Myo-inositol: Active transport by
the crystalline lens
Edward Cottier*
J1
>C myo-inositol was actively transported into rabbit crystalline lenses incubated in Tijrode's
medium. The lens/media accumulation ratios of JIiC myo-inositol reached values of 10 to 12
after 24 hr., in vitro incubations. The active transport of ^C myo-inositol into the lens was
markedly decreased by metabolic inhibitors of glycolysis and oxidative phosphorylation, and
by ouabain, or by an absence of Na+ ions in the incubation media. Kinetic studies revealed
a Km of 0.07 mM. per liter for myo-inositol transport into lens. The efflux of JiC myo-inositol
from preloaded lenses was slow, but it increased by iodoacetate. Gas chromatography determinations of myo-inositol in the lens and intraocular fluids were as follows (averages in ynM.
per kilogram of water): lens, 8835; anterior chamber aqueous humor, 135.1; posterior chamber
aqueous humor, 93.2; vitreous humor, 235.5; and serum, 40.9. The studies support the concept of active transport and slow efflux of myo-inositol into the lens, as well as the presence
of transport mechanisms specific for cyclic alcohols in tissues.
Key words: inositol, crystalline lens, transport mechanisms, ocular fluids, Tyrode's medium,
radioactivity, chromatography, rabbit.
I
adult sheep, rabbit, and human lenses are
30, 7, and 14 mM. per kilogram of lens,
respectively.1"3
Relatively few attempts have been made
to explain the reasons for such high levels
of myo-inositol in the lens. Kinoshita and
his co-workers29 found that calf lenses incubated with 14CG glucose contained a
minimal amount of radioactivity in the
inositol fraction; the main bulk of radioactivity remained as glucose, or was present in sorbitol or fructose. These experiments indicate that a slow myo-inositol
synthesis from glucose takes place in the
lens. Another possibility is that an active
transport system for myo-inositol, similar
to those existing in kidney4 and Erhlich
ascites cells,3 is functioning in the lens.
This report deals with such a system, including active transport and slow efflux
of myo-inositol from the rabbit crystalline
nositol (myo-inositol, meso-inositol) is
widely distributed in animal tissues, plants,
and microorganisms, in free and phosphorylated forms. In the bovine crystalline
lens, free myo-inositol represents more than
99 per cent of the total tissue inositol in
concentrations of 14 to 20 mM. per kilogram of wet lens. Myo-inositol averages for
From the Department of Ophthalmology and the
Oscar Johnson Institute, Washington University
School of Medicine, St. Louis, Mo.
Supported by grant NBO 7393-01 and Career Development Award of the National Institute of
Neurological Diseases and Blindness, National
Institutes of Health, Bethesda, Md.
Manuscript submitted June 2, 1970; revised manuscript accepted June 30, 1970.
"Present address: Department of Ophthalmology,
University of Illinois, Medical College, Chicago,
111.
681
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682 Cotiier
lens. The experimental results present additional evidence for the existence of myoinositol-specific, carrier transport systems
within cell membranes.
Material and methods
Preparation of tissue. Albino rabbits (Haskins
Rabbitry, Creve Coeur, Mo.), weighing 2 to 3
kilograms, were put to death by intracardiac ailinjections. The lenses were obtained by enucleation
and opening of the eye at the posterior pole. Immediately after removal, the lenses were carefully
transferred, with a Teflon-coated loop, to the incubation vessels.
Incubations and sampling
Transport into lens. Lenses (320 to 360 mg.)
were incubated in 2 or 10 ml. of Tyrode's medium
(pH 7.4), containing :14C myo-inositol (20 pM
per milliliter), in Kjeldhal round-bottom flasks
placed in a Magni-Whirl, metabolic shaker at 37°
C. The Tyrode's solution contained (in mM. per
liter) NaCl, 137; KC1, 2.68; CaCl2 • 2H,O, 2.04;
MgCl3-6H2O, 0.49; NaH2PO4, 0.416; NaHCO3,
11.9; and D-glucose, 5.50; it was gassed before incubations with a mixture of 95 per cent O2 and 5
per cent CO2. Two control flasks without tissue
were incubated simultaneously. Short-term incubations (4 hr.) took place in 2 ml. of medium;
longer incubations (8, 12, and 24 hr.), in 10 ml.
of media. A maximum of 1 fiM of medium glucose
per lens per hour was utilized, amounting to 35
per cent of the total glucose in the flask with
2 ml. of incubation media (4 hr.) and 43 per
cent of the total glucose in the flask with 10 ml.
of incubation medium (24 hr. incubations).
