Biochimica et Biophysica Acta. 1085 (1991) 343-349 © 1991 Elsevier Science Publishers B.V. All rights reserved 0005-2760/91/$03.50 ADONIS 000527609100260N

343

Effects of cholestanol feeding on corneal dystrophy in mice

K y o u n g Sook Kim t K a z u t a k a K a n o i T a k e s h i K a s a m a z, Y a s u o Ishii 3, H i d e t o s h i Y a m a s h i t a 4 and Y o u s u k e S e y a m a t

t Department of Physiological Chemistry and Nutrition. Faculty of Medicine. Unil'ersi~" of Tokyo. Hongo, Bnnkyo-ku. Tokyo (JapanL 2 Laboratory for Biochemical 4nalysis. Tokyo Medical and Denial Unit er~i.3". "Fv,L:y,, tJnpan). ~ Eye Research btstitnte of Cataract Foundation. Nishikata, Bnnkyo-ku. Tokyo (Japan) attd 4 Department of Ophthabnolog?,. Faculty of Medicbw, Unit'ersiry of Tokyo,

Hongo. Bunkyo-ku. Tokyo (Japan)

Key words: Cholestanol feeding: Cerebrotendinous xanlhomatosis: ('t~rneal opacity; Calcium deposit: (Mourn)

A cholestanol-enriched diet administered for 8 months to BALB/c mice produced in 20% two kinds of corneal opacities resembling catcific band keratopathy and Scbnyder's crystalline dystrophy in humans. The concentrations of cholestanol in serum, liver and cornea of the corneal opacity bearing mice were 30-40-times higher than those of normal mice. On the other hand, brain ¢holestanol level increased only 7-times in the opacity group as compared with that of control group. There was no significant difference in the cholesterol concentrations of serum and several tissues among opacity, non.opacity and the control group. The crystal particles were observed between epithelial basement membrane and superficial stroma by the electron microscopy. Energy dispersive analysis of the particles revealed that the deposits were composed principally of calcium and phosphorus with other crystaUine materials, which was presumed to be cholestanol. These results suggest that the cholestanol may deposit in the cornea from elevated serum levels. Deposition of cholestanol in cornea and related area may be a cause of corneal dystrophy in CTX.

Introduction

Cerebrotendinous xanthomatosis (CTX) is a rare metabolic disease that is inherited as an autosomal recessive trait. Clinically this disorder i~ characterized by progressive neurologic dysfunction (dementia, cere- beilar ataxia, spinal cord paresis), tendon xanthomas, premature atherosclerosis and juvenile cataracts [1-3], CTX patients have an impaired capacity to convert cholesterol to bile acids, due to a defect of hepatic enzyme, catalyzing the oxidation of side chains of sterol intermediates in the biosynthesis of bile acid [4,5]. The defective enzyme causes a block in bile acid synthesis, resulting in a marked decrease of bile acids (particu- larly chenodeoxyeholic acid) and brings about an accu- mulation of eholestanol, 5-dihydro derivative of choles- terol, in serum and several tissues, which result in the formation of xanthomas in the brain, lung and tendon

Correspondence: Y. Seynma. Department of Physiologica~ Chemistry and Nutrition, Faculty of Medicine, University of Tokyo, 7-3-1 Itongo, Bonkyo-ku, Tokyo 113, Japan.

[6-I0]. Although xanthomatous deposits in this disease was first described by Van Bogaert et al. in 1937, the reasons fi)r deposition and relationship to eholestanol are not yet known with certainty. The present study was designed to test ~he effect of cholestanol feeding on serum and tissue concentration and to establish its pathologic conditions resembling CTX, i.e., xanthomas or neurologic dysfunction development. After 8 months of feeding 1% cholestanol in the diet, 20% of the animals developed degenerative lesions of cornea that resemble calcific band keratopathy [11-15] and crys- talline corneal dystrophy [16-23].

