Full text of DOI:10.1007/s00417-002-0586-4

Graefe’s Arch Clin Exp Ophthalmol
2002) 240:1011–1021
(
L A B O R AT O RY I N V E S T I G AT I O N
DOI 10.1007/s00417-002-0586-4
Suketaka Kanazawa
Takashi Kitaoka
Yoshiko Ueda
Interaction of zinc and vitamin A
on the ocular surface
Huaqing Gong
Tsugio Amemiya
Abstract Purpose: The purpose of
this study was to assess morphologi- in the epithelium. The corneas and
cally the interaction between zinc
and vitamin A in their effect on the
ocular surface. Methods: Three-
week-old Wistar Kyoto rats were
decrease in the number of microvilli
Received: 17 July 2002
Revised: 30 September 2002
Accepted: 9 October 2002
conjunctivas of group A(–) Zn(+)
had a decrease in microvilli but more
microvilli than those of group A(–).
The corneas of group A(–) showed
Published online: 19 November 2002
©
Springer-Verlag 2002
divided into five groups: group A(+) keratinization, but those of group
Zn(+) was fed a diet containing both A(–) Zn(+) showed no keratiniza-
vitamin A and Zn; group A(–) was
tion. In group Zn(–) the microvilli
fed a vitamin A-deficient diet; group on the conjunctiva and cornea were
A(–) Zn(+) was given a vitamin
A-deficient diet and deionized dis-
tilled water with Zn; group Zn(–)
and group Zn(–) A(+) were fed a
Zn-deficient diet, whereby group
Zn(–) A(+) received an intraperito-
neal injection of vitamin A. Corneas Conclusion: These results suggest
and conjunctivas of each group were that a synergistic interaction exists
examined by electron microscopy.
Results: The corneas and conjuncti-
vas of group A(–) demonstrated a
sparse. Group Zn(–) A(+) had a
decrease in microvilli but more
microvilli than group Zn(–). The
conjunctivas of group Zn(–) A(+)
had more goblet cells with granules
than did those of group Zn(–).
S. Kanazawa ( ) · T. Kitaoka · Y. Ueda
✉
H. Gong · T. Amemiya
Department of Ophthalmology
and Visual Sciences,
Graduate School of Biomedical Sciences,
Nagasaki University, 1-7-1 Sakamoto,
Nagasaki 852-8501, Japan
e-mail:
Suketaka.Kanazawa@mb6.seikyou. ne.jp
Tel.: +81-95-8497345
Fax: +81-95-8497347
between vitamin A and zinc for the
maintenance of the corneal and con-
junctival epithelium.
vitamin A metabolism is manifest at many levels. Zn is
involved in alcohol dehydrogenase, which turns circulat-
Introduction
Vitamin A has many actions which are very important ing vitamin A (retinol) into active vitamin A (retinal)
for epithelial development [3], light reception [16, 33, [22, 30, 31].
3
7], skin maintenance, growth, immunity [1], anti-cancer
effect [24], hair development [20, 25], wound healing when patients have been maintained on total parenteral
12] etc. Vitamin A is necessary for normal growth and nutrition with highly purified solutions lacking adequate
Zn deficiency has been encountered sporadically
[
differentiation of epithelium; keratinization is a direct Zn [14, 28, 29]. Zn is thought to be essential for the optic
consequence of its deficiency [23]. Vitamin A deficiency nerve fibers, because papillitis, optic nerve atrophy and
remains an important cause of ocular morbidity among optic neuropathy occur under Zn-deficient conditions
patients with chronic liver disease [11, 21] and is a major [34, 38]. Zn-deficient patients complain of foreign body
cause of childhood blindness in developing countries sensation, photophobia, tearing and abnormal dark adap-
[
10, 27].
tation. Except for abnormal adaptation, these complaints
It is known that zinc is essential for the normal me- are due to ocular surface disturbances. Vitamin A defi-
tabolism of collagen [2], synthesis of DNA [36] and pro- ciency is well known to induce keratomalacia, corneal
tein and promotion of wound healing [25]. Its effect on epithelial keratinization and night blindness. Thus com-

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012
plaints of patients with vitamin A deficiency are very of retinal was measured by high-performance liquid chromatog-
raphy.
similar to those of Zn-deficient patients. The pathologi-
cal findings can easily explain the reasons for the com-
plaints due to ocular surface damage: decrease in micro-
villi and microplicae in the most superficial epithelium
of the cornea and conjunctiva and decrease in goblet
cells. These pathological abnormalities are directly relat-
ed to tearing, foreign body sensation, photophobia and
Data were analyzed by unpaired Fisher’s t-test with significant
difference. Differences were considered significant at P<0.05. All
values represent mean±SD.