Before and after incubations, 20 /iL samples of
media were obtained and plated in planchets. Following incubations the lenses were weighed on a
Roller-Smith balance, transferred to conical glass
homogenizers containing 2 ml. of 15 per cent
(weight/volume) trichloracetic acid, ground and
centrifuged at 2,500 r.p.m. Next, 20 ph samples of
the supematants were plated in planchets. All
planchets were counted at infinite thickness in a
Nuclear Chicago gas-flow counter; the radioactivity
in the samples was calculated after subtraction of
background. The ratio of counts per minute per
milliliter of tissue water to counts per minute per
milliliter of final media represented the accumulation ratio. An average of 66.6 per cent of lens
water was used for the calculations, representing
the average water for these rabbit lenses as determined by weighing, drying at 100° C , and weighing again.
Efflux from lens. Lenses were incubated in a
fashion similar to that described above with 14C
myo-inositol for 18 to 22 hr. The lenses were then
gently transferred to Kjeldhal round-bottom flasks
containing 2 ml. of Tyrode's medium (without
Investigative Ophthalmology
September 1970
any 14C myo-inositol) and incubated for 4 hr. in
the shaking bath at 37° C. At hourly intervals, 20
/*L samples from the media were obtained, plated
in planchets, and counted as described above. At
the end of the incubations, the lenses were
weighed, homogenized in 15 per cent trichloracetic acid (TCA), and processed as lenses from
transport studies. The results were calculated as
l4
C myo-inositol effluxed from the lens, a percentage of the total in the lens at each timed interval:
Eff. lens* =
EfF. mediaf
Ace. lens| + Eff. media
xlOO.
Chromatography of 14C radioactivity in lenses
and media. Rabbit lenses were weighed, homogenized in 2 ml. of 15 per cent TCA, and centrifuged at 2,500 r.p.m. The supernatant solutions
were treated with trioctylamine to remove TCA,
and lyophilized to a volume of approximately 400
u-L. Then, 5 fiL of the concentrated supematants
and of the final incubation media, and 5 juL samples containing 1 mg. per milliliter of D-glucose,
myo-inositol, D-glucuronic acid, D-fructose, and
D-sorbitol were placed at the origin of Whatman
No. 4 paper strips. All samples were run overnight in ethyl acetate-pyridine-water (W 120:
50:40) in the ascending direction, using the "pad"
technique.0 To further separate myo-inositol and
D-glucuronic acid, lyophilized lens supematants
and standards were run in phenol-NH4OH (200:
1) for 16 hr. by ascending chromatography. Chromatograms were developed with AgNO3 as described by Smith.0 Rfs values were calculated relative to glucose (Rg = 100). The paper strips were
sliced along each millimeter of their length, and
counted directly for 14C radioactivity in a Nuclear
Chicago gas-flow counter.
Distribution of 1J*C myo-inositol in the lens.
Nine lenses, incubated as described above for 1,
2, or 3 hr. in Tyrode's medium containing 11C
myo-inositol, were dissected in the following fashion: the lenses were frozen in dry ice, and trephined in the anterior-posterior direction with a
6 mm. cork bore. This central core was divided
by sectioning with a razor blade into five, approximately equal parts from the anterior to the posterior segment. The most anterior area, or No. 1,
included the epithelium of the lens. Each section
was immediately weighed and processed in a
fashion similar to that used for the entire lens.
The equatorial, or peripheral, ring of lens outside
the trephined area was processed simultaneously
"Efflux from the lens, or efflux at any time interval, as a
per cent of the total, initial amount of myo-inositol in the
lens.
f Total 14C myo-inositol efflux from the lens into the media
at any time interval = (c.p.m. per milliliter final media
x final media volume).
t Total 14C myo-inositol accumulated by the lens at the
end' of incubation ("C myo-inositol c.p.m. per milliliter
lens water x volume lens water).
