ACTA OPHTHALMOLOGICA VOL. 4 6 1 9 6 8

Ofihthalmologic Clinic of the University of Padua, Italy. Institute of Organic Chemistry of the University of Padua, Italy.

CHROMATOGRAPHIC RESEARCH ON T H E AQUEOUS HUMOR IN A CASE OF GARGOYLISM WITH CORNEAL DYSTROPHY

BY

F. More,'> C. DiBello':-'t and G. Bologna''

The name Gargoylism, (Ellis e Coll.l), or HurZer2-PfaundZer's3 syndrome, indicates an hereditary disease clinically characterized by disharmonic nanism, (dysostosis), a typical facies, dorsolombar cyphosis, oligophrenia accompained by a reduction of articular movements (dysdemosis) namely alteration of the capita, of the articular cavity and of periarticular tissues and hepatomegaly and splenomegaly. At the present time, it is believed that the disease is fundament- ally related to an alteration in the metabolism of the connective tissues in which an abnormal accumulation can be put in evidence (thesaurismosis), of mucopo- lysaccharides. (De Lunge4 and Coll., 1944; Lindsay5 and Coll., 1948; Brunt@ 1951).

Among the clinical aspects of the disease there are certain ocular symtoms which may be classified according to their frequency, in the following order:

corneal dystrophy 75"0 of cases proptosis 21 010 ,, ,, hypertelorism 17 010 1 , 7,

strabismus (squint) 10 Q / o ,, ,,

hypertension 7 010 ,, ,, congenital glaucoma,

epicanthus 4,5Vo ,, ,,

Received February 2nd 1968. * Ophthalmologic Clinic of the University of Padua (Italy)

'so Institute of Organic Chemistry of the University of Padua (Italy)

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Among these symtoms, corneal dystrophy is particularly important, for its per- centage of incidence and for its diagnostic value’).

There is a more or less uniformly diffuse turbidity, involving almost always the entire corneal surface, which loses its clear, shiny appearance and becomes opaque and greyish. The cloudiness, always bilateral, is determined by the accumulation in various parts of the membrane, especially in its third anterior, of an abnormal substance histochemically characterized by the presence of mucopolysaccharides. In a personally observed case, one of the authors (Moroa) was able to study a fragment of a corneal biopsy and to show, in accordance with the data of other authors, a severe alteration of the membrane’s structure (swelling of the basal cells of the epithelium with the formation of pseudo-cystic cavities, vacuolization of Bowman’s membrane, dissociation and disruption of the normal architecture of the parenchymal lamellae, with the appearance at various levels of histocytic type elements, full of positive McManus’ material).

This corneal turbidity is similarly interpreted, in the light of the fact that research has documented, in gargoylism, tissue deposition and urinary elimina- tion (Dorfman and Lorinczg) of acid mucopolysaccharides identified as chon- droitin-sulfate B and heparitin-sulfate. Attempts carried out in different techni- ques of fractionization of the mucopolysaccharides present in various organs, (Cifonelli and Dorfman)’o, (Prodi and Prodi)”, showed that they consist of non-homogeneous complexes, separable only with difficulty.

I n the case of gargoylism which furnished the above-mentioned biopsy, Moro also performed chromatographic research on a specimen of aqueous humor, with the dual intention of studying the composition of this material in the eye in a morbid state by means of eventual modification of its biochemistry, and to arrive at the nature of the substances implicated in the pathogenesis of the process of corneal dystrophy.2)

The first study, after acid hydrolysis, permitted the qualitative identification of a series of amino acids and revealed the presence of glucose.

The current research resumes and extends the above studies. W e will refer now to the data obtained by chromotographic analysis of the aqueous humor of a second case of gargoylism with corneal dystrophy, which we recently observed. For control and comparative study we performed the same research

1) It is maintained that corneal dystrophy represents one of the most important means of discrimination between gargoylism and Morguio’s disease, the latter being a familiar dysostosis with a benign clinical character, in many ways similar to the Hurler-Pfaundler syndrome. The observation of some cases of Morguio’s disease with corneal opacity (see Panizon and Pedrini7) leaves under discussion the real discrimina- tory value of this ocular alteration. 2) As regards the protein content of normal human aqueous humor, the reader is referred to the works of Esser12, WunderZyl3, D’ermoi4, W e i ~ e ’ ~ , Francois et RabaeylB (electrophoresis) and to those of Keup and Steiger”, Wunderby and CagianuP and of Ciusa, Cristini and B a r b i r ~ l i ’ ~ (chromatography).

1264

on samples of aqueous humor taken from the structurally normal amblyopic eyes of two five year-old patients. The presence of ocular hypertension in the child afflicted with gargoylism (see clinical study) suggested the examination for comparison, of the aqueous humor of a fourth patient of five years old afflicted with congenital glaucoma (with tension values in both eyes fluctuating between 30 and 40 mm of Hg. [Schiotz]).

