Cataracts in Galactosemia The Jonas S. Friedenwald Memorial ...

Cataracts in galactosemia

The Jonas S. Friedenwald Memorial Lecture

Jin H. Kinoshita

0,"ne purpose of this award to to pre-serve the name of a great scientist who hascontributed so much to the field of oph-thalmology. If this were the only purpose,this award would not be necessary, for Dr.Friedenwald is immortalized by his manypublications which serve as catalysts for agreat number of other investigations. Thispresentation on the cataracts in galac-tosemia is an illustration.

Human galactosemia

Congenital galactosemia is now knownto be caused by a deficiency of one en-zyme, resulting in the inability of the af-flicted individual to metabolize galactose.The main clinical findings in infants withthis disease are hepatomegaly, malnutri-tion, cataracts, and galactosemia. Mentalretardation becomes apparent with increas-ing age of the untreated infant. Becausecataracts have been observed as early asa few days after birth, they may be theearliest observable symptom.1

Previously this disease was confusedwith diabetes because of the fact that asugar was detected in the urine of these

From the Howe Laboratory of Ophthalmology,Harvard Medical School, and the MassachusettsEye and Ear Infirmary, Boston, Mass.

This study has been supported by Public HealthService Career Development Award S-K3-17082from the Institute of Neurological Diseases andBlindness, Public Health Service, and by theUnited States Atomic Energy Commission,Crant AT (30-1) -1368.

patients. However, with more specific testsnow available for glucose and galactose,this entity can easily be established. Bychecking the urine with the glucose orgalactose oxidase test, one can rapidly de-termine which sugar is present in the urine.Positive identification of galactose can bemade by paper chromatography.2

This disease can be treated simply bywithdrawing galactose from the diet. Whengalactose is withdrawn, the symptoms seemto correct themselves, provided the diseaseis detected sufficiently early.3 In most casescataracts disappear when milk—the princi-pal dietary source of galactose—is elimi-nated from the infant's diet. When affectedindividuals pass their formative stage oflife they seem able to tolerate galactose,4

and adults who had been galactosemic arecapable of ingesting the sugar without de-veloping any of the symptoms.

Galactosemia is a hereditary diseasewhich is recessive in nature and its occur-rence is approximately 1 in 18,000 births.5

The absence of, or at least the depressionin the activity of, one particular enzymeinvolved in galactose metabolism is respon-sible for the disease.6 The pathway bywhich galactose is metabolized usuallyproceeds in the following sequence of re-actions (Fig. 1): First, galactose is phos-phorylated by a reaction with ATP* to

"The following are the abbreviations used throughout thetext: ATP, adenosine triphosphate; UDPglu, uridine di-phosphate glucose; UTP, uridine triphosphate; P.P., pyro-phosphate; Gal-l-P, galactose-1-phosphate; GSH, glu-tathione; Na, sodium ion; K, potassium ion; Cl, chlorideion.

786

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Volume 4Number 5

Step I:

galactose + ATP

Step 2:

Gal-l-P + UDPGIu

Step 3:UDP Gol :

galaclokinase

G o l - t - P uridyl

transferase

UDP g o l - 4 - epimerose

• G a l - t - P + ADP

: Glu-l-P +UDP Gal

: UDP Glucose

Step 4 :UDPG pyrophosphorylase

UDP Glucose + PP . UTP + G l u - l - P

Fig. 1. A pathway of galactose metabolism. Con-version of galactose to glucose.

galactose-1-phosphate. This is followed bya transfer type of reaction in which UDPgalactose-1-phosphate and glucose-1-phos-phate are formed. The third reaction in-volves the epimerization of UDP galactoseto UDP glucose and this compound inturn reacts with pyrophosphate to give glu-cose-1-phosphate. Thus the three reactionsfollowing the galactokinase reaction arerequired to convert galactose-1-phosphateto glucose-1-phosphate—an intermediate inthe pathway of glucose metabolism.

Apparently the transferase enzyme ofreaction 2 is the target of the disease pro-cess in galactosemia, resulting in a de-ficiency of the enzyme.0-7 Since galactose-1-phosphate cannot be transferred toUDPG, the galactose derivative accumu-lates in the tissues as demonstrated in thered cells and liver.8"10 Diagnosis for galac-tosemia is made by determining whetherthere is a depression in the activity of theenzyme transferase in the blood.11'12 Thegalactosemic patients also show an abnor-mal galactose tolerance curve.2 The pe-culiar properties of the galactokinase re-action account for the elevated level ofgalactose in the blood and, consequently,in the urine. Galactokinase is inhibited bygalactose-1-phosphate; therefore the accu-mulation of the phosphorylated interme-diate, because of the enzyme defect, in-hibits the first reaction.13 Moreover, galac-tokinase is subject to substrate inhibition.Thus, galactokinase is inhibited by galac-tose-1-phosphate and, in addition, the re-sulting increase in galactose also tends

Cataracts in galactosemia 787

to decrease the rate of galactose phos-phorylation.13

The only report concerning biochemicalchanges in the lens of a galactosemic sub-ject was made by Lerman.14 In this casea 4-week-old boy with galactosemia wasfound to have a slight opacification of thelens. This patient died of complications,and analyses of the lens revealed thatgalactose-1-phosphate uridyl transferasewas completely lacking.

