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Studies on the structure of the vitreous body
ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
Studies on the Structure VIII. Endre From
Retina and Ear
the
81, 464-479
(1959)
of the Vitreous
Comparative
Body.
Biochemistry’
A. Balazs, Torvard C. Laurent, Ulla B. G. Laurent, Marcella H. DeRoche and Doris M. Burmey Foundation, Infirmary
Department and Harvard Received
of Ophthalmology of the Massachusetts Medical School, Boston, Massachusetts September
Eye
24, 1958
INTRODUCTION
The biochemical composition of the vitreous body of cattle has been studied in this laboratory with special regard to the ascorbic acid, carbohydrates, and proteins contained, and the results have been reported in earlier papers of this series (l-6). In order to obtain a broader concept of the biological role of the vitreous body, a comparative study was carried out on its chemical composition. The concentration of ascorbic acid and of certain components of macromolecules was determined in the eyes of animals of various speciesrepresenting the zoological classesof Cephalopoda, Amphibia, Reptilia, Aves, and Mammalia. A short communication on this subject was published earlier (7). Similar studies made on t’he vitreous body of fishes will be reported in a separate paper. The ascorbic acid content of the vitreous body of horses, oxen, sheep, pigs and guinea pigs was determined by Johnson (8). The hexosamine concentration of the vitreous body and the aqueous humor was studied by Meyer et al. in oxen, sheep, pigs, dogs, rabbits, cats, and humans (9). MATERIALS The following animals were used: squid (Loligo pealii), frog (Rana catesbeiana), tortoise (Pseudemys scripta elegans), chicken (Gallus gallus), turkey (Meleagris gallopavo), pigeon (Columba livia), great horned owl (Bubo virginianus virginianus), barred owl (Striz varia varia), guinea pig (Cavia porcellus), rabbit (Lepus cuniculus), sheep (Ovis Aries), and cattle (Bos Taurus). A primate belonging to the family of i This investigation was supported National Institute of Neurological and by Fight-for-Sight Grant-in-Aid Blindness, Inc., New York, N. Y. Paper No. 67, Retina Foundation.
in part by a research grant (B-1146) from the Diseases and Blindness, Public Health Service, G-180(C6) of the National Council to Combat
464
COMPARATIVE
BIOCHEMISTRY
OF
VITREOUS
465
Cebidae, owl monkey or Dourocoulis (Aotus trivirgatus), was also used. The age of the animals, if determinable, is reported in the tables. The eyes, collected immediately after the death of the animal, were dissected for analysis within 1 hr. The one exception, the squid, was packed in ice for 24 hr. prior to preparation of the vitreous body. The ascorbic acid content of eye tissues depends on the blood level and, therefore, on the diet of the animal (10). Animals kept and fed in our laboratory before they were sacrificed received a controlled diet: tortoises received lettuce; owls, cattle liver and mice; guinea pigs, Purina guinea pig chow2 containing 0.033% ascorbic acid; rabbits, Purina rabbit chow2 containing no ascorbic acid; and owl monkeys received fresh fruit rich in ascorbic acid. Thedietsof steers, sheep, pigeons, chickens, turkeys, squid, and frogs could not be controlled. Throughout these investigations the vitreous body samples used were pooled from several eyes. Unless otherwise stated, the so-called “occipital” sample (5) of the vitreous body, which was collected from the posterior section of the eyeball after it had been divided by an equatorial cut behind the lens, was used. In some experiments the gel was subdivided into “anterior,” “posterior,” “central,” and “cortical” samples, as described earlier (5). In birds, the posterior part of the vitreous body was partially liquid. This portion was collected and analyzed separately. In many cases it was difficult to completely free the retina from the vitreous body; samples containing retinal tissue are classified as “contaminated” and those free from retina are termed “clean , . ” The vitreous gels were centrifuged for 2 hr. at 105,000 X g immediately after preparation. This treatment resulted in a water-clear liquid supernatant and a sediment, both of which were used for analysis. In some cases the aqueous humor was also collected for analyses by puncturing the anterior chamber through the cornea with a needle and applying slight suction with a syringe. The samples were dialyzed for 24 hr. against a pH 7.0 M/150 phosphate buffer containing 0.1 M sodium chloride, and the hexosamine, hexuronic acid, nitrogen, and sialic acid contents were determined. Ascorbic acid was determined on undialyzed samples. The sediment after centrifugation was used for hydroxyproline determination. METHODS
Ascorbic acid was determined according to Roe et al. (11) using 2,4-dinitrophenylhydrasine. This method permits the determination of both reduced and oxidized (dehydroascorbic acid and diketogulonic acid) ascorbic acid. Hexosamine was determined by a modification of the method of Elson and Morgan (12) after hydrolysis of the vitreous body and the .aqueous humor in 6 N hydrochloric acid for 16 hr. at 96”. It has been shown previously that hexosamine determinations made on the total vitreous hydrolyzates of cattle are not subject to error because of interfering substances (6). Hexuronic acid was determined according to the carbazole method (13) using glucuronic acid as standard. Nitrogen was determined on centrifuged and dialyzed vitreous body samples by the micro-Kjeldahl method (14). Sialic acid was determined by Bial’s test (15). Hydroxyproline was determined on the sediments of the high-speed centrifugate * Ralston
Purina
Co.,
St. Louis
2, MO.
