Relationship between chondroitin sulphate–peak(a peak at 5.24 ppm)/H2O-peak ratio and Eye Changes ofGraves’ Disease of the American Thyroid Association(NOSPECS) class.

AMERICAN JOURNAL OF OPHTHALMOLOGY718 DECEMBER 1999

It is estimated that clinically detectable ocular signs arepresent in 40% of patients with Graves disease.13 Patientswith Graves disease can develop mild to severe eye disease,including eyelid retraction, lid edema, conjunctival injection,proptosis, diplopia, corneal erosion, and sight loss. It may beimportant to detect early signs of severe eye disease formanagement of Graves ophthalmopathy to the extent thatsevere eye disease might be prevented by early immunosup-pressive therapy. Ophthalmopathy, such as proptosis andextraocular muscle disorders, is assumed to result from theaccumulation of chondroitin sulphate proteoglycan in theorbit.1–3,14,15 Therefore, it may be clinically useful to evaluatethe concentration of chondroitin sulphate proteoglycan inretrobulbar tissue. In this study, chondroitin sulphate–peak/H2O-peak ratios were significantly larger in patients withNOSPECS classes 3 (patients with proptosis) and 4 (patientswith extraocular involvement) than in those of normalsubjects, whereas the ratios in patients with classes 1 and 2were within normal range. These findings suggest that thechondroitin sulphate–peak/H2O-peak ratio may represent theseverity of eye changes in Graves ophthalmopathy. 1H-magnetic resonance spectroscopy of the retrobulbar tissuemay be a new clinical tool for the evaluation of Gravesophthalmopathy.

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