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Volume 9
Number 9
with the other areas of the lens. The results were
expressed in ratios of 14C myo-inositol in lens
water of each segment of 14C myo-inositol in medium.
Myo-inositol in ocular fluids and lens. Samples
from ocular fluids, lens, and plasma were obtained
from five albino rabbits weighing 2 kilograms
each. The animals were restrained with a rabbit
wrapper, the eyes gently proptosed, and samples
of posterior chamber and anterior chamber aqueous humor withdrawn into a calibrated pipette.
Vitreous humor was aspirated with a syringe following aqueous sampling. Blood was obtained by
cardiac puncture, and the serum was separated by
centrifugation. These samples and the lenses were
frozen immediately in dry ice, they were then
chromatographed by the method of Sweeley and
his colleagues7 as follows: a 50 /*L portion of
anhydrous
pyridine-hexamethyldisilazane-trimethyl-chlorosilane (17:2:1, by volume) was
added to the freeze-dried samples of the tissue
extracts; these were kept at room temperature for
48 hr. before chromatography. Then, (iL portions
of the pyridine extracts were injected into a Hewlett-Packard F and M model 810 gas chromatograph with a 10 ml. Hamilton syringe fitted with
a Chaney adapter. Chromatography was carried
out on 15 per cent ethylene glycol succinate
(ECS) on 80 to 100 mesh Gas-Chrom P (Applied Science Laboratories, State College, Pa.)
in a 12 ft. by 0.25 in. glass tube at temperatures
between 150° and 160° C. The carrier gas was
helium with a flow of 100 ml. per minute; a flame
ionization detector was used. Measurements were
obtained from the gas chromatographic record by
comparing the peak heights of individual sugars
in tissue extracts with the heights of peaks from
the corresponding sugars in standards run under
identical conditions. Linear correlation between
peak height and weight of sugar derivatives was
demonstrated for myo-inositol by Sherman and
Stewart.8
Chemicals. 14C myo-inositol was obtained from
Nuclear Chicago, Des Plaines, 111.; sodium cyanide
and D-fructose from Mallinckrodt Chemicals, St.
Louis, Mo.; 2,4-dinitrophenol from Fisher Scientific, St. Louis, Mo.; sodium iodoacetate, ouabian,
D-sorbitol, D-galactitol, D-xylitol, and myo-inositol from Sigma Chemical Company, St. Louis,
Mo.; and D-galacturonic acid from Nutritional
Biochemicals, Cleveland, Ohio.
Saturation experiments and kinetic constants.
In saturation experiments, cold myo-inositol in concentrations ranging from 10 to 900 Mm per liter
was added to Tyrode's medium containing : | C
myo-inositol, as shown in the abscissa of Fig. 4.
The conditions of incubation and volume were
identical to those described above. Calculations of
the total myo-inositol transported into the lens
were obtained from the saturation experiments.
Myo-inositol and crystalline lens 6S3
The total net myo-inositol transported per time
unit was found as: /iM myo-inositol per lens per
hour = lens/medium 14C myo-inositol accumulation ratio x lens water x concentration of myoinositol in the medium. As a small variation in
water content occurred among lenses, this calculation was done on an individual basis. The
values for total myo-inositol transported into the
lens were plotted as a Lineweaver-Burk9 plot.
The Michaelis-Menten constant (K m ) for myoinositol (dissociation constant of the substratecarrier complex) and the maximum velocity
(Vinax) of myo-inositol transport into the lens
were found.
Ca++—free or Na+—free media.
Ca^-free
Ty-
rode's media were prepared without CaCl among
the components of the media. Na+-free media
were prepared as previously described12 substituting equimolar concentrations of sucrose for NaCl.
