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Proctor Lecture
Jin H. Kinoshita
Osmotic swelling is a common feature of many cataracts. Sugar cataracts and hereditary
mouse cataracts are two types in which osmotic changes play a prominent role. In sugar
cataracts, the initial swelling brought about by polyol accumuation leads to an imbalance in
the pump-leak equilibrium. The pump mechanism becomes unable to keep pace with the
leaky membranes. The marked increases in Na and Cl eventually results in Donnan swelling.
In the hereditary mouse cataract, the imbalance of the pump-leak system appears to be
initiated by a deficiency of Na-K ATPase, a component of the cation pump mechanism. The
inefficiency in the pump mechanism results in Na retention and osmotic swelling.
Key words: sugar cataracts, hereditary mouse cataract, aldose reductase, aldose
reductase inhibitor, deficiency of lens Na-K ATPase.
2 3 4 5 6 7 8
DAYS OF INCUBATION IN 35mM GLUCOSE
Fig. 4. The efFect of a high glucose medium. Incubation conditions were described by Chylack
and Kinoshita.10
concentration, is observed (Fig. 2). When strated while the cation pump mechanism
the rabbit lens Na-K ATPase is 50 per cent was normal. X-rays and microwave
inhibited by 2 x 10~u M ouabain there is a cataracts as observed in rabbits appear to
substantial lag period, while at 10~4 M, be in vivo examples of compromising the
where the inhibition is 90 per cent, the lag lens permeability properties.1' ll
period is considerably shorter. The reason
for the delay of swelling is that in the Sugar cataracts
ouabain-inhibited lens the initial Na gain Another example of lens swelling is that
is matched by K loss, thus no net increase caused by the cataractogenic sugars: glu-
in cation occurs. When the entry of Na is cose, galactose, and xylose. The lens
no longer matched by K exit, a net increase swelling resulting from exposure to galac-
in electrolytes results leading to an influx tose, as shown in Fig. 2, is immediate and
of H2O. The initial 1:1 exchange of Na for linear when compared to the ouabain ef-
K is illustrated in Fig. 3 which summarizes fects. Galactose, like the other two cata-
the results of incubating a rabbit lens in ractogenic sugars, is converted to sugar
10"4 M ouabain. When the gain in Na can alcohol by the enzyme, aldose reductase.1-
no longer be compensated by a loss in K, The sugar alcohol, because it is not further
chloride ions enter the lens to maintain metabolized effectively and is not able to
electroneutrality and it is at this point that readily penetrate the lens membranes, once
increase in hydration begins. As seen in formed in the lens fibers accumulates to
Fig. 3, the curve representing the increase high levels. The hypertonicity it creates is
in lens water parallels the changes in immediately corrected by an influx of
chloride rather than those of Na or K. water.
Of the three cataractogenic sugars the
Factors affecting the leak system order of decreasing effectiveness in produc-
An example of affecting the pump-leak ing changes in lens water and polyol levels
balance by the increase in lens cation is D-xylose, D-galactose, and D-glucose.13
permeability has been shown in the in vitro This is the same as the order of preference
studies with either demercarium bromide for the sugars by the lens aldose reductase
(Humorsol)," echothiophate iodide (Phos- in that the most active substrate for this
pholine Iodide),9 or surface-active agents.10 enzyme is xylose, galactose is next, and
In these cases, the increase in the leak glucose is the least active.13 In addition,
system judged by the rate of rubidium except for the complication in the xylose
leak-out from the rabbit lens was demon- cataracts, the different rates of cataract
B. Increase in Lens H 2 0
2 3 4 5 6 35 mM
GLUCOSE
DAYS IN 35 mM GLUCOSE
8 DAY INCUBATION
Fig. 5. Tetramethylene glutarate on sorbitol production. Incubation conditions in high glucose
medium were described by Chylack and Kinoshita.10
COOH—CH2—C—CH2—COOH
sorbitol and markedly reduce the increase Table I. Changes in the lenses of rats fed
in lens hydration.17 When polyol produc- galactose treated with aldose reductase
tion and the consequent osmotic change inhibitor, AY-20,263
were minimized the electrolyte and amino
acid changes did not occur.17 This is further Lens weight
Galactose level Dulcitol level change
support to the idea that in the develop- (% of control) (% of control) (% of control)
ment of sugar cataracts, electrolyte and
118±10(S.D.) 47 ±12 92 ±4
amino acid changes are secondary to the Control represents the lens of the untreated eye of a rat
sugar alcohol accumulation and the osmotic fed galactose for five days. The contralateral lens is from
the eye injected intravitreally with «,a,«,-trifluoro-N-[2-(3-
change. TMG was shown to be effective nitropryidyl)]-m-toluidine, AY-20,263. This compound was
dissolved in DMSO, 80 mg. per milliliter. Alternate eyes
in markedly delaying the changes in of 24 rats were injected intravitreally with ten microliters
of this solution. The eyes serving as controls were injected
with the same volume of DMSO without inhibitor. The
vitro.15' 1S It prevented sugar alcohol forma- values given show the per cent differences between the
lenses of inhibitor-treated eyes and controls.