Even though the enzyme defect is un-doubtedly the primary factor in galacto-semia, no satisfactory explanation is avail-able of how the absence of the enzymetransferase can account for the symptomsinvolved. Galactose is an essential com-ponent of cerebrosides and mucopolysac-charides, and interference of galactoseutilization by a tissue was thought to cre-ate a shortage in these vital macromol-ecules. However, other metabolic pathwaysare available which could provide suf-ficient amounts of galactose for this pur-pose. The accumulation of galactose-1-phosphate was considered to have possibleeffects on glucose metabolism. It has beenshown that galactose-1-phosphate is an in-hibitor for the phosphoglucomutase re-action.15 This is the reaction involving theconversion of glucose-6-phosphate to glu-cose-1-phosphate in the pathway of glyco-gen synthesis. However, in this case galac-tose-1-phosphate is a competitive inhibitor,hence even this reaction is probably notcompletely blocked in tissues of galacto-semic patients. There was one report thatgalactose-1-phosphate is an inhibitor ofglucose-6-phosphate dehydrogenase in thelens,10 but this report could not be con-firmed by others.17'1S

Experimental galactosemia and cataracts

There is a good possibility that investi-gations dealing with lens changes in galac-tosemia may lead to an understanding ofthe other symptoms of the disease. Sincea cataract can be easily induced by feed-ing rats diets enriched with galactose,many investigators have used this approach


788 Kinoshita Inoestigatioe OphthalmologyOctober 1965

in the hope of determining the etiology ofthis type of cataract.

The morphology of these experimentalcataracts seems to differ from those ob-served in human patients. In young ratsthe cataracts first appear as vacuoles inthe equatorial region of the lens and, asthe cataract progresses, the vacuoles be-come more numerous and eventually adense nuclear opacity develops. In con-trast to these morphological changes inexperimental animals, cataracts in humangalactosemia are mostly of the nuclearlamellar type. However, in our laboratorywe have confirmed the observation thatnewly born rats, whose mothers weremaintained on a galactose diet, developa nuclear lamella type of cataract closelyresembling that observed in the galacto-semic infants.19 Therefore, it seems reason-able to assume that the factors initiatingthe galactose cataract in young rats arevery similar to those involved in the hu-man galactose cataract. The manifestationsof the cataract, however, may differ, de-pending on the developmental stage of theanimals. The lens opacities in rats that arefed galactose, like those in human galac-tosemic subjects, slowly disappear whenrats are placed on diets free of galac-tose.20' 21

Studies on the pathogenesis of the cata-ract of galactose-fed rats have led to thespeculation that the overabundance ofgalactose in the lens in some way inter-feres with carbohydrate metabolism.22 Butno definitive evidence indicating that glu-cose utilization or lactate production wasaltered sufficiently to cause the cataractcould be provided.28 Directly bearing on

this point is the determination of ATPlevels in the lenses of galactose-fed ratswhen opacities first become visible. At thisinitial vacuolar stage of cataract, the ATPlevel was depressed by only 10 per centof the control, whether the comparison wasmade on a per lens or a dry weight basis(Table I). These results are in good agree-ment with those of Mandel and Klethi24

who observed only a 12 per cent drop inthe lens ATP level when opacities becomevisible. Sippel25 showed that in the 50 gramSprague-Dawley rats the appearance ofopacities preceded any change in the ATPlevel. These findings, showing that no sig-nificant change in lens ATP level occursat the time when opacities first becomevisible, make it appear doubtful that lackof biological energy is the causative factorin the initiation of the cataractous process.However, since the ATP level is markedlydepressed in the later stages of cataractdevelopment,18 a lack of energy may bea contributing factor in producing changesobserved later.

Osmotic changes in the lens

A new lead into the pathogenesis ofgalactose cataract was provided by Dr.Friedenwald in his study on the histopathol-ogy of this type of cataract.20 He observedthat the earliest morphologic change wasthe appearance of hydropic lens fibers. Hepresented evidence indicating that the ac-cumulation of fluid was intracellular andnot extracellular. As the cataract pro-gressed, the hydropic fibers ruptured anddissolved, leading to interfibrillar cleftswhich were filled with precipitated pro-teins.

Table I. Changes in ATP levels in the initial vacuolar stage of galactose cataract

No.

26

Glucose-fed

ATP content of lens(mjimoles/mg. dry

weight lens)

7.3 ± 0.6

rats

ATP content of lens(mumoles/lens)

72 ± 0.7

No.

24

Galactose-fed

ATP content of lens(nvfimoles/mg. dry

weight lens)

6.8 ± 0.7

rats

ATP content of lens(mjj-moles/lens)

64 ± 0.8Analyses were made on lenses from 100 gram rats which were kept on a high galactose diet37 until lens opacities firstbecame visible.For controls, rats were kept on a glucose diet17 for the same period. The firefly luminescence assay for ATP was used.40