Results
Accumulation of "C myo-inositol by the
lens. Rabbit lenses accumulated 14C myoinositol against the concentration gradient
when incubated in vitro (Fig. 1). The
lens/medium accumulation ratios were
found to be 2.31 ± 0.32 at 4 hours (n = 36);
4.30 ± 0.58 at 8 hours (n = 4); 5.54 ± 0.72
at 12 hours (n = 4); 9.30 ± 0.98 at 19
hours (n = 8); and 12.60 ±1.2 at 24 hours
(n = 4). As shown in Fig. 1, the active
transport of "C myo-inositol was linear
with time, and at 24 hr. had not reached
saturation.
These tissue/media accumulation ratios
reflected only in part the gradient against
which 14C myo-inositol was taken up by
the lens, as the rabbit lenses contained 5
to 7 mM per liter of myo-inositol, while
the concentrations of 14C myo-inositol in
the medium were 3 to 5 /.iM per liter. In
studies on kidney transport of myo-inositol,
tissue slices were preincubated in medium
to decrease the tissue levels of inositol;
this also served to increase the accumulation ratios of labeled myo-inositol.4 Attempts to decrease the endogenous levels
of myo-inositol in the lens were made, but
preincubation in Tyrode's medium for 2
to 24 hr. at 37° or 4° C. did not result in
significant losses of lens myo-inositol.
14
C myo-inositol in lens supernatants and
media. By paper chromatography in ethyl
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Investigative Ophthalmology
September 1970
684 Cotlier
.5 12
| 6
I4
S.D.
£ 2
8
12 16
20
24
HOURS
Fig. 1. The 14C myo-inositol accumulation ratios
of normal rabbit lenses at various time intervals.
Each point represents the number of lenses described in the text.
500
acetate-pyridine-water, myo-inositol spots
(Rf = 28) separated from D-glucuronic
acid (Rf = 12), sorbitol (Rf = 84), Dglucose (Rg — 100), and D-fructose (Rf
= 120). After precipitation with TCA, the
14
C radioactivity of aliquots of lens supernatants and final incubation media was distributed in the chromatograms as shown in
Fig. 2. By this procedure, as well as by
chromatography in phenol-ammonia-water,
100 per cent of the 14C counts in the supernatants and media fractions were found in
myo-inositol, indicating that no further
metabolism of the cyclic alcohol had taken
place after entering the lens, and that no
other 14C labeled compounds had diffused
into the media.
Protein and lipoprotein in the TCA precipitates were washed twice with water,
plated, and counted for 14C radioactivity.
Only 0.6 to 0.9 per cent of the total 14C
counts in the lens were found in the precipitates. Of the 14C myo-inositol found in
the lens, 99 per cent or more was in the
TCA supernatants as free myo-inositol.
The possibility that a 14C myo-inositol
bond to protein or lipid was broken by the
vigorous TCA extraction was explored by
extracting single lenses with 6 ml. of chloroform-methanol (2:1). The precipitate, or
14
C myo-inositol in lens
400
§300
^
14
Cmyo-inositol in final media
200
100
10
20
d-glucuronic
40
myo-inositol
50 Rf 60
70
80
90
d-sorbitol
100 110
d-glucose
120
d-fructose
Fig. 2. Distribution of 14C counts per minute in the chromatograms in ethyl acetatepyridine—water (12:5:4) of lens supernatants and final media. The locations of spots corresponding to d-glucuronic acid, myo-inositol, d-glucose, and d-fructose and the corresponding
Rf are shown along the abscissa.
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Volume 9
Number 9
Myo-inositol and crystalline lens 685
2.5
68
102
137
171
mEq/£ Na + in media
Fig. 3. The 14C myo-inositol accumulation ratios
of rabbit lenses incubated in Tyrode's media containing various Na+ ion concentrations. To preserve osmolarity, wherever Na+ ions were omitted,
equimolar concentrations of sucrose were added to
the media. Each point represents the mean of
four lenses, incubated for 4 hr. as described in the
section on methods and materials.
inositol bound to phosphoprotein, contained
only 0.6 per cent of the total 14C radioactivity of the lens. These supernatants
were separated after addition of 2 ml. of
H..O, into an upper, or water, phase (free
myo-inositol) and a lower, or organic,
phase (lipid myo-inositol), and contained
98.5 and 0.5 per cent respectively of the
total lf C counts in the lens.
Metabolic and Na+ ions requirements.