466
BALAZS,
LAURENT,
LAURENT,
DEROCHE
AND
BUNNEY
(16, 17). In warm-blooded animals collagen was calculated from the hydroxyproline content using the multiplication factor 7.46 (16). The viscosity of the vitreous body of some animals and the viscosity of the aqueous humor of owls was studied on the supernatant after high-speed centrifugation (105,000 X g) and was measured at 37” in an Ostwald capillary viscometer. The results are expressed as specific viscosity (q., = qr - 1). RESULTS
State of the Vitreous Body and the Aqueous Humor Only two animals were found to have an entirely liquid vitreous body, viz., the squid and the owl monkey.3 In hens, turkeys, and pigeons the vitreous body behind the lens is a solid gel, while the part next to the retina is liquid. In frogs, tortoises and owls, and in all mammals except the owl monkey, the entire vitreous body is a gel. The vitreous body of the owl monkey is a viscous liquid with a specific viscosity of 8.20. After 1 hr. of centrifugation at 105,000 X g, the specific viscosity dropped to 6.86. When the centrifuged sample was dialyzed against 0.15 N NaCl, the viscosity dropped further to 5.41. After treatment for 20 min. at 37” with testis hyaluronidase (57 units U.S.P. (TR)/~IL),~ the specific viscosity dropped to 0.09. The specific viscosity of the liquid vitreous of the squid was 0.06. There was some difference between the viscosity of the liquid and the gel part of the vitreous body of birds. The liquid and the gel of pooled samples of the vitreous body of turkeys were centrifuged at, 105,000 X g for 2 hr. and subsequently dialyzed against 0.14 N NaCl. The specific viscosity of the liquid vitreous body was 0.02 and that of the liquid supernatant of the vitreous gel was 0.06. The aqueous humor of all the animals investigated was a liquid of low specific viscosity (
reported that another (Galago crassicaudatus analyses were made Inc.,
Philadelphia,
primate, agisym[Luck, C. Pa.
COMPARATIVE
BIOCHEMISTRY
TABLE Ascorbic
Acid
Content
of the Vitreous Number of eyes pooled
Stat’
OF
467
VITREOUS
I Body
in Different
of,$~,,vltreo”s
Animal
Total
ascorbic
Species acid
mg./lOO ml.
Squida
50
Tortoise Chicken (I-yr.old hens) Chicken (13-15 days old) Pigeon Great horned owl Guinea pig Rabbitb Rabbit embryos (26 days old) Sheep Steer
78 20
a Det,erminations death. b Determinations to 2 years.
Liquid Gel Gel Liquid Gel
20
0.3 0.3 0.3
2.2 0.5 15.0 6.8 f 3.4 2.8
50 61
Gel Gel
16.5 13.7
24 hr.
after
were
made
on ten
groups
death.
Animals
of animals
were
ranging
ml.