Volume 4Number 5


The problem was, then, to find an ex-planation of how these hydropic lens fibersdevelop. It would seem that there are twomain, reasons for the swelling of a cell.Osmotic changes can result either fromthe accumulation of abnormal metabolitesor of electrolytes. In the galactose cataractan obvious abnormal metabolite is galac-tose-1-phosphate. Although galactose-1-phosphate accumulates in the lens of agalactose-fed rat,17 it does this slowly, andthe level attained would probably exertonly a minimal osmotic effect. A morelikely abnormal metabolite to effect an os-motic change was uncovered by VanHeyningen.27 By chromatographic methodsshe was the first to demonstrate the pres-ence of dulcitol, the sugar alcohol formof galactose, in the lens of a galactose-fedrat. The nature and factors concerning thesynthesis and accumulation of this com-pound strongly suggest that the early os-motic changes can be explained by theretention, of dulcitol.28"31 First of all, thelevel of dulcitol found in the lens of galac-tose-fed rats is about equal to that of theK ion—the normal lens constituent whichis present in the highest concentration.The retention of dulcitol in the lens to sucha level must obviously have some osmoticconsequence, for it creates a hypertonicityand the obligatory response is the move-ment of water into the fibers. These os-motic changes were shown to occur bothin vivo and in vitro. In both cases, asshown in Figs. 2 and 3, the accumulationof dulcitol in the lens is paralleled by anincrease in lens water.

The study of the enzymatic mechanisminvolved in the reduction of galactose todulcitol has revealed that the lens is aparticularly favorable site for the accumu-lation and production of dulcitol.31 Galac-tose concentration, must be fairly high be-fore the enzyme aldose reductase can con-vert significant amounts of the sugar tothe alcohol form. In other tissues, eventhough the organ may be exposed to highlevels of galactose, the phosphorylationmechanism is sufficiently active to keep

4 6 8 10 12DAYS ON GALACTOSE DIET

Fig. 2. Increases in dulcitol and in water in thelens of galactose-fed rats. From Kinoshita et al.:Nature 194: 1085, 1962.

20

20 2412 l(

HOURS

Fig. 3. Increases in dulcitol and in water in therabbit lens incubated in high levels of galactose.The incubation medium contained either 30 mM.fructose + 5 mM. glucose (control), or 30 mM.galactose + 5 mM. glucose. Paired lenses wereused, one lens placed in the control medium andthe other in the aldose medium. Data republishedfrom Kinoshita, Hayman, atod Merola: J. Biol.Chem. 240: 310, 1965.

the sugar at low levels. On the other hand,since the lens has a sluggish system tophosphorylate galactose, there is a ten-dency for the sugar to increase when highlevels of it are made available to this oc-ular tissue. Furthermore, the lens has anactive shunt mechanism which providesTPNH—the co-enzyme necessary for the


790 Kinoshita Investigative OphthalmologyOctober 1965

reduction of galactose. Thus because ofthe distribution of enzymes characterizedby a low activity of galactokinase, and ahigh shunt activity, coupled with a rela-tively active aldose reductase, the lens isa favorable site for dulcitol synthesis.Furthermore, in contrast with other sugaralcohols, dulcitol is not a suitable sub-strate for polyol dehydrogenase—the en-zyme responsible for the further oxidationof polyols. Sorbitol and xylitol are activelyoxidized to their keto sugar form, but dul-citol is not.30 Another important factor isthat sugar alcohols do not easily penetratethe lens membrane. Since dulcitol does notreadily leak out of the lens nor is it me-tabolized, its continued synthesis can leadto unusually high levels within the lensfibers.

We believe that the sequence of eventsin galactose cataract leading to the ap-pearance of vacuoles is as follows: Theinitiating factor is the increased level ofgalactose in the aqueous humor which,in. turn, increases the concentration of thissugar in the lens. The increased availabil-ity of galactose stimulates the synthesis ofdulcitol. A hypertonic condition is createdas dulcitol accumulates, but this is imme-diately corrected by movement of waterinto the lens fibers. The resulting osmoticswelling of the lens fibers probably ex-plains the histological appearance of thehydrops. As the swelling continues, someof the fibers rupture, and vacuoles prob-ably represent areas in the lens where con-siderable osmotic dissolution of fibers hasoccurred.

Friedenwald20 has shown that the fibersat the equator which are immediately su-perficial are not first affected, but thatthose deeper in the lens are the most sus-ceptible. No experimental evidence isavailable to explain these observations.

Defect in the amino acid-concentratingmechanism

There are other changes which occur inthe development of galactose cataractwhich must be related directly either to

Table II. Changes in the level of aminoacids in the rabbit lens incubated in thepresence of galactose

RabbitNo.

Amino acidsin controlmedium

(jwioles/lens)

Amino acidsin galactose

medium(^moles/lens)

%Change

123456

3.843.573.853.733.584.35

Average increase

2.392.25

4 2.432.241.892.75

in lens weight =

-38-37-37-40-47-38

20.9 mg.

Paired lenses from rabbits were used, one lens incubatedin a control medium,33 30 mM. fructose plus 5 mM. glu-cose, and the other in a medium containing 30 mM.galactose and 5 mM. glucose. After 21 hours the lens washomogenized in 10 per cent trichloroacetic acid. Thevalues given are for the total ninhydrin-reactive materialmeasured in the trichloroacetic acid filtrates.