The active transport of 1IC myo-inositol
into the lens was dependent on both glycolytic and oxidative phosphorylation. In 4
hr. experiments the addition of the following inhibitors resulted in the following XIC
myo-inositol lens/media accumulation ratios as percentages of control lenses: iodoacetate (1 mM. per liter), 10 per cent;
cyanide (1 mM. per liter), 73 per cent;
2,4-dinitrophenol (1 mM. per liter), 50
per cent; and ouabain (0.1 mM. per liter),
40 per cent. In 19 hr. experiments the
effects of these inhibitors were augmented.
The incubation of lenses in glucose-free
media did not alter the 1JC myo-inositol
accumulation ratios compared to controls
in the four hour experiments but decreased those of lenses incubated for 19
hr. to 50 per cent of controls. Thus, in the
absence of glucose, the endogenous lens
substrates appeared to provide the necessary energy for myo-inositol transport in
short-term incubations.
The absence of Ca++ resulted in 14C myoinositol lens/media accumulation ratios of
38 per cent of controls. The removal of Na+
ions from the media resulted in decreases
in the transport of "C myo-inositol into the
lens (Fig. 3), proportional to the concentration of Na+ in the media.
Kinetics of myo-inositol transport into
lens. The addition of nonlabeled myo-inositol to the media resulted in saturation
of the transport system and decreased l l C
myo-inositol accumulation ratios (Fig. 4).
From the values of total myo-inositol transported into the lens at various external concentrations, the maximum velocity (Vmax)
and half-saturation concentration (Km)
were calculated. The Vmnx was 28 /.iM per
kilogram lens water per hour, and the Km
= 62 fiM per liter. At lower Na+-media
concentrations (Na+ = 68 mEq. per liter
or Na+ = 34 mEq. per liter), the total myoinositol transported into lens decreased
(Fig. 5) with an increase in the Kln to 350
ju,M per liter but without appreciable
change in the Vmax (Fig. 6). These findings were suggestive of a common transport
system for inositol and Na+ ions, each sharing similar carriers, as has been previously
postulated for the Na+-linked transport of
myo-inositol by Ehrlich cells.5 It is currently accepted that a majority of the active
transport systems for carbohydrates, amino
acids, and other organic compounds in
various tissues,17- 21 including lens,12 depend
on the concentration of Na+ ions in the
media.
Distribution of 14C myo-inositol in lens.
The distribution of 1IC myo-inositol in the
lenses after 1 and 2 hr. of incubation is
shown in Fig. 7. The equatorial area and
the anterior part of the lens containing the
epithelium showed much higher accumulation ratios of the cyclic alcohol than other
areas of the lens. These findings indicated
that the active transport system for myoinositol took place mainly through the
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Investigative Ophthalmology
September 1970
686 Cotlier
0
13
5 7
10
50 100
Myo- inositol^i/moles /I x I01
1000
Fig. 4. Effect of various myo-inositol media concentrations on the lens/media 14C myoinositol accumulation ratios. Each dot represents the mean of three or more rabbit lenses incubated for 4 hr. as described in the section on methods and materials.
Naf'l49mEq/£
14
12
\
10
Na*=34mEq/fL
Km =350/2. moles/&
1/V 8
Na*=l49mEq/£>
Km=62p moles/(L
I
0
1 2
3
x10
5
7
10
12
moles I*> Myo-inositol
Fig. 5. Total myo-inositol accumulated by lenses
incubated in complete Tyrode's media with Na+
= 149 mEq. per liter and in media containing
Na+ = 68 mEq. per liter and Na+ = 34 mEq.
per liter. Each dot represents the mean of three
or more rabbit lenses incubated for 4 hr. as described in the section on methods and materials.
epithelium of the lens, a characteristic of
other active transport systems of the lens
such as those for SGRb, 4-K, or 14C a-aminoisobutyric acid.10'12
Ages of lenses. The 14C myo-inositol
lens/media accumulation ratios by rabbit
lenses decreased with the age of the rabbits. The accumulation ratio values at 4
hr. for rabbit lenses of various ages were:
10
I/S
Fig. 6. Lineweaver-Burk plot of the data shown
in Fig. 5. The reciprocal of the myo-inositol concentration in the media (1/s) in millimoles per
liter is on the abscissa, and the reciprocal of the
initial velocity (1/v) in millimoles of myo-inositol
accumulated per lens per hour on the ordinate.