Materials and Methods

A n i m a l s and diets Male BALB/c mice (final body weight of 25-30 g)

obtained from a single breeding parents colony were used throughout the experiments. Mice were fed for 8 month~ with a diet containing 1% cholestanol. The control mice were fed with the standard diet, The parent~ mice and diets were obtained from Clea Japan (Tokyo). The standard mouse diet (CE-2) coi~tained

344

24.8% protein, 4.4% fat, 3.5% fiber and all necessary vitamins and minerals at recommended level, besides, 0.018% (w/w) cholesterol and trace amounts (below 0.003%~ of eholestanol. The diet containing cholestanol was prepared by mixing the standard diet with 1% cholestanol. All mice were housed in two animals per cage. The animal room was maintained at a tempera- ture of 26°C and a relative humidity of 50-60%. Light- ing was provided by artificial illumination for 12 h per day. The mice received experimental diet and drinking water ad libitum for 8 months.

Biochemical analysis Cholestanol (5a-cholestan-3/3-ol) and epicopro-

stanol (5/3 cholestan-3a-ol) as an internal standard were purchased from Sigma Chemical (St. Louis, MO). All other chemicals and solvents used were the highest grade available unless otherwise stated. Sample prepa- ration and analysis of sterols by HPLC were performed as described previously [26]. The serum (0.1 ml) was diluted with 10 volumes of 1 M ethanolic KOH and hydrolyzed at 80°C for 1 h, followed by extraction twice with n-hexane. The solvent was evaporated under a stream of nitrogen and derivatized with benzoyl chlo- ride reagent, and analyzed by HPLC using 5fl-choles- tan-3a-ol as an internal standard. The liver tissue (50 mg) was hydrolyzed with 1 ml of 1 M ethanolie KOH, and cerebellum (50-60 rag, in bulk), cerebrum (120-150 rag, in a half bulk) and cornea (5-6 rag, a pair) were hydrolyzed with 2 ml, 5 ml and 1 ml of 1 M ethanolic KOH, respectively, and processed by the same proce- dure as described above. The column was packed with SBC-ODS (0.25 X 15 cm, Shimadzu) and maintained at 50°C during analysis.

Histopathology The eyes of affected and control mice were frozen

by liquid nitrogen, then sectioned at 5/xm and stained with hematoxylin and eosin (HE). Calcific keratopathy lesions were stained with periodic acid-Schiff, yon Kossa's S. and Alizarin red S. For identification of lipid, frozen sections of crystalline lesions were also stained with Oil red O, Sudan black B, and OsO 4. Stainings specific for cholesterol were performed t~y both the methods of Schultz [27] and Adams [28]. Cholcstanol was not stained by the latter method [28].

Transmission electron microscopy The eyes from control and affected mice with corneal

dystrophy were fixed in 3% glutaraldehyde solution. The corneas were removed, cut into l-mm pieces, post-fixed with osmium tetroxide, dehydrated through a series of concentrations of alcohol. They were then embedded in epoxy resin (Epok 812R). Thick sections (I /zm) were stained with Toluidine Blue O, and thin sections (70 nm) were stained with uranyl acetate and

lead citrate and examined in electron microscope (JEM 100).

Quafitatire elemental analysis The energy dispersive X-ray spectrometer installed

on a transmission electron microscopy (TEM/EDX) was used for analysis of minerals on the eyes of two affected mice. The eyes were postfixed in 2.5% glu- taraldehyde, then also fixed at 4°C 2 h in 1% osmium tetroxide. Samples were then dehydrated with ethanol, embedded in epon araldite, sectioned and mounted on nickel mesh. They were analyzed by an H-7000 trans- mission electron microscope integrated with an EMAX-3770 energy dispersive X-ray microanalyzer (Hita. hi, Japan).

Statistical analysis Data are presented as mean + S.D. unless otherwise

indicated. The significance was evaluated by Student's t-test [29].

o.6-

O.S-

E 0.4" E" 2 o3-

~ o.2,

~ o.1,

g, o.o

[] opacity I~l non-opacity • control

Serum Liver Cerebellum Cerebrum Cornea

20"

~ 15

lO

~ s

o

~1 opacity [ ] non-opacity ~ ~ • control ~

Serum Liver Cerebellum Cerebrum Cornea

Fig. I. Variations of sterols in serum and several tissues. Cholestanol (A) and cholesterol (B) in cholestanol-fed and control diet mice. The sterols were determined by the procedures described under Materi- als and Methods. Each value and bar represent the mean_+S.D. tn = 5). Analysis of data was done by Student's t-test (P < 0.001,

P < 0.05).