Table III. Accumulation of AIB in mediacontaining various aldoses

Medium

Increasein sugaralcohol

(limoles/lens)

Increasein water

(mg./lens)(AIB)Lem/

(AIB)Medium

ControlGalactoseGlucoseMannose

6.94.91.2

±0.5±0.7±0.2

19.5 ±12.5 ±4.2 ±

1.51.20.9

24.6 ±3.57.6 ±1.2

10.5 ± 2.017.2 ±1.6

(128)(28)(14)(7)

In all experiments, paired lenses from a rabbit were used,one lens placed in the control medium,33 30 mM. fructoseplus 5 mM. glucose, and the other in a medium contain-ing 30 mM. aldose in addition to 5 mM. glucose. Eachmedium contained 0.1 mM. 1-IC-AIB and had an initialtonicity of 290 to 293 milliosmolal. The changes in sugaralcohol level and water content of the lens incubated for21 hours in the aldose solution are given as the increasesabove those of the lens incubated in the control medium.The results are given as the mean ± the standard devia-tion. The number of experiments are indicated in paren-theses.

dulcitol formation or to the resultant os-motic changes. One change which occursearly in the course of cataract develop-ment is the depression of the amino acidlevel in the lens.32 Since many of the earlychanges which occur in the lens of a galac-tose-fed rat can be reproduced by simplyincubating the lens in a medium contain-ing high levels of galactose, the amino acidproblem was studied in vitro. As shown inTable II, there is a 40 per cent depressionof total amino acids in these lenses incu-bated in a high galactose medium com-pared with those incubated in a control


Volume 4Number 5


medium. As shown by Kinsey and Reddy,32

further insight into this problem can begained by studying the uptake of a-amino-isobutyric acid (AIB)—a nonmetabolizableacid. AIB is taken up by an active processwhich concentrates it in the lens to a levelmuch higher than that of the medium. InTable III, we see that a lens incubated for21 hours concentrates AIB 25 times abovethe concentration in the medium.33 In thepresence of galactose, however, the lensis able to concentrate AIB to about 30per cent of normal. Other aldoses, glucoseand mannose, have a similar effect. Theextent of the inhibition by these aldoses onAIB uptake seems to have an inverse re-lationship to the amount of sugar alcoholand gain of water in the lens. For example,the lens exposed to galactose accumulatesthe highest level of sugar alcohol, has thegreatest increase in water, and is least ef-fective in concentrating AIB. On the otherhand, in the presence of mannose the lensaccumulates very little sugar alcohol, hasthe slightest gain in water, and also pro-duces the slightest inhibition of AIB up-take. The eflfect of glucose is intermediatebetween that of galactose and mannose.Apparently, either the sugar alcohol itselfor the concomitant osmotic change seemsto have an adverse effect on the ability

of the lens to concentrate amino acids. Thepossibility that the effect was osmotic wasfirst studied. Changes in the state of hydra-tion can also be achieved by anothermeans, that is, by incubating the lens eitherin a hypo- or hypertonic condition. Thelens behaves as an osmometer, in that ifthe lens is placed in a medium which ishypotonic it tends to take up water, andwhen placed in a medium which is hyper-tonic it loses water. As shown in Fig. 4,one can see that in a range from 64 mil-liosmolal hypotonic to about 32 millios-molal hypertonic to the normal mediumthere is a linear relationship between thechange in water and that of the tonicityof the medium. Thus, in this limited rangeof tonicity the lens behaves as an osmom-eter. Furthermore, the lens is a sensitiveosmometer in that it responds to a 5 percent change in the tonicity of the medium.The effect of changes in tonicity on theability of the lens to concentrate AIB isshown in Fig. 5. When exposed to hypo-tonic media the lens is less able to concen-trate AIB, and, up to a point, the morehypertonic the solution, the more effec-tively is the lens able to concentrate theamino acid. Thus, tonicity plays a veiy im-portant part in determining the effective-ness of the amino acid-concentrating mech-

•I-40 +20 0 -20 -406 MILUOSMOLALITY

-60 -80

Fig. 4. The effect of varying tonicity of the medium on the hydration of the lens. Rabbitlenses were incubated for 21 hours. Equivalent amounts of NaCl were added1 to or subtractedfrom the control medium to vary the tonicity. The results are given as the mean ± the standarddeviation of the mean. Data republished from Kinoshita, Hayman, and Merola: J. Biol. Chem.240: 310, 1965.



anism in the lens. In view of these re-sults, the osmotic change may be the ex-planation of what occurs in the lens whenexposed to galactose. In this case the dul-citol-induced osmotic change would sim-ulate the swelling induced by a hypotonicmedium. The osmotic swelling may be thereason for the lowered efficiency of theAIB-concentrating mechanism in the galac-tose-exposed lens. If this is the case, thenpreventing the swelling from occurring,despite the accumulation of dulcitol, shouldallow the lens to concentrate AIB to nor-mal levels. To investigate this possibility,the lens was incubated in a medium con-taining galactose. However, during thecourse of the experiment the tonicity ofthe medium was gradually increased sothat even though dulcitol accumulated,the movement of water into the lens wasprevented. The incubation of the lenswhich was exposed to galactose in theosmotically compensated medium for 24hours maintained the normal water volumeof the lens (Table IV). Furthermore, un-