CLINICAL CASE

The essential anamnestic and objective data in the case of gargoylism which gave origin to the current research is as follows:

O.G., two and a half years old, from Mestre. (Card 7632). Third child, born in a normal delivery of parents who were first cousins, in good health. At the age of four months a tumefadion of the vertex was noticed, as well as an umbilical hernia and deformation of the spinal column. From that time on, the child was under regular care of pediatricians and treated with calcium and multiple vitamins. A month ago a vision deficit was revealed. Objective examination: The diagnosis of gargoylism was derived on the basis of objective and radiological data: characteristic facies with scaphoencephalia; a marked thickening of the skin of the extremities; psychic deficit; cyphosis of the spinal column; large umbilical hernia; hepatomegaly and splenomegaly; reduction of falangeous articulation, rendering the hands claw-like. Radiology: Roundish heart with irregular increase of the convexity of the left ventricle hooked ribs; cranium with precocius synthesis of the longitudinal and lambaoideae sutures; conspicuous enlargement of the sella with swelling of its floor, and disappea- rance of the anterior clinoidal processes. Column: Slight S-shaped curvature of the spinal column; vertebral bodies only slightly developed with aspects of biconvex lens. Lumbal column with cyphotic inversion of the physiologic lordosis and serious alterations of the cranial vertebrate bodies which appear squat and shortened. The posterior arches are distant from one another in this area. ECG: N.N. EEG. Diffuse signs of encephalic suffering. Ocular Report: Slight hypertelorism, with some degree of bilateral palpebral ptosis. The cornea is slightly enlarged (R.E.; horizontal diameter 13 mm, vertical 12 mm). L.E.; horizontal diameter 13.50 mm, vertical 12 mm. Slight and diffuse turbidity comprising uniformly all the optic section, with some thickening in the third anterior of the paren- chyma. Thickness of the optical section: normal. Absence of newly formed vasa. Slight hypoesthesia. Anterior chamber slightly more ample. Lens: N.N. Iris; N.N. Pupil rounded normally reacting to the light. Fundus: The papilla is seen normally with clear borders. Tension [Schiotz] : R.E.; fluctuating between 18.5 and 30.4 mm Hg.; L.E.; fluctuating between 20.5 and 49.8 mm Hg. Electronometry (in narcosis): R.E. C = 0, 19; F = 1,53 mm3JM0. L.E.C. = 0,18; F = 1 rnmVM’0. ERG: see the following diagram.

1265 Acta Ophthalmol. 46, VI 84

I 1 I 1 [ 1 I b

C H R O M A T O G R A P H I C S T U D Y

In the four samples of A. H. examined, (1 case gargoylism, 1 case hydrophthal- mus and two amblyopic subjects taken as controls) we performed:

(a) Qualitative analysis of Ninhydrine reactive substances present in the A. H. before hydrolysis. Samples of A. H. were chromatographed at different concentrations:

1) On unactive Silica Gel G20 thin layers, using as solvent system n-butanol- glacial acetic acid-water (60-20-20). The ascending technique was used and the plates were removed when the solvent point had reached 10 cm. Temperature during the runs was kept at 18 +_ 2OC.

2) On NO 1 Whatman paper, using as solvent system n-butanol-glacial acetic acid-water (80-20-100). The descending technique was used and the sheets were removed when the solvent point had reached 35-40 cm. Spots were developed by an 0.40/0 acetonic solution of Ninhydrine. Data are shown in Table I below.

(b) Quantitative amino-acids analysis in the A. H. after hydrolysis. Hydrolysis: Known A. H. amounts were treated in a Pyrex tube with 6N

HC1 (1 ml). The tube was sealed in vacuo and kept in a thermostatic bath at 110°C for 22 hours. Then the tube was opened and the residue brought to

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Table 1.

x

E

4

30- 28-

2 6 .

2 4.

22-

20-

ia 16- 14.

12.

10-

8.

6 -

4 .

2 .

I Normal 1st I Normal 2nd I Gargoylism Hydrophthalmus I

0-

Lysine + + + - Glucosamine + + + + Threonine + + + -

+ Serine + - + + Alanine - - + + Valine -

- -

dryness under reduced pressure, dissolved in pH 2.2 citric buffer (4 ml) and automatically analyzed according to Moore and Stein21.

Results are reported in Table 2 and in figs. 1 and 2. ( c ) Qualitative sugars determination. A. H. samples were chromatogxaphed

on silica Gel G thin layers saturated with 1/15 Molar phosphate buffer (pH 8). This method, besides being in a quick procedural framework, allows the sugars to be put in evidence in the presence of appreciable quantities of salts.22

Repeated tests in the above-mentioned conditions produced results sum- marized in Table 3.