Results

Determination of sterols by HPLC The mice were divided into three groups, designated

'opacity group'; cholestanol-fed with corneal opacity, 'non-opacity group'; cholestanol-fed without corneal opacity, and 'control group'; fed with standard diet. Fig. 1 shows the contents of sterols in serum and various tissues. The variations of cholestanol concen- trations are shown in Fig. 1A. When a diet containing 1% eholestanol was fed for 8 months, the cholestanol contents in serum and liver of opacity group were increased almost 34- and 38-fold from those of control group (0.005 to 0.17 m g / m l and 0.01 to 0.38 mg/g , respectively), whereas there was no significant differ- ence between opacity and non-opacity group. The cholestanol level in cerebellum and cerebrum of opac- ity group was about 7- and 4-times higher than those in control group (0.37:0.05 m g / g and 0.22:0.05 mg/g , respectively), but in the case of cerebellum cholestanol content, there was a significant difference between opacity and non-opacity group (0.37 _+ 0.14 and 0.26 + 0.02 mg/g , respectively, P < 0.05). The concentration of corneal cholestanol in opacity gronp was increased 36-fold as compared with control (0.18 and 0.005 mg/g), though there was no significant difference between opacity and non-opacity group (0.18 + 0.06 and 0.12 + 0.01 mg/g , respectively). Fig. IB shows the changes of cholesterol contents. The serum cholesterol levels were not significantly different among the groups. However,

345

the hepatic cholesterol concentration was significantiy decreased by 25% in opacity group as compared with control (2.00 + 0.30 and 2.68 + 0.34 mg/g , respectively, P < 0.05), though the cholesterol content of non-opac- ity group was similar to that of control group (2.61 + 0.30 and 2.68 + mg/g). In the brain which contains large amounts of cholesterol which seems to be charac- teristic of nervous tissue, cholesterol concentration of cerebellum in opacity group was increased 45% as compared with that of control (15.72 + 3.46 and 10.83 + 1.52 mg/g , respectively). On the other hand, the concentrations of cerebrum cholesteroI in the opacity and non-opacity groups were similar to those of control and cholesterol level of cornea similar to that of liver showed no remarkable variations among three groups.

Fig. 2 shows the HPLC chromatogram of the corneal sterols. The chromatography profles of corneal sterols revealed that the peak of cholestanol (P3) was detected markedly in opacity group (Fig. 2A), but not prominent in control gr~mp (Fig. 2B).

Histological findings Corneal opacity was observed in about 20% (23/118)

of the cholestanol-fed animals. The lesions occurred in both sexes, and two kinds of lesions were observed; one was zonular shaped in mineralized deposit (data not shown) and the other was oval shaped opacity with crystalline-like deposits together with new vessel for- mation. Corneal opacities never occurred in the control mice (Plate D.

I

,, tin 210 ~11 ' 40rain

Fig. 2. HPLC chtomatogram of corneal sterol. Opacity group (A) and control group (B). Samples were analyzed on an SBC-ODS (0.25 × 15 cm) column with a rnnning solvent of acetonitrile/water/acetic acid (97:3:0.2) at a flow rate of 0.5 ml/min. Ultraviolet absorbance was monitored

at 228 nm. Peak PI; cholesterol, peak P2; epicoprostanol (internal standard), peak P3; cholestanol.

346

Fig. 3. Electron microscopy of a region epithelium (Ep) and stroma (St) from a case of band keratopalhy. The dense conglomerates (arrow heads) seen between subepilhelium and superficial slroma appear to be composed of a fusion of the individual spherules (arrows) such as those observed in keratncyte (K) (8000x). Bm;

basemenl membrane. N; nucleus.

Band kcratopathy with mineralized deposits was ex- amined by electron microscope (Fig. 3). The electron dense crystal particles were not in epithelial cell.

The crystal par t ic les of the eye of two af fec ted mice and one normal mouse were examined by a t ransmis- sion e lec t ron m i c r o s c o p y / e n e r g y dispersive X-ray analysis ( T E M / E D X ) . Energy dispersive X- ray analy- sis revealed p r o m i n e n t peaks o f ca lc ium a n d phospho- rus in opaci ty g r o u p (Fig. 4). The peak des igna t ed Ni shows a presence of nickel which was used as a gr id.