tion and osmotic changes that occur in the
lenses incubated in medium enriched with
either galactose, glucose, or xylose.1317 It DMSO was diluted and the resulting pre-
also prevented the loss of amino acids, cipitate served as a depot source for the
myoinositol, and the changes in electrolyte inhibitor. The yellow color also was helpful
distribution as well as the morphologic in indicating when another injection of the
changes that occur in simulating the condi- inhibitor was necessary. In these experi-
tions for sugar cataract development.13'17 ments, usually no more than two injections
TMG, however, did not prevent these were required during the course of galac-
changes from occurring in rats fed galac- tose cataract development. The results
tose. show that administration of AY-20,263 by
The first aldose reductase inhibitor that this manner reduced the level of dulcitol
was effective in vivo was AY-20,263, a formed and the lens swelling was propor-
compound discovered by Dr. D. Dvornik tionately less in the treated eye (Table I).
of Ayerst Laboratories (Fig. 6). This In these galactosemic rats the initial
toluidine derivative is yellow in color and vacuoles are usually observed five days
has a low aqueous solubility. Advantage after the initiation of the galactose diet. In
was taken of both these properties. A con- the treated eye the lens change was delayed
centrated solution was made up in dimethyl- by a median value of eight days so that
sulfoxide (DMSO) which was injected into the initial vacuoles occurred on Day 13
the vitreous cavity of one of the eyes of a (Fig. 7). In the untreated eyes the dense
rat fed high galactose. Into the other .eye, nuclear opacity appears at about 19 days
the same volume of DMSO was injected after feeding. In the treated • eyes the
to serve as a control. As soon as AY-20,263 median delay in the appearance of the
was injected it precipitated out as the nuclear opacity was two weeks. In many
16
14
12
in
5 10 Median
u. 8
o
o 6
4
2
0 2 4 6 8 10 12 14 16 18+ 2 4 6 8 10 12 14 16 18+
DELAY PERIOD IN DAYS DELAY PERIOD IN DAYS
INITIAL NUCLEAR
VACUOLES OPACITY
Fig. 7. Delay in the onset of galactose cataract formation by intravitreal injection of AY-20,263.
Rats were fed a 50 per cent galactose die. AY-20,263 was injected into the vitreous of one
eye of the galactosemic rat.
animals nuclear opacity was not observed Dr. Varma, in our laboratory, has been
even, after a delay of 18 days. It is impor- able to show a delay in the galactose
tant to note, however, that the opacity cataract formation in rats when the eyes
eventually develops in all rats. Thus, the are topically treated with AY-22,284. In the
action of this aldose reductase inhibitor eyes treated with the inhibitor the nuclear
delays the onset of sugar cataract, but does opacity was delayed by approximately a
not prevent it. week. However, the delivery mechanism
The development of AY-22,284 by Ayerst must be improved before the topical ap-
Laboratories is another advance in obtain- plication can be considered an effective
ing a more effective aldose reductase in- means of controlling this type of cataract.
hibitor (Fig. 6). The actual potency of all It is of significance that the three in-
three aldose reductase inhibitors is approxi- hibitors of aldose reductase (Fig. 6) are
mately the same in that 10~r> M concentra- structurally vastly different. However, each
tions of these inhibitors cause a 50 per cent has a hydrophobic group and an acidic
inhibition in the lens aldose reductase group. The acidic group in AY-20,263
activity. However, AY-22,284 has a high resides in the aromatic nitro group. The
aqueous solubility and it has low toxicity. fact that so many compounds inhibit aldose
In lens culture in high galactose medium reductase probably is related to the fact
it appeared effective in preventing the that many aldehyde-containing compounds
vacuoles from appearing after three days serve as its substrate. It also indicates that
of incubation and blocking dulcitol ac- other compounds with the necessary struc-
cumulation and minimizing lens hydration tural requirements may turn up as even
(Fig. 8). This inhibitor was the first to be more potent aldose reductase inhibitors.