Solvent system: n-butanol-acetone-water (40-50-10).

GargayLlsm

lltNorrnal 2a Normal

Hydrophthalmus

L y s H y s Arg A S P Thr Ser lieu Leu T y r Phe

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1 .a

0.9

I 0.8

: 0.7 5 0.6

; 0.1

4 0.1

i 0.:

4 0.: 4'

0.1

Gargoylism

1 5 ' Normal

2@Norrnal

68 Hydrophthalrnus

Fig. 2.

Detection reagent: Aniline phosphate-diphenylamine. (d) Qualitative determination of lipids'). W e followed the method described

by Randerathzs for the analysis of unknown lipid mixtures. The samples examined gave negative tests with the reagents used.

RESULTS

The presence of substances reactive to Ninhydrine in the normal A. H. have been shown and identified for comparison as follows: lysine, glucosamine, threonine, serine (the latter was present in dosable quantities in only one of two samples tested). The samples of A. H. show pathologically a different composition from the normal because of the presence, in the case of hydroph- thalmus, of alanine, in the case of gargoylism of alanine and valine, whereas lysine present in all the other three samples is absent.

2) After acid hydrolysis in the normal A. H. we were able to identify ten aminoacids in a percentage almost equal in the two samples and precisely in decreasing quantitative order: glutamic acid, alanine, leucine, serine, aspartic acid, glycine, valine, lysine, threonine, isoleucine. In gargoylism the amino-

1 ) Only in the A. H. of gargoylism.

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Tabl

e 2.

Nor

mal

1st

Cas

e A

min

oaci

ds

Nor

mal

2nd

Cas

e H

y dro

phth

alm

us

Gar

go yl

ism

p m

oles

p m

oles

O/o

p m

oles

p m

oles

O/o

p m

oles

p m

oles

O/o

Lys

ine

His

tidi

ne

Val

ine

Isol

euci

ne

Arg

inin

e A

spar

tic A

cid

Thr

eoni

ne

Seri

ne

Glu

tam

ic A

cid

Prol

ine

Gly

cine

A

lani

ne

Cys

tine

Leuc

ine

Tyr

osin

e Ph

enyl

alan

ine

Cys

teic

Aci

d M

ethi

onin

e

- t.9 0, (0

3,lO

X 1

0-2

7,58

3,O

O X

1e

2

7,51

14

,90

X I

03

13

,35

2,90

X 1

03

2,60

2,

20 x

I0

9

2,86

7,9 x

10-2

8,

32

6,8

X 1

0”

7,16

4,19

X l

o4

10,2

5 3,

90 X

10”

9.

78

13,O

O X

104

11

,60

8,1

X 1

0-?

8,53

1,82

X

3,95

x I

03

9,

47 x

10

3

1,76

X 1

0”

4,OO

X l

o2

9,40

X l

o-?

7,lO

X 1

03

6,35

7,O

O X

6,

24

20,O

O X

10”

17

,90

4,70

9,

65

23,2

0

4,41

10

,lO

23,5

5

4,8

X 10

3 5,

06

6,3

X 1

0”

6,63

14

,O X

10”

14

,75

3,87

X 1

03

5,

42 X

1k

2

9,45

13

,20

3,80

X 1

0”

5,30

X 1

09

9,52

13

,25

4,lO

X 1

0”

3,66

12

,Ol X

109

10

,72

4,2

X

4,42

13

,2 X

le

e

13,9

0

3,12

X

1,42

x 1

0”

4.44

x 1

04

7,62

3,

49

10.8

5

3,lO

X 1

03

1,

36 X

4,

30 X

7,74

3,

40

10.7

6

1,02

x 10

-2

0,89

2,

60 X

109

2,

32

12,4

0 X

104

11

,lO

4,70

X l

os

4,20

4,

81 X

109

4,

29

6,6

X

6,95

2,

7 X

lo2

2,

84

10,3

X 1

0“

10,8

2

A.A

. pre

sent

s in

p m

oles

A

.H.

A.H

. A

.H.

A.H

. 40

,90

X I

Os

in m

g 18

2 of

39

,92

X I

F in

170

mg

of

108,

74 X

10-

2 in

mg

200 of 8

4,90

X 1

0-2

in m

g 11

2 of

Comparison substances Gargoylism

acids determined were eleven for the presence of arginine, which was absent in the normal samples.

In hydrophthalmus the aminoacids determined were fourteen for the presence of histidine, arginine, tyrosine and phenylalanine.

