Reports 301
Volume 15
Number 4
7.6, the reaction in epithelial cells was almost
absent, but at pH 8.0 to 9.0, the cells demonstrated strong reaction. On the other hand, the
optimal pH value of the peroxidase in the exorbital
lacrimal gland is 6.5. These peroxidases may differ
in their function.
I thank Mrs. H. Ueno for her help in preparing
this paper.
From the Departments of Bacteriology and
Cytochemistry, Chest Disease Research Institute,
and Department of Ophthalmology, Faculty of
Medicine, Kyoto University, Kyoto, 606, Japan.
Submitted for publication Nov. 7, 1975. Reprint
requests: Dr. Takeshi Iwata.
Key words: peroxidase, epithelium, conjunctiva,
cornea, goblet cell, cytochemistry.
REFERENCES
1. Morrison, M., and Allen, P. Z.: Lactoperoxidase: Identification and isolation from
Harderian and lacrimal glands, Science 152:
1626, 1966.
2. Essner, E.: Localization of endogenous peroxidase in rat exorbital lacrimal gland, J.
Histochem. Cytochem. 19: 216, 1971.
3. Herzog, V., and Miller, F.: The localization
of endogenous peroxidase in the lacrimal
gland of the rat during postnatal development, J. Cell Biol. 53: 662, 1972.
4. Iwata, T., Ohkawa, K., and Uyama, M.: The
fine structural localization of peroxidase activity in goblet cells of the conjunctival epithelium of rats, INVEST. OPHTHALMOL. 15:
40, 1976.
5. Graham, R. C , and Karnovsky, M. J.: The
early stages of absorption of injected horseradish peroxidase in the proximal tubules of
mouse kidney: Ultrastructural cytochemistry
by a new technique, J. Histochem. Cytochem.
14: 291, 1966.
6. Novikoff, A. B.: Visualization of peroxisomes
(microbodies) and mitochondria with diaminobenzidine, J. Histochem Cytochem. 17:
675, 1969.
7. Virchow, H.: Mikroskopische Anatomie der
au/?eren Augenhaut und des Lidapparates,
in: Graefe-Saemisch Handbuch der Gesamten
Augenheilkunde, Ed. 2. Leipzig, 1910, Wilhelm Engelman, Vol. I, Part I, Chap. 2, pp.
575-618.
8. Bindner, R.: Beitrag zur Kenntnis der
Schleimzellen in der Conjunctiva bulbi bei
Macacus rhesus, v. Graefes Archiv Ophthalmol. 153: 477, 1953.
9. Fahimi, H. D.: The fine structural localization of endogenous and exogenous peroxidase activity in Kupffer cells of rat liver, J.
Cell Biol. 47: 247, 1970.
10. Novikoff, A. B., Beard, M. E., Albala, A.,
et al.: Localization of endogenous peroxidases
in animal tissues, J. Microscopie 12: 381,
1971.
Distribution of hexos amines in bovine
cornea. FREDERICK A. BETTELHEIM AND
DENNIS GOETZ.
Excised bovine cornea were sectioned from
epithelium to endothelium into 6 to 8 fractions.
The glucosamine/galactosamine ratio steadily increases from epithelium to endothelium in all
bovine corneas investigated. The glucosamine/
galactosamine ratio reflects the keratan sulfate/
chondroitin-4-sulfate ratio in the cornea. The significance of this topographic distribution is discussed in terms of the different hydration properties of proteoglycans containing predominantly
keratan sulfate or chondroitin-4-sulfate chains.
About 60 per cent of the corneal glycosaminoglycans is keratan sulfate and the remaining 40
per cent is mainly chondroitin-4-sulfate.1 Small
amounts of dermatan sulfate and chondroitin-6sulfate2 have also been reported. It has been
alleged3 that although the glycosaminoglycans are
largely responsible for the hydration of the cornea,
the keratan sulfate plays a different role than the
chondroitin-4-sulfate in the hydration process.