Electron microscopy
Following enucleation under pentobarbital anesthesia, corneas and
dry eyes and can be easily observed by scanning and palpebral conjunctivas were excised and fixed with 4% glutaralde-
hyde in 0.05 M cacodylate buffer for 1 h and postfixed in 1%
transmission electron microscopy. Biochemistry suggests
an interaction between vitamin A and Zn, but it is diffi-
cult to prove this interaction morphologically. It would
be very interesting to know whether the effect of either
of the two nutrient deficiencies can be prevented by sup-
osmium tetroxide in veronal acetate buffer for 1 h after an over-
night washing with 0.05 M cacodylate buffer containing 0.44 M
sucrose.
Scanning electron microscopy
plementation of the other nutrient. We paid special atten-
tion to the similar abnormalities in deficiencies of vita-
min A and Zn and easily obtained data with observable
procedures. The purpose of this study is to assess the being treated in a Hitachi HCP2 (Tokyo, Japan) critical point dry-
interaction between Zn and vitamin A in their effect on
the ocular surface morphologically. For this purpose
we devised an experiment to demonstrate the effect of
vitamin A on the conjunctival and corneal epithelium of graphs at a magnification of 600. The ratio of light cells to light
Zn-deficient rats and the effect of Zn on that of vitamin
The fixed materials were dehydrated through a series of ethanols.
Whole corneas and conjunctivas were placed in amyl acetate after
ing apparatus and then shadowed with gold in an Eicho ion coater
and examined with a Hitachi S2360 scanning electron microscope
2
(
Tokyo, Japan). The light and dark cells per 36,000-µm field in
the cornea and conjunctiva of each group were counted in micro-
cells plus dark cells was calculated for each group. The number of
2
goblet cells in a 30,000-µm field of the conjunctiva of each group
A-deficient rats.
was counted in micrographs at a magnification of 300.
Transmission electron microscopy
Materials and methods
The central parts of other corneas and conjunctivas were cut into
Three-week-old normal weanling Wistar Kyoto rats were divided
small pieces. After dehydration with a series of ethanols following
randomly into five groups:
fixation with 4% glutaraldehyde and 1% osmium tetroxide, the
●
Group A(+) Zn(+) was fed a solid diet containing both vitamin specimens were embedded in Luveak 812. Ultrathin sections were
cut with a Porter-Blum MT2 microtome, stained with uranyl ace-
A and Zn. Deionized distilled water was provided freely.
●
Group A(–) and group A(–) Zn(+) were fed a commercially tate and lead citrate and examined with Hitachi H300 (Tokyo,
Japan) and JEOL JEM-1210 (Tokyo, Japan) transmission electron
microscopes.
prepared vitamin A-deficient solid diet obtained from Nippon
Clea (Tokyo, Japan). This diet contains less than 0.01 mg of
retinol per 100 g of diet and all other nutrients. Deionized dis-
tilled water was provided freely to group A(–) rats, while the
A(–) Zn(+) group was given deionized distilled water with
Results
3
0 mg Zn/100 g.
●
Group Zn(–) and group Zn(–) A(+) were fed a commercially
prepared Zn-deficient solid diet and all other nutrients obtained Overt characteristics
from Nippon Clea. This diet contains 0.07 mg Zn/100 g of
diet. Deionized distilled water was provided freely to both The bodyweights of rats in groups A(–) and A(–) Zn(+)
groups. Group Zn(–) A(+) rats received an intraperitoneal in-
were lower than those of group A(+) Zn(+) rats. The dif-
ference was significant between groups A(–) and A(+)
Zn(+) and between groups A(–) Zn(+) and A(+) Zn(+)
jection of vitamin A (3000 IU retinol palmitate) once a week
from 3 to 5 weeks after birth.