0.3
Gel Gel Gel Gel Gel
made
ascorbic
acid
mg./lOO
1.3 6.0 0.7 0.7 1.2
24 2 12 2-20 16
were
Oxidized
0.1 0.4 1.1 0.5-1.2 1.0 3.2 1.2
kept
on ice
in age from
after 5 days
in all birds and in squid. Higher values (in the order of 10 mg./lOO ml.) were found, essentially, in all mammals and in tortoises. The amount of oxidized ascorbic acid is usually low. In mammals it constitutes about 10 % of the total ascorbic acid; in the other animals investigated, the values obtained were unreliable because of low concentration. The ascorbic acid concentration of the aqueous humor of the great horned owl was determined and was found to be 0.7 mg./lOO ml. The total ascorbic acid concentration of the aqueous humor of the owl monkey was 5.7 mg./lOO ml. and that of the oxidized ascorbic acid was 1.1 mg./lOO ml. Ascorbic acid determinations on the rabbit vitreous body were carried out on ten groups of animals of different ages (from 5 days to 2 years old). No age dependence was found. However, there was a large variation in the analysis values (Table I) despite the fact that the animals were kept on an ascorbic acid-free diet. The ascorbic acid concentration of the vitreous body of 26-day-old rabbit embryos6 was lower (2.8 mg./lOO ml.) than the corresponding value in the mother (4.2 mg./lOO ml.). 6 The
gestation
period
of rabbits
is approximately
31 days.
468
BALAZS,
LAURENT,
LAURENT,
DEROCHE
AND
BUNNEY
Hexosamine and Hexuronic Acid Content of the Vitreous Body and the Aqueous Humor Determinations of nondialyzable hexosamine and hexuronic acid in the vitreous body of different animal species are shown in Tables II, III, V, VI, VII, and VIII. For comparison, some determinations of the aqueous humor are listed in Tables IV, V, VI, and VIII. The vitreous body of the squid contained approximately equivalent amounts of hexuronic acid and hexosamine (Table II). The frog vitreous body contained about one-third of the hexosamine concentration of the squid, but there was no detectable amount of hexuronic acid (<0.05 mg./lOO ml.). In the vitreous body of birds the hexosamine and the hexuronic acid concentration is low, with the hexosamine usually higher than the hexuronic acid (Table III). The only exception was the owl vitreous body, where the hexuronic acid content was about equivalent to the hexosamine (Table V). In fowls, the values are not influenced by the age or the sex of the animal. Both the liquid and the gel contained about the same amounts of hexosamine and hexuronic acid. The aqueous humor of birds contained hexosamine and hexuronic acid in the same concentrations as the vitreous body (Tables IV and V). In owls, however, the anterior part of the aqueous humor contained 20 times more hexosamine and hexuronic acid than the posterior part. The hexosamine and the hexuronic acid concentrations of the total aqueous humor, collected with a syringe, were in the same magnitude as those of the vitreous body. The results of determinations carried out on some mammals are given in Tables VI, VII, and VIII. The rabbit vitreous body contained hexosamine and hexuronic acid in the same low concentrations as the bird (Table VI), with the hexosamine somewhat higher than the hexuronic acid value. The concentrations of hexosamine and hexuronic acid are not appreciably affected by age (Table VII). The values in the guinea pig were similar to those found in the rabbit TABLE Analysis
of the Vitreous
II
The volume of the vitreous body collected 0.6 ml. and from each frog eye 0.1-0.2 ml. Number of eyes pooled
Animal
Hexosamine mg./lOO ml.
Squid Squid Frog
I II
50 60 72
15.2 13.6 4.6
of Squid
Body from
and
each squid
Hexuronic
acid
mg./lOO ml.
11.8 16.0 <0.05
Frogs eye was approximately
Nitrogen mg./1OOml.
13.8 12.2 12.5
Sialic
acid
ntg./lOO ml.
2.8
COMPARATIVE
BIOCHEMISTRY
TABLE
OF
469
VITREOUS
III
Analysis of the Vitreous Body of Birds Hens I and II were 2- to 4-year-old animals. Hens III-A were animals with active ovarial function, and Hens III-B were animals with inactive ovarii. Turkeys I were 22-week-old males. The ages group II are unknown. Roosters I, II, and III are animals between The volume of the liquid portion of the vitreous body collected eye was approximately 0.9 ml., and the volume of the gel was 1.3 Number of eyes pooled
Animal
State
ofb~~yvitreous
Hemsamine mg./lOO
Hen
I
20
Hen
II
Hen
III--A
20
Hen
III-B
20
Rooster
I
Rooster
IIQ \
Rooster Rooster
IF III
50
Chicken days Turkey
(15-17 old) Ia
50 20
56 Turkey
Ib
Turkey
II
1
Pigeon
5 “Clean” samples. b “Contaminated”
44 24
Gel Liquid Gel Liquid Gel Liquid Gel Liquid Gel Liquid Gel Liquid Gel Gel Liquid Gel Liquid Gel Liquid Gel Liquid Gel Liquid Gel Liquid
1.7 2.7 3.4 7.8 1.0 1.1 4.6 5.0 2.5 2.1 3.4 0.8 0.5 0.7 1.1 1.5 2.8 1.7 3.8 3.8 2.6 1.7 3.0
ml.