For one, the corneal wound healing shows reduced keratan sulfate and increased chondroitin4-sulfate synthesis and injury to the Descemet
membrane and endothelium transforms keratccytes to dermatan sulfate-producing cells.4 Furthermore, non-swelling shark cornea contains little keratan sulfate and considerable amount of
chondroitin-4-sulfate.5 The water vapor absorption
properties of these two glycosaminoglycans are
also quite different. While keratan sulfate absorbs large amounts of water, it retains very
little in the dehydration process6; the reverse is
true for chondroitin-4-sulfate.7
However, the glycosaminoglycans in the cornea
are in the form of proteoglycans. These are
heterogenous macromolecules carrying keratan
sulfate and chondroitin-4-sulfate in different proportion. In our laboratory we isolated from cornea
one proteoglycan with predominantly keratan sulfate and one with predominantly chondroitin-4sulfate side chains.8
In characterizing the proteoglycans we also
found that the water absorption and retention
power of these proteoglycans were different, reflecting the behavior of the side chains. If the
different proteoglycans differ so much in their
hydration properties, it is possible that there is
some specific distribution in the cornea reflecting
these roles. Anseth1 reported that there was no
difference in the distribution of glycosaminoglycans in the central or peripheral parts of the
cornea. The glucosamine/galactosamine ratio
"seemed to be higher in the anterior than in the
posterior part. This difference, however, was not
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302 Reports
Investigative Ophthalmology
April 1976
52
48
44
FRACTION
No.
Fig. 1. Separation of glucosamine and galactosamine in Section 2 of cornea A. The total
hexosamine content was 3.26 mg. per gram wet weight or 14.8 mg. per gram dry weight.
22 h
18
1.648
1.863
1.977
1.904
1.935
2.240
1.0
0.2
0.4
Fig. 2. Histograms of glucosamine/galactosamine ratio as a function of distance from the
anterior surface in four bovine corneas.
significant." On the other hand, Borcherding and
co-workers9 found that in human cornea the
keratan sulfate content decreases steadily proceeding from the center to the periphery. They
attribute this specific distribution to the collagen
fiber organizing ability of acidic glycosaminoglycans. In view of the data on the role of proteoglycans in the hydration of the cornea,8 it was
deemed necessary to analyze the topographic distribution of proteoglycans from epithelium (anterior) to endothelium (posterior).
It was decided to use the techniques of
Antonopoulos10 to determine quantitatively glucosamine and galactosamine on a microgram scale.
Bovine eyes (two years old) were obtained from
a slaughterhouse less than 12 hours postmortem.
Corneal epithelium and endothelium were removed by scraping with a scalpel. Two incisions
were made in the cornea so that the whole cornea
could be flattened on a planchet and sectioned
parallel to the endothelium. The cornea was
sectioned in a frozen state by microtome and the
wet weight of each section was determined. The
sections were hydrolyzed for eight hours in sealed
Pyrex tubes in 6 N HC1. After hydrolysis the
samples were evaporated to dryness by opening
the tubes and placing them in a desiccator over
NaOH pellets. The residues were taken up with
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GLUCOSAMINE/GALACTOSAMINE
GLUCOSAMINE /GALACTOSAMINE
GLUCOSAMINE/GALACTOSAMINE
O
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CD
Investigative Ophthalmology
April 1976
304 Reports
0.1 ml. H2O and chromatographed on a Dowex
50 by 8 250 mesh column under air pressure of
100 torr. The material was eluted by 0.3 N HC1
and the effluent was collected in 0.25 ml. fractions in a fraction collector. The amino sugars
were analyzed after acetylation with acetylacetone
and using p-dimethylaminobenzaldehyde. The
chromophore absorbance was read at 535 nm.
Fig. 1 shows the separation of glucosamine
and galactosamine with this method. The glucosamine/galactosamine ratios are calculated from
such graphs. These ratios are presented in a
histogram form in Fig. 2 for four corneas.
All corneas investigated showed the same trend,
namely, there was a gradual increase in the
glucosamine/galactosamine ratio when one proceeds from the epithelium to the endothelium of
the cornea. No particular trend was observed regarding the total hexosamine content.