The e lec t ron dense crystal par t ic les were also pre- sent be tween subepi the l ium a n d superf ic ia l s t roma (Fig. 5A), analyzed by X-ray analysis a n d p roved to be a deposi t con ta in ing ca lc ium (Fig. 513).

O.lOO 5.220

©a

.10o 5.220 nORIBA Et.Ig× F, l a p 200see }is : IOKev ( lOeV/ch } VFS: 1279

Fig. 4. Computerized energy dispersive X-ray anabsis of corneal crystal depnsits (arrows). The height of the peaks rt:presents the relative quantity of minerals. B*; background, Ca: calcium. P: phos-

phorus, Os; osmium, Ni; nickel (grid).

Co rnea l dys t rophy with crystal l ine-l ike depos i t s we re s e a r c h e d by light microscopy (Pla te 11). Ext race l lu la r crystal l ine a r r ays were c o n c e n t r a t e d a long the ep i the-

Plate 1. 8 month-old mouse with corneal opacity. Oval shaped opacity (asterisk) is observed in the center of cornea and new vessel formation (arrows) is also ~,bserved.

Plate 11. The light microscopic examination of the corneal opacity mouse. Rectangular plate shaped crystalline array (arrows) was concentrated alt)ng tile epithelial basement membrane and superficial stroma (2400x). New vessels were also observed in stroma but not in the unaffected

mice. Ep; epithelium, V: vessel, St: stroma. Inset = enlargement of crystalline (6000×).

347

348

(A)

Fig. 5. Computerized picture of X-ray of corneal crystal deposils. The cry. stal particles (A, arrows) between subepithelium and superficial stroma were coincided with a series of dot arrays (B) indicating the ,~resence of calcium in the region. Ep; epithelium, St; stroma.

lial basement men, brane and superficial stroma and new vcsscls were al, o observed in the superficial layer of the stroma. Crystalline-like deposits in some af- fected mice wcrc plcscnt in the deeper stroma layer, near to Descemcnt's membrane (data not shown). At- tempt to stain the frozen sectioned tissue by Schultz, Adam's methods specific for cholesterol was done to identify the composition of crystalline material, but was unstained.

Discussion

In the previous study, we have found that the cholcstanol level in serum, liver and cerebellum was increased markedly in ~he mice fed cholestanol-rich diet for 32 weeks [3(I]. The new results presented in this paper are the development of ophthalmic abnor- malitlcs in mice fed cholestanol-rich diet. When the diet containing 1% cho!cstanol was fed to mice for 8 months, the ct':~lestanol concentrations in serum and liver o1 the opacity group were higher than that of control ~roup, 34- and 38qold, respectively. However, it was notable that the hepatic cholesterol content in the opacity group, unlike non-opacity group, decreased 25q~. as compared with that of control gl,~up (Fig. IB).

These resuhs agree with the previous finding [24], that a 2% cholcstanol fed to rats for 2 weeks, resulted in incrcascd serum (6-fold) and liver ',35-fold)

cholestanol, whereas hepatic cholesterol concentration decreased 8% as compared to the control, coinciden- tally HMG-CoA reductase activity rose almost 3-fold. Thus, exogenously absorbed large amounts of cho- lestanol stimulated the hepatic HMG-CoA reductase and despite structural similarity between cholesterol and its 5a-saturated derivative, cholestanol does not exert feedback inhibition on the hepatic cholesterol synthesis.

On the contrary, Buchmann and Clausen [25] have reported that the cholestanol concentrations of serum and liver in rabbits f~:d 2% cholestanel diet increased significantly and the cholesterol content in the liver was also increased.

Our results may support the former finding and indicate that the high cholestanol diet leads to replace- ment of cholesterol by cholestanol in the liver. In the cholestanol fed mice, cholestanol deposited in cerebel- lum, with both cholesterol (Fig. IB) and cholestanol levels (Fig. IA) markedly increased as compared to that of the control mice. Of importance is the fact that both cholesterol and eholestanol concentrations were higher in the cerebellum of opacity group. It could be speculated that the abnormalities in the transport sys- tems for sterol influx and efflux from tissues [31], or unknown defects of blood-brain barrier [25,32] might contribute to tli,, excessive cholestanol accumulation in brain.