2- to 3-year-old of the same age of the turkeys in 1 and 3 years old. from each turkey ml.
Hexuronic
ng./lOO
acid
ml.
Nitrogen ng.
/lOO ml.
16.9 16.3 33.4 6.5
0.7 1.2 1.0 1.5 1.3 1.6
6.0 9.1 17.7 10.1 6.0 6.1 21.3 23.8
22.4 7.2 5.3 8.7 42.2
samples.
(Table VIII). In the owl monkey the concentrations of these two monosaccharides was much higher. In calves the hexosamine concentration was considerably higher than the hexuronic acid. In adult steers the concentrations of hexosamine and hexuronic acid were the highest of all animals investigated. The hexosamine and the hexuronic acid concentrations were compared in anterior and posterior, as well as in cortical and central, samples of the vitreous body of owls, rabbits, sheep, and owl monkeys (Tables V and
470
BALAZS,
LAURENT,
LAURENT,
DEROCHE
Analysis
of the Aqueous
TABLE Analyses
were
made
on pooled
Allima
samples
mg./lOO
n The
I II 1.
volume
of the
of
Humor of 4569
Hexuronic ml.
humor
the Aqueous
7
Number of eyes pooled
Species
Nitrogen
ml.
mg./lOO ml.
1.0 2.0 0.8 collected
Humor
-
acid
nzg./lOO
TABLE
of
Anal@s
Birds
eyes.
4.6 2.5
aqueous
BUNNEY
IV
Hexosamine
Rooster Rooster Turkey
AND
each
turkey
eye was 0.2 ml.
V and
-
from
13.2 57.0 36.6
the Vitreous
Body
of Owls
-
Analysis made on
-
rng./lOO
Barred
horned
Before
After
ml.
1.3 Total Total Anterior Posterior Total
owl
viscosity
hyaluronidase treatment
-Great owl
Specific
2.0-J 1.8 19.4
2.6 1.8 12.4
0.7
0.7
2.9 5.4
0.84
0.05
0.80
0.02
2.93 0.12
2.3
1.1
1.3 2.5 1.8
1.7 9.2
1.2
1.7
0.45
0.11
0.13
0.02
a value
of 21.5
.
V itreous
Great owl
Barred
horned
owl -
0 Hexosamine mg./lOO ml.
hod:
Central Cortical Occipita Occipita Occipita. determination
1.2 2.1 2.1 1.0
-
made
on the undialyzed
0.6
-
sample
gave
VIII). In owls, sheep, and owl monkeys the anterior or the central sample showed a significantly lower concentration than the posterior and cortical samples. This difference was much less in rabbits. The distribution of hexosamine and hexuronic acid was studied in the vitreous body of the owl monkey in the following way: The eye was frozen in Dry Ice-acetone immediately after the death of the animal. The sclera,
COMPARATIVE
BIOCHEMISTRY
OF
TABLE Analysis
of the Vitreous Age of animal
Sample
Body
months
Vitreous body Occipital Vitreous body Anterior Posterior Aqueous humor
1 12
VI
and the Aqueous
Number of eyes pooled
471
VITREOUS
HeXOSamine ?&I00
ml.
Humor
of Rabbits
Hexu&nic
Nitrogen
mg./lOO ml.
mg./lOO
Sialic acid ml.
mg./loo
20 10
2.3 2.2
1.7 1.9
16.5 8.3
0.8 0.6
8
2.9 2.4 0.3 0.4
2.1 2.3 <0.3 <0.3
1.3 0.7 4.5 6.0
0.9 0.8
?nl.