0
2
5 days old (52 mg.*), 3.58; 15 days old
(97 mg.), 2.74; 30 days old (140 mg.),
2.46; 3 months old (329 mg.), 1.87; 6
months old (475 mg.), 1.50; and 1 year old
(545 mg.), 0.902 (Fig. 8).
0
Average weight of the rabbit lenses.
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Volume 9
Number 9
Myo-inositol and crystalline lens 6S7
1.8
Incubationo 1 hr.
•2 hr.
1.6
1.2
1.0
0.8
0.6
•S 0.4
«* 0.2
5
Post.
E.R.
Ant.
LENS
AREAS
Fig. 7. The n-'C myo-inositol lens/media accumulation ratios in various areas of the lens at 1 and
2 hr. of incubation. ER = equatorial ring. The
central core has been divided into five areas from
the anterior (Ant) to the posterior (Post) pole of
the lens. Each clot corresponds to averages of
three lens sections.
Efflux of ™C myo-inositol from lens. The
efflux of 14C myo-inositol proceeded at a
maximum rate of 4 per cent of the total in
the lens. As seen in Fig. 9, these findings
agree with those of van Heyningen, who
found only traces (0.06 mg.) of lens inositol
in the media after 5 hr. incubations in a
Krebs-phosphate-Ringer media containing
glucose.-' The lens efflux of 34C myo-inositol
was not significantly modified by additions
to the efflux media of nonlabeled inositol
in various concentrations up to 30 niM. per
liter, 1 mM, ouabain, or into Ca4"1"- or
Na++- free Tyrode's medium.
The addition of 1 mM. sodium iodoacetate to the efflux media resulted in increased
effluxes of 14C myo-inositol as shown in
Fig. 9. This effect of iodoacetate on " C
myo-inositol efflux from the lens could result from a decreased uptake (reconcentration) by the tissue as the initial 30 minute
rate of efflux was not affected.
Discussion
At steady state, the concentrations of
myo-inositol in the lens exceed many times
.5
1 4,00
Wt.52mg
i
3.00
Wt. 97mg
7. 140mg
2.00
m. 329 mg
Wt. 475 mg
Wt.545mg
1.00
0u
5d 30d
3mos
AGE
6mos
OF RABBITS
1yr
Fig. 8. The 14C myo-inositol lens/media accumulation ratios at various ages (weight) of the
lens. Each dot corresponds to the average of five lenses, incubated for 4 hr. as described in
the section on methods and materials.
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Inve.itillative Ophthalmology
September 1970
688 Cotlier
Immole// [AA
Fig. 9. Efflux of 14C myo-inositol from rabbit
lenses into Tyrode's media. Each dot corresponds
to average of samples obtained from 18 control
lenses and 6 lenses incubated in media containing
1 mM. per liter of sodium iodoacetate (IAA).
that in the surrounding fluids. Average
ratios of myo-inositol lens water to posterior
chamber aqueous humor are 95; the lens
water/vitreous humor ratios are 37 (see
Table I). Thus the active transport, or
"pump," for myo-inositol must be operative
in vivo. In addition, in rabbits, myo-inositol
concentrations in aqueous and vitreous humor are above plasma levels. Either active
transport of myo-inositol occurs into intraocular fluids, or these high concentrations
represent myo-inositol that has leaked from
the lens and accumulated in the aqueous
and vitreous humor compartments of the
eye. In human cerebrospinal fluid, Bunuel
and Bunuel13 found a threefold level of
myo-inositol as compared to plasma levels.
The "pump" and "leak" concepts, proposed as the means of active transport of
K+, Rb+, or amino acids into the lens,14 may
be equally applicable to the active transport and efflux of myo-inositol. The lens
accumulation ratios for 14C myo-inositol in
the experiments followed for 24 hr. indicate
that the actual transport rate was linear
and slow, and that, presumably, saturation
had not been reached at that time. Saturation would be reached after longer incubation periods in vitro, and when the tissue/
media ratios approach those found in vivo
between the lens and its surrounding fluids.