The animals were treated according to the ARVO Statement for
the Use of Animals in Ophthalmic and Vision Research. All rats
were maintained at a constant room temperature of 21°C and 12 h
(
P<0.05). However, there was no significant difference
in bodyweight between group A(–) and group A(–)
light/12 h dark cycles under normal room conditions. Corneas and Zn(+) (Table 1). Group A(+) Zn(+) rats were in normal
palpebral conjunctivas from rats of groups A(–), A(–) Zn(+) and health with no hair loss. Groups A(–) and A(–) Zn(+)
A(+) Zn(+)were examined in the 12th and 16th weeks of the ex-
had coarse hair and hair loss starting at 3 months of the
experiment.
periments. Corneas and palpebral conjunctivas from group Zn(–),
group Zn(–) A(+) and group A(+) Zn(+)were examined in the 1st,
2
nd and 3rd weeks of the experiments. Zn-deficient rats rarely live
The bodyweights of rats in groups Zn(–) and Zn(–)
more than 4 weeks on a Zn-deficient diet, and signs of deficiency A(+)were lower than those of group A(+) Zn(+) rats.
appear rapidly and are severe. The changes in vitamin A deficien-
There was a significant difference between groups Zn(–)
cy, however, occur more slowly. Therefore the time of examina-
and A(+) Zn(+) and between groups Zn(–) A(+) and
tion differs between the Zn and vitamin A deficiency experiments.
Plasma immediately before the enucleation was analyzed for Zn
content by atomic absorption spectrophotometry. The serum level difference between groups Zn(–) and Zn(–) A(+)
A(+) Zn(+) (P<0.05); however, there was no significant

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Table 1 Bodyweight, serum vitamin A levels and serum zinc levels of rats in the five experimental groups. The number of animals in
each group was five
Group
Weeks after start of experiment
1
3
12
16
Bodyweight (g)
Group A(+) Zn(+)
Group A(–)
Group A(–) Zn(+)
Group Zn(–)
Group Zn(–) A(+)
77.0±2.6a
110±2.0a
–
350±7.07c
356±11.4c
c, d
c, d
–
–
288±14.8
298±13.0
c, d
c, d
–
320±25.5
302±19.2
a, b
a, b
a, b
51.3±1.1
51.0±1.0
56.0±1.0
55.7±1.1
–
–
–
–
a, b
Serum vitamin A level (ng/ml)
Group A(+) Zn(+)
Group A(–)
Group A(–) Zn(+)
Group Zn(–)
Group Zn(–) A(+)
392±15.0E
360±37.4e
403±14.9
399±10.3
–
–
–
–
<30*
<30*
–
<30*
<30*
–
e, f
e, f
216±25.4
227±32.6
194±10.1
208±17.8
e, f
e, f
–
–
Serum Zn level (ng/ml)
Group A(+) Zn(+)
Group A(–)
Group A(–) Zn(+)
Group Zn(–)
Group Zn(–) A(+)
106.5±7.9g
113.6±7.9g
109±7.0i
i, j
106±3.8
i
i
–
–
–
–
85±5.0
80±3.3
i, j
i, j
106±4.2
–
–
100±3.6
–
–
g, h
g, h
45.8±1.7
39.3±1.7
39.8±2.2
g, h
g, h
42.0±1.8
d
*
The serum vitamin A levels in group A(–) and group A(–) Zn(+)
There is no significant difference between group A(–) and group
A(–) Zn(+)
are lower than 30 ng/ml, the limit of assay
a, e, g
i
There is a significant difference between group Zn(–) and
The difference is significant between group A(–) and group A(–)
group A(+) Zn(+) (P<0.05) and between group Zn(–) A(+) and Zn(+) (P<0.05) and between group A(–) and group A(+) Zn(+)
group A(+) Zn(+) (P<0.05)
(P<0.05)
b, f, h
j
There is no significant difference between group Zn(–) and
There is no significant difference between group A(+) Zn(+) and
group Zn(–) A(+)
group A(–) Zn(+)
c
The difference is significant between group A(–) and group A(+)
Zn(+) (P<0.05) and between group A(–) Zn(+) and group A(+)
Zn(+) (P<0.05)
(
Table 1). Groups Zn(–) and Zn(–) A(+) had coarse hair
Groups Zn(–) and Zn(–) A(+) had significantly lower
serum Zn levels than did group A(+) Zn(+) (P<0.05)
and hair loss starting in the 2nd week of the experiment.
(
Table 1).