effective by oral administration in delaying
the onset of cataract formation as judged Possible clinical implications
by the appearance of the dense nuclear The possible clinical use of the aldose
cataract.1S Fig. 9 shows that all the rats reductase inhibitor appears obscure. Even
fed galactose have developed cataracts by the galactosemic cataracts in infants are of
the twenty-ninth day on the galactose diet. infrequent occurrence. In addition, the
However, in those rats fed galactose along galactosemic subjects can be treated simply
with AY-22,284, only 20 per cent developed by withdrawing galactose from the diet.
cataract by 29 days.ls Oral administration The true diabetic cataracts are also un-
of this aldose reductase inhiibtor was thus common. Moreover, surveys made thus far
shown to effectively delay the onset of seem to indicate that the incidence of
galactose cataracts. cataracts in diabetic subjects is no different
20
100 -
10.
• O.I 0.3
MILLIMOLAR AY-22, 284
Water
40
Cataract
.-#--•
Normal
10 h
10 20 30 40 50 60 70 80 over
100
Mouse Age ( days )
Cations
600
500
400
300
200
100
10 20
this situation, the result may be that the trolyte and osmotic changes occur approxi-
two cataractogenic factors together en- mately 20 clays after birth, about the time
hance the maturation process of the cata- the pinhead opacity is observed. At this
ract. If this is the case, the action of an stage, the other changes are not remarkable
effective aldose reductase inhibitor may be in that slight decreases are observed in
helpful in minimizing the diabetic effects potassium, glutathione, and ATP levels.
and delaying the cataractous process. A With time, the nuclear opacity becomes
significant delay in the maturation of the more prominent and eventually the opacity
cataract would have obvious beneficial ef- covers the entire lens. At this advanced
fects for the patient. stage a dramatic increase in sodium
chloride and loss in potassium are observed,
Hereditary mouse cataract along with marked overhydration, typical
Another type of cataract involving an of many cataracts, as mentioned previously.
osmotic change occurs in a strain of mice The changes early in the course of the
that develops a pinhead nuclear opacity cataract prior to the appearance of the pin-
three weeks after birth.'1 These mice were head opacities were examined in hopes of
discovered in Japan and are called the uncovering the initiating factor in this type
Nakano Cataract Strain. These mice were of cataract. Since the electrolyte distur-
crossed with the Charles River Strain and bance occurs early, the cation pump mecha-
a new colony of the Nakano Strain was nism in these cataracts was evaluated. As
developed in this country. Except for the early as 13 days after birth the rubidium
cataract, these defective mice seem to be as ion uptake, which measures the ability of
healthy and grow as well as the control the lens to concentrate potassium ion, is
mice. The most obvious change in these less effective by 50 per cent in these
cataracts is a sudden increase in lens cataracts than in the control mouse lenses.8
hydration just about the time the pinhead The inability of the cataract to concentrate
opacity is observed (Fig. 10). It appears rubidium is substantial and persists
that concomitant with the increase in lens throughout the cataractous process. The
hydration there is also a sudden increase failure of the cation pump is further mani-
in the lens sodium."1 It does, therefore, ap- fested in the cataract by loss of effective-
pear that the sodium increase is directly ness in extruding Na.M The defect is ob-
related to the osmotic swelling. The elec- served as early as 13 days. There is a
100 i—
o 60 —
•0 5 1.0 15 2 0
tive against the Na-K ATPase from rat not present, or at least not detectable with
lens, calf lens, or calf retina. The inhibition the methods used, in the normal mouse
was less than that observed for the mouse lens. Whether this inactivator is responsible
lens ATPase since the activity of the en- for initiating the cataractous process is an
zyme was much higher from the other intriguing possibility that requires further
sources. study.
We decided to use calf lens as the source
REFERENCES
of Na-K ATPase for all subsequent studies
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