The most characteristic and striking observations in the present study is two kinds of corneal dystruphy histologically resembling the ealcific hand keratopathy [ 1 1 - 1 5 ] a n d S c h n y d c r ' s c rys ta l l ine d y s t r o p h y [ 16-23] in

h u m a n . In the c a s e o f ca lc i f ic k e r a t o p a t h y , t he les ion is

a s s o c i a t e d w i t h a v a r i e t y o f s p o n t a n e o u s s t a t e s c a u s e d by a l t e r e d c a l c i u m m e t a b o l i s m [12] i n c l u d i n g hyper -

p a r a t h y r o i d i s m [13], v i t a m i n D in tox ica t io | l [14] a n d

h y p e r c a l c e m i a o f m a l i g n a n c y [15]. T h e l c d o n s pre- s e n t e d in th i s r e p o r t m a y h a v e s o m e rc c v a n c e to c h o l e s t a n o l a c c u m u l a t i o n as c v i d e n c c d by an i n c r e m e n t in c o r n e a l c h o ! e s t a n o l c o n c e n t r a t i o n (F ig . I A , 2A) a n d t h e c a l c i u m d e p o s i t i o n as v e r i f i e d by T E M / E D X {Fig. 4, 5). R o t h et al. [23] h a v e r e p o r t e d tha t the owd

c o r n e a l o p a c i t y o f b e a g l e s in w h i c h t h e r e is no e l eva -

t ion o f s e r u m c h o l e s t e r o l o r t r i acy lg lyceroL e x c e p t i n g c a s e s w h e r e the m o s t a d v a n c e d m o r p h o l o g i c type ( w h i t e a rc ) , w a s f o u n d . A l t h o u g h we w e r e no t yet ab le to

e l u c i d a t e the f ine m e c h a n i s m o f f o r m a t i o n s o f t he

d e p o s i t i o n o f c a l c i u m in c o r n e a , it is poss ib l e to specu -

la te tha t t h e i n c r e a s e d s e r u m c h o l c s t a n o l c o n c e n t r a -

t i on c a u s e s d i s t u r b a n c e o f c a l c i u m m e t a b o l i s m in

c o r n e a , r e s u l t i n g in local d e p o s i t i o n o f ca l c ium. R e -

cen t ly , m a n y i n v e s t i g a t o r : h a v e r e p o r t e d t h a t t he crys- t a l l ine d e p o s i t s in t h e S e h n y d e r ' s d y s t r o p h y w a s c o m -

p o s e d o f c h o l e s t e r o l a n d tha t t h e ba s i c fau l t l e a d i n g to i ts local d e p o s i t i o n is a local d e f e c t in l ipid m e t a b o l i s m

[21,22]. T h e d i s t u r b a n c e o f l ipid m e t a b o l i s m resu l t s in

o v e r l o a d i n ~ by s t e ro l d e r i v e d f r o m the b lood s t r e a m

[18-21]. Our results suggested that the crystalline-like de-

posits in the cornea are probably cholestanol. Although t h i s r e p o r t is t h e r e su l t o f p r e l i m i n a r y s t u d i e s on the

e f f e c t s o f c h o l e s t a n c l f e e d i n g on t i s sue s t e ro l s ar id

c o r n e a l o p a c i t y in m i c e , it m a y be o f g r e a t i n t e r e s t ir~

e o n n e c t i o u w i t h m e p a t h o l o g y in C T X w h i c h is a l ipid

s t o r a g e d i s e a s e wi th p r e d o m i n a n t a c c u m u l a t i o n o f

c h o l e s t a n o l in s e r u m a n d t i s sues .

Acknowledgements

W e t h a n k the s t a f f o f H i t a c h i E n g i n e e r i n g Inc. K~r

t h e i r c o - o p e r a t i o n in c a l c i u m ana lys i s by X - r a y m i c r o -

ana lyze r . T h i s w o r k w a s s u p p o r t e d by g r a n t s - i n - a i d f r o m the

M i n i s t r y o f E d u c a t i o n , S c i e n c e a n d C u l t u r e o f J a p a n (02044044) a n d by g r a n t s f r o m t h e J a p a n e s e F o u n d a - t i on o f M e t a b o l i s m a n d D i s e a s e to Y. S c y a m a .

349

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