Serum vitamin A levels
Scanning electron microscopy findings
in the conjunctiva
In the 12th and 16th weeks of the experiments, the serum
vitamin A level was significantly lower in group A(–)
and group A(–) Zn(+) than in group A(+) Zn(+). The
serum vitamin A level in groups A(–) and A(–) Zn(+)
was lower than 30 ng/ml, the limit of assay of serum
vitamin A (Table 1).
Serum vitamin A levels were lower in group Zn(–)
and group Zn(–) A(+) than in group A(+) Zn(+). There
was a significant difference in serum vitamin A levels
between groups A(–) and A(–) Zn(+) and group A(+)
Zn(+) (P<0.05) (Table 1).
In group A(+) Zn(+), microvilli and microplicae were
numerous and well developed in the most superficial
conjunctival epithelium and goblet cells with well-devel-
oped granules were seen frequently in the epithelium
(
Fig. 1A). On the other hand, in group A(–) the surface
of the conjunctiva showed no microvilli and microplicae
but keratinization and desquamation of the superficial
epithelium (Fig. 1B). There were no goblet cells in the
conjunctiva of group A(–) rats. Conjunctival microvilli
and microplicae of group A(–) Zn(+) rats were fewer
than in group A(+) Zn(+), but more numerous than in
group A(–). In group A(–) Zn(+) rats, there were no gob-
Serum zinc levels
The serum Zn level was significantly lower in group let cells in the conjunctiva (Fig. 1C). Group A(–) Zn(+)
A(–) than in groups A(+) Zn(+) and A(–) Zn(+) (P<0.05) rats had more dark cells in the conjunctiva than did
(
Table 1).
group A(+) Zn(+) rats, and the ratio of light cells to light

1
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Fig. 1 SEM pictures of the conjunctival epithelium of rats from
group A(+) Zn(+) (A), A(–) (B), A(–) Zn(+) (C), Zn(–) (D) and
Zn(–) A(+) (E). A In a group A(+) Zn(+) rat, numerous fine
microvilli are seen in the conjunctival epithelium. There are many
goblet cells with well-developed granules (arrows). B The con-
junctiva of a group A(–) rat demonstrates almost no microvilli, but
keratinization (arrows) is present in most superficial epithelial
cells. C The conjunctiva of a group A(–) Zn(+) rat has light cells
with microvilli (asterisks) but no goblet cells. The conjunctiva in
both group A(–) and group A(–) Zn(+) shows complete absence of
goblet cells. D In a group Zn(–) rat, the superficial epithelium of
the conjunctiva shows decrease in microvilli. The goblet cells
show hole-like structures of granules (arrows). E The conjunctiva
of a group Zn(–) A(+) rat has goblet cells with granules (arrows).
The goblet cells of group Zn(–) A(+) have more granules than do
those of group Zn(–). The goblet cells of group Zn(–) A(+) have
fewer granules than do those of group A(+) Zn(+). Bar 10 µm
(
A–E)

1
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Fig. 2 SEM pictures of corneal epithelium of rats from group
A(+) Zn(+) (A), A(–) (B), A(–) Zn(+) (C), Zn(–) (D) and Zn(–)
A(+) (E). A There are many light cells (asterisks) in the most
superficial corneal epithelium of a group A(+) Zn(+) rat. These
have a high density of microvilli and microplicae. B The corneal
epithelium of a group A(–) rat shows absence of microvilli. Kera-
tinization (asterisks) is observed in a group A(–) rat. C The super-
ficial corneal epithelium of a group A(–) Zn(+) rat has a lower
number of light cells (asterisks). The cornea of a group A(–)
Zn(+) rat shows no keratinization. D In a group Zn(–) rat, the most
superficial corneal epithelium shows many dark cells with scarce
microvilli (asterisks). E In a group Zn(–) A(+) rat, the microvilli
and microplicae in the epithelium are less numerous than in a
group Zn(+) A(+) rat, but more numerous (asterisks) than in a
group Zn(–) rat. Bar 10 µm (A–E)

1
016
and dark cells was significantly higher in group A(+) Table 2 Ratio of light cells to light and dark cells in the conjuncti-
vas and the corneas of groups A(+) Zn(+), group A(–), group A(–)
Zn(+) than in group A(–) Zn(+) (P<0.05) (Table 2).
However, the ratio in group A(–) could not be calculated
because of extensive keratinization of the epithelial cells.