Table I. Concentration and distribution
ratios of myo-inositol in lens and intraocular fluids of rabbit eyes
Samples"
Lens
Lens water
Anterior chamber (AC)
aqueous humor
Posterior chamber (PC)
aqueous humor
Vitreous humor
Serum
Myo-inositol
(imoles/Kg. of fresh
tissue or iimoles/Kg.
of water (± S.D.)
5890 ±208
8835 ±312
136.1 ±25.1
93.2 ± 8.2
235.4 ± 28.5
40.9 ± 6.9
Distribution ratios
Lens water:PC aqueous
Lens water:AC aqueous
PC aqueous:serum
Lens water:vitreous
humor
Vitreous humor:serum
AC aqueous:serum
95
65
2.28
37.5
5.7
3.33
"Average ± S.D. of five individual samples.
For such studies, systems for prolonged
lens cultures, like those developed by Kinsey,14 must be used. The slow initial velocity for myo-inositol transport is also reflected in the calculated Vmax of 28 /AM per
kilogram of lens water per hour, a value considerably lower than the Vmax for accumulation of 8GRb or 14C a-AIB by the lens of
10.5 and 2 mM. per kilogram of lens water
per hour respectively.12-14> 15 The affinities
of the transport "carriers" for myo-inositol,
however, are higher than those of other
compounds actively transported into lens.
The Km for myo-inositol is 62 ^M per kilogram of lens water; that of Rb+ is 2.3 mM.
per kilogram of lens water; and that of
a-AIB is 2.5 mM. per kilogram of lens
water.12 These kinetics actually favor the
active uptake of myo-inositol by the lens
in physiologic conditions as the concentration in the posterior chamber is 93 JUM
per kilogram of water, a value slightly
above the Km for transport into lens.
In the lens, the efflux of 14C myo-inositol
into media is slow, at a maximum rate of
4 per cent of the total in the lens. The
efflux is not increased by 1 mM. ouabain
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Volume 9
Number 9
nor 10 to 30 mM. "cold" myo-inositol in
the media. According to Heinz,10 the addition of high concentrations of a nonlabeled
substance to the efflux media, while measuring the runout from the tissue of such
a compound in the labeled isotope form,
is a measure of (1) exchange-diffusion
(carrier-mediated diffusion out of a tissue),
and (2) saturation on the outer side of the
membrane blocking re-entry of the isotope
into the tissue. The difficulties in demonstrating outside saturation, or exchangediffusion, for lens 14C myo-inositol could
represent the different affinities of the carriers for influx and efflux. Helmreich and
Kipnis'17 nomenclature for these influx and
efflux carrier affinities is Kx and K2, respectively. They have proposed that exchangediffusion is more likely to occur in a system where the K2, or Km, in the inner surface of the membrane is of similar magnitude to that of the K1} or Km, in the outer
surface of the membrane. When Kj > Kt
the affinities of the carrier for efflux are
considerably smaller than the affinities for
active uptake; consequently a slow efflux
takes place.
If that were the case with 14C myoinositol in the lens, increasing the Kt by
lowering the Na+ concentration in the
media should increase the efflux of myoinositol. However, increases of the Kt for
myo-inositol active transport from 62 to
350 M. per liter by lowering the Na+ concentration in the media from 149 mEq. per
liter to 34 mEq. per liter did not affect the efflux rate during a 5 hr. efflux period. Thus, the mechanisms for efflux of myo-inositol from the lens do
not appear to be explained on the basis of
"carriers," nor kinetic constants for uptake
or efflux. It might be possible that undirectional carriers, transporting myo-inositol into the lens, are the only operational carriers.
The relative impermeability of the lens
membranes to linear sugar alcohols has
been pointed out previously by Kinoshita
and co-workers,29 and by Pirie and van
Heyningen.1 These authors found high accumulations in the lens of sugar alcohols,
Myo-inositol and crystalline lens 639
such as galactitol or sorbitol, produced
through metabolism of high levels of galactose or glucose, respectively. The main differences between these linear alcohols and
the cyclic alcohol, myo-inositol, is that only
the latter is actively transported into the
lens.