The glucosamine/galactosamine ratio reflects
the keratan sulfate/chondroitin-4-sulfate ratio in
the cornea thus implying that relatively more
chondroitin-4-sulfate chains bearing proteoglycans
are in the anterior part of the cornea than in
the posterior part. If proteoglycans play a vital
role in the transparency of cornea by maintaining
the proper hydration then such a distribution
makes sense. We have shown that proteoglycans
bearing predominantly keratan sulfate side chains
absorb two to three times as much water as
proteoglycans with moderate or high chondroitin4-sulfate content.s Proteoglycans with predominantly keratan sulfate side chains make up about
25 per cent of the total proteoglycans in the
cornea. According to the topographic distribution
presented in Fig. 2, these keratan sulfate proteoglycans are concentrated near the posterior of
the cornea near the endothelium. The proteoglycans not only absorb water to a great extent
but also transfer them with ease since little is
retained by them in the dehydration process.8
Thus, their positioning near the endothelium
hence the near aqueous humor serves the purpose
of facilitating the movement of water into the
cornea. In essence, we propose that the "active
pump" mechanism of the membranes is reinforced with a thermodynamic concentration gradient of the proteoglycans that sets up a hydration gradient from corneal endothelium to epithelium.
Correspondingly, proteoglycans with predominantly chondroitin-4-sulfate side chains or even
with moderate chondroitin-4-sulfate content (3
keratan sulfate chains/chrondroitin-4-sulfate chain)
are positioned more conceitedly near the epithelium. These proteoglycans have lesser capacity
to absorb water than the keratan sulfate proteoglycans but on the other hand, once they are
solvated they retain water eight to nine times
better in the molecular matrices than predom-
inantly keratan sulfate proteoglycans. Thus, their
positioning near the epithelium enhances the
proper hydration by preventing water loss through
dehydration.
We thank Adriel Bettelheim for his help in the
preliminary part of this study and Dr. Howard
Grob for the use of the microtome.
From the Chemistry Department, Adelphi University, Garden City, N. Y. 11530. This research
was supported by a research grant of the National
Eye Institute EY 00501-07 United States Public
Health Service. Submitted for publication Nov.
1, 1975.
Key words: bovine cornea, galactosamine, glucosamine, hydration, proteoglycans.
REFERENCES
1. Anseth, A.: Studies of corneal polysaccharides.
III. Topographic and comparative biochemistry, Exp. Eye Res. 1: 106, 1961.
2. Handley, C. J., and Phelps, C. F.: The biosynthesis in vitro of keratan sulphate in
bovine cornea, Biochem. J. 128: 205, 1972.
3. Hedbys, B. O.: The role of polysaccharides
in corneal swelling, Exp. Eye Res. 1: 81,
1961.
4. Anseth, A., and Fransson, L.: Studies on
corneal polysaccharides. VI. Isolation of
dermatan sulfate from corneal scar tissue,
Exp. Eye Res. 8: 302, 1969.
5. Praus, R., and Goldman, J. N.: Glycosaminoglycans in the nonswelling corneal
stroma of dogfish shark, INVEST. OPHTHALMOL. 9: 131, 1970.
6. Plessy, B., and Bettelheim, F. A.: Water
vapor sorption of keratan sulfate, Mol. Cell.
Biochem. 6: 85, 1975.
7. Bettelheim, F. A., and Ehrlich, S. H.: Water
vapor sorption of mucopolysaccharides, J.
Phys. Chem. 67: 1948, 1963.
8. Bettelheim, F. A., and Plessy, B.: The hydration of proteoglycans of bovine cornea,
Biochim. Biophys. Acta 381: 203, 1975.
9. Borcherding, M. S., Blacik, L. J., Sittig, R.
A., et al.: Proteoglycans and collagen fiber
organization in human corneoscleral tissue,
Exp. Eye Res. 21: 59, 1975.
10. Antonopoulos, C. A.: Separation of glucosamine and galactosamine on the microgram
scale and their quantitative determination,
Ark. Kemi 25: 243, 1966.
Corneal membrane water permeability as
a function of temperature. KEITH GREEN
AND SUSAN J. DOWNS.
Osmotically driven water flow across the corneal epithelium and endothelium has been measured as a function of temperature. For both
membranes deviations from a single straight-line
relationship are found in a logarithmic plot of
hydraulic conductivity against 1/T. Both mem-
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