Zn(+), group Zn(–) and group Zn(–) A(+) (n number of fields
examined)
The conjunctivas of group Zn(–) demonstrated a de- Group
crease in microvilli in the superficial epithelial cells,
Conjunctiva (n=12)
Cornea (n=16)
Group A(+) Zn(+)
0.558±0.095a
0.645±0.059
0.447±0.098
b
while the conjunctivas of group Zn(–) A(+) had a certain
decrease in microvilli but displayed more microvilli than
did the conjunctivas of group Zn(–) rats. In the conjunc-
tivas of group Zn(–) rats, goblet cells were decreased.
The conjunctivas of group Zn(–) A(+) had more goblet
cells than did those of group Zn(–) rats. However, the
conjunctivas of group Zn(–) A(+) rats had less goblet
cells than did those in group A(+) Zn(+). The number of
goblet cells in the conjunctiva was significantly higher in
rats of group A(+) Zn(+) than in group Zn(–) A(+)
b
Group A(–)
Group A(–) Zn(+)
*
0.486±0.095a
b
0.529±0.085
Group A(+) Zn(+)
Group Zn(–)
Group Zn(–) A(+)
0.588±0.059c
0.675±0.089
0.465±0.059
0.577±0.054
d
d
d
c
0.417±0.05
0.473±0.07c
*
The ratio in group A(–) could not be calculated because of ex-
tensive keratinization of the epithelium
a
There is a significant difference between group A(+) Zn(+) and
group A(–) Zn(+) (P<0.05)
b
The difference is significant between group A(+) Zn(+) and
(
P<0.05), and the difference between group Zn(–) A(+) group A(–) Zn(+) (P<0.05) and between group A(–) Zn(+) and
group A(–) (P<0.05)
and group Zn(–) was also significant (P<0.01) (Table 3).
c
There is asignificant difference between group A(+) Zn(+) and
The goblet cells of group Zn(–) rats showed hole-like
group Zn(–) A(+) (P<0.001) and between group Zn(–) A(+) and
structures of granules (Fig. 1D). The granules of goblet group Zn(–) (P<0.01)
d
cells of rats in group Zn(–) A(+) (Fig. 1E) were smaller
and more shrunken than those of group A(+) Zn(+) rats.
However, the goblet cells of group Zn(–) A(+) had more
granules than did those of group Zn(–). The ratio of
light cells to light and dark cells in the conjunctiva was
There is a significant difference between group A(+) Zn(+) and
group Zn(–) A(+) (P<0.01) and between group Zn(–) A(+) and
group Zn(–) (P<0.05)
significantly higher in group A(+) Zn(+) than in group Table 3 Number of goblet cells in the conjunctiva. The number of
A(–) Zn(+) (P<0.001), and the difference between group fields examined in each group was ten
Zn(–) and group Zn(–) A(+) was also significant
4
2
Group
Number of goblet cells/3×10 µm
(
P<0.01) (Table 2).
Group A(+) Zn(+)
Group A(–)
Group A(–) Zn(+)
Group Zn(–)
Group Zn(–) A(+)
17.9±1.287a
*
*
Scanning electron microscopy findings in the cornea
There were many light cells on the corneal epithelium of
a
12.1±1.912
15.5±2.461a
group A(+) Zn(+) rats, which had a high density of * The number of goblet cells in groups A(–) and A(–) Zn(+) could
microvilli and microplicae in the most superficial epithe- not be counted because of epithelial damage
a
There is a significant difference between group A(+) Zn(+) and
lial cells (Fig. 2A). The corneal epithelium in groups
A(–) and A(–) Zn(+) rats showed more dark cells than
did that in group A(+) Zn(+) rats. In group A(–) rats
group Zn(–) A(+) (P<0.05) and between group Zn(–) A(+) and
group Zn(–) (P<0.01)
(
Fig. 2B) there were more dark cells in the epithelium
than in group A(–) Zn(+) rats (Fig. 2C). In group A(–)
rats microvilli were absent in the most superficial epithe-
The corneas of group Zn(–) rats demonstrated ab-
lial cells of the cornea (Fig. 2B). The density of micro- sence of microvilli in the most superficial epithelial cells
villi and microplicae was lower in the light cells of (Fig. 2D). The density of microvilli and microplicae in
group A(–) rats than in those of group A(+) Zn(+) rats. the light cells of group Zn(–) rats was lower than in
On the other hand, the corneas of group A(–) Zn(+) rats those of group A(+) Zn(+) rats, while the corneas of
(
Fig. 2C) had a certain decrease in microvilli but exhibit- group Zn(–) A(+) rats (Fig. 2E) had a certain decrease in
ed more microvilli than did the corneas of group A(–) microvilli but showed more microvilli than those of
rats (Fig. 2B). The corneas in group A(–) showed kerati- group Zn(–) (Fig. 2D).