3) Comparing the complessive quantities of the aminoacids identified after hydrolysis it is documented that, while the two samples of normal A. H. contain an almost equal quantity of aminoacids, the A. H. of the gargoylism has a quantity of aminoacids three or four times superior to that of the normal one and of about 1/4 superior to that of hydrophthalmus. This means that the proteic amount of the A. H. of the gargoylism is higher than that of the hydrophthalmus in which the amount is in turn higher than that of the normal A. H.

The examination of the percentage of the singular aminoacids still shows that A. H. in gargoylism has a quantity of serine, glutamic acid and glycine of about half in comparison to that of the normal A. H.; the arginine absent in the normal A. H. and very scarce in the hydrophthalmus is, on the other hand, present in a considerable amount in the A. H. of gargoylism.

In hydrophthalmus, histidine, tyrosine, phenylalanine and argine are present in an almost equal amount. 4) In the samples of A. H. tested whether normal or pathologic the results

showed the presence of glucosamine, while no sugar different from glucose were seen.

5 ) The lipid research, carried out in the A. H. from the case of gargoylism resulted negative.

Hydrophthalmus

CONCLUSION

The most important data of this research can be summarized as follows: a) in the A. H. of gargoylism and of hydrophthalmus we state after hydrolysis a

quantitative and qualitative increase of proteic aminoacids in comparison to the normal A. H.

The hypothesis can be made that the hypertension, present in the two patho- logical cases, can influence the aminoacids composition of the A. H. but the fact that the quantitative amount of proteic aminoacids is considerably higher in gargoylism could retain that in this disease the occurrence of a factor not related to the hypertension, but possibly connected to the dysmetabolic condition could play an important role.

b) In the increase of proteic aminoacids of the gargoylism the presence of arginine that occupies the sixth place in the decreasing order of the eleven aminoacids is of particular importance.

c) The negativity of the lipid research in the case of gargoylism goes against the hypothesis, which is already put aside in recent literature, of a lipid the- saurismosis~~~5. On the contrary, our data agree with the actual concept of an alteration of the metabolism of the mucopolysaccharides in gargoylism.

SUMMARY

The authors have made a chromatographic study of the aqueous humor in one case of gargoylism with ocular hypertension, and for comparison, one case of congenital glaucoma and two normal cases (amblyopic eyes).

With regard to the normal cases, and after hydrolysis, the protein amino acids appeared increased qualitatively and quantitatively, both in gargoylism (with hypertension), as well as in congenital glaucoma, more markedly in the first case than in the second.

In the increase of protein amino acids, there was an especially noticeable rise in the rate of arginine. All research on fats remained negative. In each case examined, the presence of glucose and glucosamine was observed.

REFERENCES

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5. Lindsay, S., Reilly, W. A., Gotham, T. I., SRahen, R.: Anner. J. Dis. Child., 76,

6. Brante, G.: Fette u. Seif. 53, 457 (1951). 7 . Panizon, F . and Pedrini, V.: Acta paed. latina 15, 353 (1962). 8 . Moro F.: Annali ottalm. e C1. Ocul., 83, 283 (1957). 9. Dorfman A. and Lorincz A. E.: Proc. Nat. Acad. Sc., 43, 443, (1957).

10. Cifonelli /. A . and Dorfman A,: J. Biol. Chem., 235, 3283 (1960).

31, 398 (1944).

239 (1948).

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11. Prodi G. and Prodi M . P., G. Biochimica: 5, 382 (1965). 12. Essere H., Heizler F. and Pan H.: Graefes Arch. Ophth. 155, 1 1 (1954). 13. Wunderly Ch., Steiger R. and Bohringer H . R.: Experientia, 10, 432 (1954). 14. D’Ermo F.: Bull. SOC. Franc. Ophtal. 68, 66 (1955). 15. Weise H.: Docum. Ophth. 11, 311 (1957). 16. Francois J . and Rabaey: A.M.A. Arch. Ophth. 63, 836 (1960). 17 . Keulj and Steiger R.: Brit. J. Ophth. 39, 503 (1955). 18. Wunderly Ch. and Cagianut B.: Brit. J. Phth. 38, 357 (1954). 19. Ciusa W., Cristini G. and Barbiroli G.: Min. Oftal. 7, 95 (1965). 20. Stahl E.: Diinnschicht-Chromatographie, Springer-Verlag (1962). 21. Moore S., Sflackman D. H . and Stein W . H.: Anal. Chem. 30, 1185 (1958). 22. Ragazzi E., and Veronese G.: I1 Farmaco N . 3, 152 (1963). 23. Randerath K.: Thin layer Chromatography, Academic Press - New York (1963). 24. Straus, R., Merliss R. and Reiser R.: Amer. J. Clin. Path. 17, 671 (1947). 25. Henderson J . L., Macgregor A. R., Thannhauser S. J . and Holden R.: Arch. Dis.

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