3 4

3 2

3 0

2 8

2 6

2 4

(AIB

) M

ediu

m

1 4

\ ,

2 10

Q) 8S3 6

4

2

0

-

-

-

y

r

( J

i i i i i i i-100 - 8 0 - 6 0 - 4 0 - 2 0 0

a MILLIOSMOLALITY

Fig. 5. The effect of varying tonicity of the me-dium on the AIB uptake by the rabbit lens. Thedata were obtained from analyses of the lensesincubated in media as described in Fig. 4. Theresults are given as the mean ± the standard de-viation. Data republished from Kinoshita, Hay-man, ajnd Merola: J. Biol. Chem. 240: 310, 1965.

der these conditions, the lens was able toconcentrate AIB as well as the lens in thenormal medium. To illustrate that this ef-fect is not limited to a specific amino acid,the lenses exposed to galactose but incu-bated in an osmotically compensated me-dium were able to retain normal levelsof amino acids (Table V). Thus, fromthese experiments it would appear thatthe loss of amino acids in the lens whenexposed to galactose is primarily due tothe osmotic swelling of the lens" broughtabout by the retention of dulcitol.

In human galactosemia one of the in-teresting clinical findings is that these pa-tients have an amino aciduria.3 All of theamino acids are found in excessive quan-tities in the urine. This problem was studiedin the rat by Rosenberg and associates,34

who found that maintaining rats on a high

Table IV. Accumulation of AIB by rabbitlens in osmotically compensated medium

Medium

Distributionof AIB

(AIB)LY(AIB)M (mg./lens)

Sugaralcohol

(jimoles/lens)

Control 24.3 ± 2.9Calactose 22.7+1.5 -1.3±1.2mg. +7.8 ± 0.2The tonicity of the incubating media containing 30 mM.galactose plus 5 mM. glucose was progressively increasedduring the incubation in a manner previously described.33

The level of "C-AIB was maintained at 0.1 mM. Theresults are given as the mean ± the standard deviation. Atleast 12 pairs of lenses were \ised in each experiment.

Table V. Changes in the level of aminoacids in the rabbit lens incubated in anosmotically compensated medium contain-ing galactose

Rab-bitNo.

In controlmedium

Amino acids(jimoles/lens)

In osmotically compensatedgalactose medium

Ammo acids(p.moles/lens)

Change inlens weight

(mg.)

123456

3.733.503.252.633.052.87

3.643.433.362.503.043.16

+1.6+1.4+1.9+0.7

0-0.2

Descriptions are the same as those given in Table II andTable IV.


Volume 4Number 5

Cataracts in palactosemia 793

galactose diet does produce an amino aci-duria. In addition, it has recently been re-ported that dulcitol was found in the urineof human galactosemic patients85 and inthe kidney of galactose-fed rats.is'3F> Thesefindings indicate that studies to determinewhether the same factors are affectingboth the lens and kidney in the galacto-semic patients may prove informative.

Changes in lens hydration during thecourse of galactose cataract

The discussion thus far has dealt withchanges which occur prior to the appear-ance of vacuoles in the lens of a galactose-fed rat. As the cataract progresses, thesevacuoles which first appear at the equa-torial region become more numerous andspread over the anterior surface of thelens. This continues for a period of about

STAGE OF CATARACT

Fig. 6. Changes in hydration of the lens duringvarious stages of galactose cataract. Data repub-lished from Kinoshita and Merola: INVEST.OPHTH. 3: 577, 1964.

14 to 15 days after the onset of the galac-tose diet. Any time after this period,usually within a few hours, a dense nu-clear opacity develops.21 Along with theappearance of nuclear cataract there is an-other and a most dramatic osmotic change.To illustrate this point, the changes in hy-dration during various stages of galactosecataract are shown in Fig. 6. The initialvacuolar stage is marked by a 25 to 30per cent increase in lens water. Betweenthe initial and the late vacuolar stage—theperiod just prior to the dense nuclear opac-ity—there is essentially no increase in lenswater. However, the animals are 2 weeksolder than those at the initial vacuolarstage and consequently the lenses normallywould have a lower water content. Thus,in comparison with the lens of a controlrat, the late vacuolar stage cataracts showa 50 per cent increase in lens water. Themajor change in lens water occurs, how-ever, when the lens develops the nuclearopacity (mature cataract). A three- to four-fold increase in lens water is observed atthis stage. These changes in hydration dur-ing the development of a galactose cataractare very striking and, although a completeexplanation is not available, there are somefindings which undoubtedly bear upon thisproblem.

Electrolyte changes

It is generally accepted that inorganicelectrolytes are the constituents of the lensor of any cell which regulate the normalcell volume. Thus, it seemed important tofollow the changes in the Na, K, and Clcontents during cataract development. Asshown in Fig. 7, there is a dilution of cat-ions in the initial vacuolar stage. The totalcation (Na+K) concentration on a waterbasis of the lens from a galactose-fed ratis lower than that of the normal lens. How-ever, this does not mean there is a decreasein the total Na and K ion, for actually thetotal amount remains the same, as shownby the results expressed on a dry weightbasis. The dilution of the cation is ex-plained by the fact that the dulcitol reten-



- L£NS WATER BASIS

it

DRY WEIGHT BASIS

<fiM

240 Q

CONTROL INITIAL LATE NUCLEAR CONTROL INITIAL LATE NUCLEAR

VACUOLAR VACUOLAR

STAGE OF CATARACT STAGE OF CATARACT

Fig. 7. Electrolyte changes during the variousstages of galactose cataract. Details of the dataare given in Kinoshita and Merola: INVEST.OPHTH. 3: 577, 1964.