nization (Fig. 2B), but those in group A(–) Zn(+) showed
In the corneas of groups Zn(–) and Zn(–) A(+), dark
no keratinization (Fig. 2C). The ratio of light cells to cells were more numerous on the surface than in those
light and dark cells in the cornea was significantly higher of group A(+) Zn(+). The ratio of light cells to light
in group A(+) Zn(+) than in group A(–) and group A(–) and dark cells in group Zn(–) was significantly different
Zn(+) (P<0.01), and the difference between group A(–) from that in group A(+) Zn(+) and group Zn(–) A(+)
and group A(–) Zn(+) was also significant (P<0.05) (P<0.01). There was a significant difference between
(
Table 2).
group Zn(–) and group Zn(–) A(+) (P<0.05) (Table 2).

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Fig. 3 TEM pictures of conjunctival epithelium of rats from
group A(+) Zn(+) (A), A(–) (B), A(–) Zn(+) (C), Zn(–) (D) and
Zn(–) A(+) (E). A In a group A(+) Zn(+) rat, there are fine micro-
villi and goblet cells with well-developed granules (asterisks).
B A group A(–) rat demonstrates keratinization (arrows). Many
keratohyalin granules are seen in the epithelial cytoplasm. There
are no microvilli and goblet cells in group A(–) rats. C A group
A(–) Zn(+) rat has a few microvilli (arrow) but no goblet cells.
Keratinization is not observed. D In the group Zn(–) rats, granules
of goblet cells were hard to find (asterisks). E The conjunctiva of
a group Zn(–) A(+) has goblet cells with granules (asterisks). No
keratinization is observed in the conjunctivas of group Zn(–) and
Zn(–) A(+) rats. Bar 2 µm (A–E)

1
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Fig. 4 TEM pictures of corneal epithelium of rats from group
A(+) Zn(+) (A), A(–) (B), A(–) Zn(+) (C), Zn(–) (D) and Zn(–)
A(+) (E). A A group A(+) Zn(+) rat has many microvilli (arrows)
in the superficial epithelial cells of the cornea. B In the cornea of a
group A(–) rat, keratinization (arrows) is present and microvilli
and microplicae are not observed in the epithelial cells. C Group
A(–) Zn(+) rats have a slight decrease in the number of microvilli
(
arrows) in the superficial epithelial cells. However, there is no
keratinization in the cornea. D In a group Zn(–) rat, the cornea has
few microvilli (arrow) in the superficial epithelial cells. E The
cornea of a group Zn(–) A(+) rat shows some decrease in micro-
villi. However, the corneal epithelium of the group Zn(–) A(+) rat
has more microvilli (arrows) than that of the group Zn(–) rat. No
keratinization is found in the corneas of group Zn(–) and group
Zn(–) A(+) rats. Bar 2 µm (A–E)

1
019
Transmission electron microscopy findings
in the conjunctiva
Discussion
Many reports on interaction between vitamin A and Zn
In group A(+) Zn(+) rats, there were fine microvilli and at various levels have been published [8, 32]. With re-
goblet cells with well-developed secretory granules on gard to the biochemical level, previous studies have sug-
the conjunctival epithelium (Fig. 3A). Group A(–) rats gested that there are two common mechanisms to explain
demonstrated almost no microvilli and microplicae, but the potential dependence of vitamin A metabolism on
showed keratinization and epithelial cells containing Zn. One relates to the obligatory role of Zn on the syn-
keratohyalin granules. There were no goblet cells in thesis of its carrier protein, retinol-binding protein
group A(–) rats (Fig. 3B). Group A(–) Zn(+) rats had a (RBP). Zn maintains the serum vitamin A level and
few microvilli but no goblet cells (Fig. 3C).