Although it is known that myo-inositol is
an essential requirement for replication of
such yeasts as Schizosaccharomyces pombe,
Kloeckera brevis and S. carlsbergens,^' 1<J
the cellular function of this cyclic alcohol
in higher vertebrates has remained largely
undefined. It appears now that myo-inositol
is an essential requirement for the growth
of normal and cancer cells in culture,-0 and
for the preservation of the transport of
amino acids by cells cultured in vitro.21
Only that part of the transport system for
a-AIB, serine, and glycine dependent on
extracellular Na+ ions (or the Na+-K+
ATPase) is absent from inositol-deficient
cells.21 A similar function can be tentatively
assigned to myo-inositol in lens, as lenses
of animals fed high galactose diets that
have lost 70 to 80 per cent of the free amino
acids of the lens have markedly decreased
levels of myo-inositol (3 per cent of control
lenses).22 Lembach and Charalampous21
have considered, among other things, the
effects of myo-inositol on cell membranes
as playing either a structural role in the
formation of the membrane or an intermediate role in the phosphatidic acid cycle
of Hokin and Hokin.23 This concept would
be applicable to tissues such as kidney,
brain, or liver where a high percentage of
injected 14C myo-inositol is incorporated
into phospholipids and cell membranes.21
However, it does not appear valid for the
lens as, in agreement with previous investigators,2' 30 we failed to demonstrate a
significant binding of myo-inositol to lipids
or proteins of the lens membranes. It is
likely that the functions of free inositol are
altogether different from those of bound
myo-inositol.
The adult rabbit lens does not appear to
metabolize any significant amounts of myoinositol. Radioactive myo-inositol recovered
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Investigative Ophthalmology
September 1970
690 Cotlier
from the lens supernatants after 4 hr. of
incubation accounted for 98 per cent, or
more, of the radioactivity missing from the
media. In the incubation experiments with
5-day-old lenses, however, there was a discrepancy between "C myo-inositol radioactivity in media and lens water, with 30
per cent of the media counts remaining
unaccounted for. The possibility of myoinositol conversion to CO2 through various
routes, such as described in intact rats,32
must be explored in newborn rabbit lenses.
In rat kidney, myo-inositol is metabolized
to D-glucuronic acid,25 and kinetic studies
of such an enzyme system indicate a high
Km for inositol, 2.21 x 10 2 M. per liter. It
thus appears doubtful that the kidney
enzyme system has a major biological significance in vivo. Free myo-inositol in the
kidney is actively taken by the tubules arid
accumulated against the concentration
gradient in vitro.4 The alterations of these
transport systems result in excessive urinary inositol in diabetes mellitus, or following glucose loads in man.20
The conversion of glucose to myo-inositol
through cyclization was first suggested by
Fischer,27 and found in testicular tissue.2S
On the other hand, myo-inositol formation
through condensation of 2 and 4 carbon
fragments of glucose has led Charalampous
and Lyras33 to postulate an alternative
hypothesis for glucose conversion to myoinositol by yeast and tissues. The rates of
n
'C glucose conversion to myo-inositol are
not remarkably high. Brain slices incubated
in Krebs-Ringer with 2 mM. D-glucose form
myo-inositol at a rate of 10 /.iM per kilogram
of wet brain per hour.31 Assuming a similar rate of myo-inositol formation for rabbit
lenses, metabolizing at a rate of 3 mM.
glucose per kilogram lens per hour, only
0.3 per cent of lens glucose would be converted to myo-inositol. Kinoshita and his
associates29 found minimal inositol labeling
(65 to 83 c.p.m. per micromole), following
calf lens incubations for 24 hr. with 10
mM. per liter of [14CG] glucose with specific
activity of 1,050 c.p.m. per mole. Most of
the radioactivity was found in lens glucose,
lens sorbitol, and lens fructose. The reverse
reaction, namely, the formation of glucose,
or glucose intermediates, from myo-inositol,
may take place but, from the results of the
present experiments, does not appear of
major significance in the normal lens.
Whether lenses deprived of glucose utilize
endogenous inositol to any significant extent remains to be determined.
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