tion is responsible for drawing water intothe lens. Apparently the movement ofwater is not accompanied by electrolytesat this stage of cataract development. Thedifference in the total cation concentrationexpressed on a water basis between theearly cataracts and control lenses indicatesthat a 24 per cent dilution of the cationshad occurred. This value is in fair agree-ment, considering the different techniquesemployed, with the actual measurement ofthe net increase in water. In these lensesthe increase in water was 5.3 mg. per 10mg. dry weight of the lens, representing anincrease in hydration of about 30 per centover that of the normal lens. It is also ofinterest to compare the amount of dulcitolfound in the lens with the increase in waterobserved. At this stage of cataract theamount of dulcitol recovered was 2.5 mg.per 10 mg. dry weight of the rat lens. Thus,the ratio of the amount of dulcitol foundto the increase in water was 0.472 /xmoleper milligram water. In other words, withthe amount of dulcitol found, a larger in-crease in lens water would be expected.Part of this discrepancy is explained bythe loss in amino acids and glutathione. Inaddition, even though some dulcitol ac-cumulates in the nucleus of the lens, never-theless, because of existing hydrostaticforces, this area of the lens may not takeup theoretical amounts of water. In con-

trast to the rat lens, incubation of rabbitlens in a high galactose medium has led toa more favorable ratio of the amount ofdulcitol found to the increase in water.-9

During a three-day incubation, this ratioranged from 307 to 373 /̂ moles per milli-gram of water increase.

In the late vacuolar stage there seemsto be a dilution of the total cation concen-tration on a water basis but, in contrastwith the initial stage cataract, there is alsoa slight increase in total cations (Fig. 7).Therefore, it appears at this stage that theincrease in hydration is due in part todulcitol retention as well as to electrolyteincrease. Furthermore, there seems to besome obvious change in the distribution ofNa and K, in that the concentration of Naand of K is decreasing. This change is alsotaking place in the cataract at the initialstage, but is most apparent in the latevacuolar stage. The cation changes areparalleled by the changes in concentrationof chloride ion.21 In the initial stage thereis a dilution of chloride without a netchange in the total amount of this anion.In the late vacuolar stage the increase inchloride content corresponds roughly tothat in total cations.21 Up to the appear-ance of the dense nuclear cataract the in-crease in electrolytes is not quantitativelystriking and amounts to approximately an18 per cent increase in total electrolytes.21

However, with the onset of the nuclearcataract a tremendous increase in theamount of salt results (Fig. 7). Thismarked change in electrolyte content ischaracterized by extremely high levels ofNa and Cl, with low levels of K. The largeincrease in salt can account for all of thewater gained by these cataracts. At thisstage the lens has lost its ability to main-tain normal permeability characteristics, inthat Na and Cl, which are normally ex-cluded, find ready access into the intra-lenticular spaces, and dulcitol is no longerretained. The accumulation of salt is ac-companied by a movement of water sothat the situation becomes a classical dem-onstration of colloidal osmotic or Donnan


Volume 4Number 5


swelling. It has been shown in other cellsthat this type of osmotic swelling is an un-stable condition in which equilibrium isnever established unless an external force,such as a hydrostatic pressure, is im-posed.30*30 Otherwise, the cell continues toswell until it eventually bursts. Since mostcell membranes are permeable to electro-lytes and impermeable to proteins, thequestion may be raised why cells do notgenerally undergo Donnan swelling. Theexplanation is that all cells are apparentlyendowed with a cation pump. It is by thismechanism that a cell prevents the freemovement of cations by excluding Na andretaining K. Poisoning the pump withouabain or interfering with the metabolismso that energy is not adequate to maintainthe pump are two examples of conditionswhich would eventually lead to Donnanswelling.

The effect of galactose on cation pumpmechanism

One reason for the striking osmoticswelling observed in the nuclear cataractstage may be that the cation pump failsto function. This possible explanation wastested by studying in vitro the uptake of42K and 2lNa by the rat lens at the variousstages of cataract development.21 In theinitial stage there is a slight drop in the42K uptake and a further drop in the latevacuolar stage (Fig. 8). However, it is in-teresting to note that the nuclear cataractis equally effective in concentrating 42K asis the cataract of the late vacuolar stage,so there is no sudden decrease in the ac-tivity of the K pump to account for themarked Donnan swelling. The data ob-tained on the 2lNa uptake mechanismillustrate the ability of the normal lens toexclude Na (Fig. 9). Only a small fractionof 21Na is taken by the rat lens during theincubation. The cataract in the initial vacu-olar stage appears to exclude Na as wellas the normal lens. However, in the cata-ract of the late vacuolar stage there is adefinite uptake of 2lNa. As the cataractprogresses to the nuclear opacity stage, the

lens seems unable to exclude Na, as evi-denced by the marked uptake of 2lNa.