plays a role in the release of vitamin A from the liver, by
In groups Zn(–) and Zn(–) A(+) rats, the conjunctivas influencing either the synthesis of RBP or the release of
demonstrated a decrease in microvilli in the superficial the RBP–vitamin A complex from the liver. Zn deficien-
epithelial cells. The conjunctivas of group Zn(–) A(+) cy can depress the synthesis of RBP in the liver and lead
had some decrease in microvilli but more microvilli than to a lower concentration of RBP in the plasma [22, 30,
did those of group Zn(–). In the group Zn(–) rats, gran- 31]. The other postulated mechanism for the interaction
ules of goblet cells were hard to find, buried under the between Zn and vitamin A is explained by alcohol dehy-
superficial epithelial cells (Fig. 3D). The conjunctivas of drogenase (ADH). Zn is necessary for the oxidative con-
group Zn(–) A(+) rats had more goblet cells with gran- version of retinol to retinaldehyde (retinal). ADH activi-
ules than did those of group Zn(–) rats (Fig. 3E). No epi- ty declines significantly and retinal oxidase activity in-
thelial cells exhibiting keratinization or containing kera- creases significantly in Zn deficiency [15].
tohyalin granules were seen in the conjunctivas of group
Zn(–) and group Zn(–) A(+) rats.
In the present study, we noted a decrease in the plas-
ma vitamin A concentration in Zn-deficient rats. Earlier
reviews [5] reporting responses of vitamin A concentra-
tions in plasma and liver to experimental Zn deficiency
Transmission electron microscopy findings in the cornea have shown inconclusive evidence of an interaction be-
tween vitamin A and Zn, mainly because of inadequate
Group A(+) Zn(+) rats had a high density of microvilli control of the secondary effects of food and growth re-
and microplicae in the superficial epithelial cells of the striction in Zn deficiency in most animal studies. While
cornea (Fig. 4A). In the cornea of group A(–) rats, kera- in the rats with inadequate dietary Zn no change oc-
tinization was present and microvilli and microplicae curred in liver Zn and ADH levels, the retina was more
were not observed in the most superficial epithelial cells sensitive to lack of dietary Zn [15]. Newly absorbed vi-
(
Fig. 4B). In the corneas of group A(–) rats, epithelial tamin A is mobilized and utilized in preference to that
cells contained keratohyalin granules and thick dense previously stored in the liver in Zn-deficient rats [6]. In-
membranes, indicating keratinization. Intercellular spac- jection of vitamin A in the group Zn(–) A(+) rats may be
es were wide, but in spite of decreased desmosomes epi- useful for preventing corneal and conjunctival change,
thelial cells appeared as light cells because of decreased because despite the fact that there was no significant dif-
fibrils in the cytoplasm. Group A(–) Zn(+) rats had a ference in the plasma vitamin A and Zn levels between
slight decrease in microvilli in the superficial epithelial groups Zn(–) and Zn(–) A(+) in the present study, group
cells. The density of the corneal epithelial cell layer in Zn(–) A(+) rats showed some improvement in the ocular
group A(–) Zn(+) rats was less than that in group A(+) surface.
Zn(+), but more than that in group A(–) rats because of a
Although the corneas in group A(–) rats showed de-
slight increase in fibrils in the cytoplasm, although inter- creased microvilli and keratinization, group A(–) Zn(+)
cellular spaces were present. However, there was no rats showed a slight decrease in microvilli but no kerati-
keratinization in the cornea of group A(–) Zn(+) rats nization. These results may be related to vitamin A me-
(
Fig. 4C).
tabolism. Zn deficiency may reduce ADH activity, there-
In group Zn(–) rats, the cornea had few microvilli and by disturbing vitamin A metabolism. Thus, in vitamin A
microplicae in the superficial epithelial cell (Fig. 4D). deficiency, excessive Zn may promote vitamin A metab-
The corneas of group Zn(–) A(+) rats (Fig. 4E) showed olism by stimulating ADH activity to release liver stores
some decrease in microvilli. However, the corneal epi- of vitamin A. The replenishing of the Zn in the group
thelium of group Zn(–) A(+) rats had more microvilli A(–) Zn(+) would cause the mobilization of remaining
and microplicae than did those of group Zn(–) rats. No liver vitamin A, some of which would be used for pre-
keratinization was found in the corneas of group Zn(–) venting corneal and conjunctival change. The decreased
and group Zn(–) A(+) rats.
plasma Zn concentrations in group A(–) rats may impair
the corneal wound healing effect of Zn. Supplementation
of Zn may accelerate wound healing or delay the onset

1
020
of corneal and conjunctival changes due to vitamin A cytology decreased significantly in the groups receiving
deficiency in group A(–) Zn(+).
Zn, vitamin A, and Zn plus vitamin A.
Recent studies at the cellular level showed that the
The present study showed that decrease and loss of
effects of retinoic acid, the biologically active form of microvilli in the superficial layer of the corneal and con-
vitamin A in cells, are mediated by members of the junctival epithelium could be prevented by vitamin A in
superfamily of nuclear receptors. The retinoic acid re- Zn deficiency and by Zn in vitamin A deficiency.
ceptors (RARs) and the retinoid X receptors (RXRs) Although the effect of Zn is incomplete, it is surprising
bind to retinoid response elements on DNA, thereby that Zn could prevent keratinization of the corneal and
controlling gene transcription [13, 26]. Bossenbroek et conjunctival epithelium in vitamin A deficiency. TEM
al. [4] demonstrated the expression of RARs and RXRs also showed rather good, though incomplete, mainte-
in the rabbit cornea and conjunctiva. The DNA-binding nance of the cytoplasmic organelles with supplementa-
domain of the RAR which contains zinc finger is sus- tion of Zn or vitamin A.
ceptible to intracellularly relevant oxidizing agents [7].
In Zn deficiency excessive vitamin A may accelerate
Zn plays a functional role in regulating DNA protein in- epithelial development to maintain the structure of
teractions of zinc finger proteins. In Zn deficiency the microvilli. That is, there is an interaction between vitamin
structure of these proteins could be abnormal, yielding a A and Zn. Since corneal and conjunctival epithelial cells
possible explanation for the resemblance of signs of Zn are sensitive to deficiencies of vitamin A and Zn which
deficiency to those of vitamin A deficiency. The find- cause similar easily observable changes, electron micro-
ings in the group A(–) Zn(+) rats in the present study scopic findings of the present experiment provides rather
showed that excessive Zn may prevent epithelial dam- clear evidence of an interaction between vitamin A and
age due to vitamin A deficiency. This may be related to Zn. Either vitamin A or Zn deficiency caused a decrease
the Zn finger of retinoic acid in the nucleus via the or loss of goblet cells in conjunctiva. These nutrients ap-
RARs and RXRs activated by excessive Zn supplemen- pear to be necessary for the maintenance of goblet cell
tation. The concept of Zn finger proteins may explain structure and the production of mucous substances. Since
the intriguing possibility of interaction between vitamin the number of goblet cells was greatly decreased in
A and Zn.
groups A(–) and A(–) Zn(+), comparisons were very dif-
There are reports suggesting a nutritional relationship ficult. The role of vitamin A in goblet cell development
between Zn and vitamin A in humans [32]. A positive as- and maintenance may be stronger than that of Zn.
sociation exists in malnourished populations, in which de-
There have been no ultrastructural studies showing
ficiencies of both micronutrients often coexist [9, 17, 18]. that a deficiency of one nutrient can be compensated by
Kraft et al. [19] examined electroretinogram (ERG) supplementation with another nutrient. In the present
changes in both Zn- and vitamin A-deficient rats. Their re- study, we demonstrated that morphological impairment
port suggested that retinal function could be reversed with in rats with a deficiency of either nutrient could be com-
both vitamin A and Zn or with vitamin A, but not with Zn. pensated for by supplementation with the other nutrient,
In their light-microscopic examination, Zn supplementa- although the compensation was incomplete. Fluctuation
tion alone did not prevent ongoing retinal degeneration in the status of one micronutrient can reasonably alter
and corneal keratinization in several rats with both Zn and the metabolism of the other with functional consequenc-
vitamin A deficiency. Udomkesmalee et al. [35] reported es. Electron-microscopic findings indicate a synergistic
the findings of interaction between vitamin A and Zn sup- interaction between vitamin A and Zn for the mainte-
plementation in Thai children with both Zn and vitamin A nance of corneal and conjunctival epithelium. To our
deficiencies. After 6 months of supplementation, the pro- knowledge, this is the first report to demonstrate mor-
portion of children with abnormal conjunctival impression phologically the interaction between vitamin A and Zn.
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