The results of the cation uptake studiesare expressed as ratios of the concentrationof 42K or 24Na in the lens against that inthe medium. The values may be somewhatmisleading. For example, the uptake ofJ2K by the initial vacuolar stage cataractappears to be significantly lower than thatof the control lens. However, if the in-crease in hydration of the cataract is takeninto consideration, the amount of 42K actu-ally taken up is very close to that of thecontrol. A more meaningful analysis ofchanges in cation pump activity is to mea-sure the rate of exchange of cations or theturnover rate. This type of flux measure-ments for K in the lens has been accom-

Initial vacuolar

150

Fig. 8. 42K uptake by the lens in various stagesof galactose cataract. Data republishecl fromKinoshita and Merola: INVEST. OPHTH. 3: 577,1964.



^ o.e

Si ./^Mature

S ^Late vacuolar

Initial vacuolar. — O

Control

150

Fig. 9. 2<lNa uptake by the lens in the various1

stages of galactose cataract. Data republished fromKinoshita and Merola: INVEST. OPHTH. 3: 577,1964.

plished by Thoff10 and is now being madeto determine changes in the K-concentrat-ing mechanism in the lenses of galactose-fed rats, as well as in rabbit lenses incu-bated in a high galactose-containing me-dium. The results have shown that the up-take of K by the lenses in the early cataractstage was at normal rate or slightly high-er.iS Even in the nuclear cataract stage theK pump is 50 per cent of normal; there-fore, the changes in the cation pump donot appear to be responsible for the majoralteration occurring in the nuclear cataractstage.1S In the rabbit lens exposed to highlevels of galactose for 20 hours, the rate ofK exchange was found to be higher thanthat of the control lens.is The increase inK flux observed in the lens incubated ingalactose indicated that the rate of bothuptake and leakout of K is increased. In all

probability the increase in K flux is ini-tiated by an increase in the leakout of K.This fact would be consistent with the AIBuptake studies. The lens exposed to galac-tose undergoes osmotic swelling, the resultof which is an increase in permeability.

When exposed to a high level of galac-tose, the lens appears to be much more ef-fective in preserving the normal distribu-tion of cations than of the amino acids.This is illustrated in the initial vacuolarstage cataract where there is a slight lossof K and gain of Na, but the 40 to 50 percent decrease in the amino acid content ismuch more striking.1 s Furthermore, therabbit lens incubated in galactose lost 40per cent of its free amino acids but in-curred only a slight change in the distri-bution of cations. The AIB uptake in vitroby the rabbit lens is depressed by 70 percent in the presence of galactose, while theK flux is increased. The cation pump ap-pears much more effective than the aminoacid pump, in that its rate can be acceler-ated to compensate for the increase in lenspermeability, and thus is less vulnerableto the toxic effects of galactose.

Since the cation pump is sufficiently ac-tive in the nuclear cataract stage, it isprobably not the factor responsible for themarked loss in K, gain in Na and Cl andwater which characterize the changes ofthe terminal stages of the cataract. Aprobable explanation for these changes isa continual increase in lens permeabilityduring the entire course of the cataractousprocess. The early swelling caused by dul-citol retention initiates the changes in lenspermeability. It accounts for loss in aminoacids and also begins to affect the cationdistribution, resulting in a Na increase andK decrease. The shift in cations does notnecessarily cause an increase in hydration.As long as the rate of Na entry is matchedby the rate of K loss, no gain in cationsoccurs and consequently no increase intonicity. This is illustrated in the experi-ments in which the lens was exposed tocold temperatures. Under these conditionssubstantial changes in Na and K occurred


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without an increase in lens water.41 How-ever, when a stage is reached where thegain in Na is no longer compensated forby the loss in K, then an increase in hydra-tion occurs. Not only is there a net increasein Na but, since electroneutrality must bemaintained, Cl also gains access into thelens. The increase in osmotically activecomponents would, have to be compen-sated for by an increase in water. This kindof situation might be involved at the nu-clear cataract stage. At the onset of thisstage of cataract, additional changes, suchas the loss in ATP, may affect the permea-bility still further.

The object here was to point out the fac-tors involved in the osmotic swelling thatoccurs during the development of galac-tose cataract. I have neglected to delineatethe metabolic changes which are undoubt-edly important, especially as they affectthe later stages of the cataract. The recentfinding of Becker and Cotlier42 that thelens permeability is dependent upon me-tabolism is particularly pertinent. Anotherfinding related to metabolism which re-quires further study is the observation ofPatterson and co-workers,43 who foundthat a diet rich in fats and proteins delaysthe nuclear cataract stage. Although somestudies on protein metabolism in galactosecataract"1 *15 have been reported, an at-tempt must be made to integrate thesefindings in order to account for the ap-pearance of the dense nuclear opacity. Ob-viously, additional work is needed beforewe can have a more definitive picture con-

cerning the pathogenesis of galactose cata-ract.

Summary

A summary of the events occurring ingalactose cataract is presented in Fig. 10.The primary factor initiating this type ofcataract is the high concentration of galac-tose in the aqueous humor. The abnormallevel of galactose in the lens triggers theenzyme aldose reductase to convert galac-tose to dulcitol. Since the lens membranesare relatively impermeable to sugar alco-hols, dulcitol once formed begins to ac-cumulate, creating a hypertonic condition.To maintain osmotic equilibrium, water isdrawn into the lens fibers. Unless, at thispoint, the swelling is checked by with-drawing galactose, the viability of the lenssteadily declines. Even before any lenschanges are grossly visible—the prevacuolestage—the resulting osmotic swelling hasdeleterious effects on the lens. The increasein hydration markedly affects the aminoacid transport mechanism, accounting forthe decrease in amino acid content. In theprevacuole stage, osmotic changes proba-bly are also responsible for the appearanceof hydrops, which later, with further in-crease in water, disintegrates to form vacu-oles.

In the late vacuolar stage the dulcitolcontent is maintained at the level observedin the initial stage. This probably does notmean its synthesis has stopped, but sincethe permeability properties are sufficientlyaltered the rate of exit is equal to that of

High galactose level

Water

galactose galactose

NORMAL LENS PRE-VACUOLESTAGE

INITIAL VACUOLARSTAGE

LATE VACUOLARSTAGE

NUCLEAR CATARACTSTAGE

Fig. 10. Changes that occur during development of a galactose cataract.



its formation. There is only a slight in-crease in water, but changes in cations re-veal a dangerously abnormal condition.The Na content is approaching the level ofK, indicating that the lens is having diffi-culty excluding Na. The significant lower-ing of ATP may be contributing to themarked changes in cation. In spite of thesechanges there is no major change in hy-dration during the period just prior to theappearance of the nuclear opacity.

The nuclear cataract stage is character-ized by another and more pronounced in-crease in lens hydration. The swelling isdue to the accompanying increase in elec-trolytes. Apparently a complete loss in theselective permeability results because dul-citol is no longer retained. Moreover, theelectrolytes are so freely diffusible thatDonnan swelling develops.

The author wishes to express his appreciationto Mr. Lorenzo O. Merola, Mr. Elias Dikmak, andMr. Bill Tung for their technical assistance duringthe course of these studies.

REFERENCES1. Cordes, F. C : Galactosemia cataracts: A

review, Am. J. Ophth. 50: 1151, 1960.2. Bickel, H., and Hickmans, E. M.: Paper

chromatographic investigations on urine ofpatients R. T. and R. R., Arch. Dis. Child-hood 27: 341, 1952.

3. Guest, G. M.: Hereditary galactose disease,J. A. M. A. 168: 2015, 1958.

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1-phosphate in liver nucleotid'es in congenitalgalactosemia, Science 125: 113, 1957.

10. Schwarz, V., Holzel, A., and Komrower, G.M.: Laboratory diagnosis of congenital galac-tosemia at birth, Lancet 1: 24, 1958.

11. Anderson, E. P., Kalckar, H. M., Kurahashi,K., and Isselbacher, K. J.: Specific enzymaticassay for diagnosis of congenital galactosemia,I. Consumption test, J. Lab. & Clin. Med. 50:469, 1957.

12. Robinson, A.: Assay of galactokinase andgalactose-1-phosphate uridylyl-transferase ac-tivity in human erythrocytes, J. Exper. Med.118: 359, 1963.

13. Cuatrecasas, P., and Segal, S.: Mammaliangalactokinase: Developmental and adaptativecharacteristics in the rat liver, J. Biol. Chem.240: 2382, 1965.

14. Lerman, S.: The lens in congenital galac-tosemia, Arch. Ophth. 61: 88, 1959.

15. Sidbury, J. B., Jr.: Molecular genetics andhuman disease, Springfield, 111., 1961, CharlesC Thomas, Publisher, pp. 61-80.

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18. Kinoshita, J. H.: Unpublished results.19. Segal, S., and Bernstein, H.: Observations on

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20. Gifford, S. R., and Bellows, J.: Histologicchanges in the lens produced by galactose,Arch. Ophth. 21: 346, 1939.

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22. Patterson, J. W.: Effect of blood supply ondevelopment of cataracts, Am. J. Physiol.180: 495, 1955.

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24. Mandel, P., and Klethi, J.: From paper readat Seventh Conference on Ophthalmic Bio-chemistry, Dedham, Mass., Feb. 22-24, 1963.

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27. Van Heyningen, R.: Formation of pblyolsby the lens of the rat with sugar cataract,Nature 184: 194, 1959.

28. Kinoshita, J. H., Merola, L. O., Satoh, K.,


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and Dikmak, E.: Osmotic changes caused bythe accumulation of dulcitol in the lenses ofrats fed with galactose, Nature 194: 1085,1962.

29. Kinoshita, J. H., Merola, L. O., and Dikmak,E.: The accumulation of dulcitol and waterin rabbit lens incubated with galactose,(Biochim. et biophys. acta 62: 176, 1962.

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34. Rosenberg, L. E., Weinberg, A. N., andSegal, -S.: The effect of high galactose dietson urinary excretion of amino acids in therat, Biochim. et biophys. acta 48: 484, 1961.

35. Wells, W. W., Pittman, T, A., Wells, H. S.,and Egan, T. S.: The isolation and identifica-tion of galactitol from the brains of galac-tosemia patients, J. Biol. Chem. 240: 1002,(1965.

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42. Becker, B., and Cotlier, E.: The efflux ofS(!rubidium from the rabbit lens, INVEST.OPHTH. 4: 117, 1965.

43. Patterson, J. W., Patterson, M. E., Kinsey,V. E., and Reddy, D. V. N.: Lens assayson diabetic and! galactosemic rats receivingdiets that modify cataract development,INVEST. OPHTH. 4: 98, 1965.

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Cataracts in Galactosemia The Jonas S. Friedenwald Memorial ...

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