12
6 12 TABLE Analysis
of the Vitreous
Age
Number of eyes pooled
2 weeks 4 weeks 7 weeks 8 weeks 6 months 1 year 2 years
12 12 4 12 20 10 4
Body
VII of Rabbits
Volume obtained from one eye ml.
0.15 0.3 0.5 0.6 0.8 1.1 1.3
of Different
Hexosamine mg./loo
2.7 1.8 2.4 2.1 2.3 2.4 2.5
Ages
Hexuronic acid ml.
mg./lOO ffll.
0.6 1.6 1.0 1.4 1.6 1.6
Nitrogen mg./loo
ml.
6.0 3.4 2.7 3.8
and retina were removed from the still-frozen vitreous body. Then the liquid which dripped from the surface of the thawing vitreous body was collected in four 0.3-0.7-ml. fractions. The hexosamine concentration in these fractions, representing concentric portions starting from the retina toward the center and the lens, was as follows: 27.8, 26.1, 18.7, 11.8 mg./lOO ml., respectively. The hexuronic acid concentration in the same sampleswas 36.1, 35.7, 24.8, 16.4 mg./lOO ml., respectively. This indicates that the concentration decreasestoward the center and the lens as in all other mammals. The aqueous humor of all of the mammals investigated had a very low hexosamine content. In steers the hexosamine content of the aqueous humor was nearly 100 times lower than that of the vitreous body. This difference was not so great in calves, owl monkeys, and sheep. As a rule, the hexuronic acid concentration was lower than the hexosamine in the aqueous humor. choroid,
472
BALAZS,
LAURENT,
LAURENT,
DEROCHE
AND
BUNNEY
TABLE VIII and the Aqueous Humor of Mammals A volume of 0.2 ml. vitreous body was collected from each guinea pig. From 4 to 5 ml. vitreous b%ly and 0.8 ml. aqueous humor were collected from each sheep eye. From each owl monkey eye were collected 2.0 ml. vitreous body and 0.4 ml. aqueous humor. Analysis
of the Vitreous
Body
-
Animal
Sample
Number of eyes pooled
w./~OO
Guinea pig Sheep
Steer (1-3 yr. old) Calf (2~preeks old) Owl monkey I
Owl monkey II
Vitreous body Vitreous body Posterior Anterior Aqueous humor Vitreous body Aqueous humor” Vitreous body Aqueous humoru Vitreous bodyc Cortical Central Aqueous humor Vitreous bodyc
7 50
14 8
2.5
14.6 4.4 1.7 29.6 0.3 9.6 0.5 19.ld 10.9 6.0 0.5 18.7
Iexuronic acid
Nitrogen
W./O0
W&/pJ
1.9
14.6
14.4 3.3 0.9 29.9 0.1 4.0 0.1 21.7 13.7 8.5
11.1 10.1 47.1 7.9
Sialic acid
mg;.,oo
2.8 1.6 1.1 3.4
10.0 7.lb
4.2
1.5
a Before analysis the aqueous humor was concentrated 7X by dialysis against polyvinylpyrrolidone (Plasdone type ND-K-30, General Aniline and Film Co., N. Y.). The values are calculated for the unconcentrated aqueous humor. b The nitrogen content before dialysis was 20.0 mg./lOO ml. c The vitreous body was withdrawn with a syringe using a 22-gage needle. d The hexosamine determination gave a value of 27.0 mg./lOO ml. in the undialyeed sample. I s!.
,‘7
Nitrogen Content of the Vitreous Body
.The:&trogen content of the dialyzed vitreous body of different, animals was also, determined, and the results are tabulated in Tables II, III, V, VI,
VII, an$,VIII. Concentrations in the order of 10 mg./lOO ml. were found in all animals. This is only about 1% of the nitrogen concentration of dialyzed serum. The nondialyzable nitrogen values, however, showed considerable variation within each animal species,especially in birds. This is due to the difficulties encountered in the separation of the vitreous body from adhering tissues. Even the slightest retinal contamination increases the nitrogen value consideral$y. In two experiments the vitreous body of roosters (Rooster Prepn. II) and of turkeys (Turkey Prepn. I) was divided into two parts: one contaminated and one macroscopically uncontaminated. The
COMPARATIVE
BIOCHEMISTRY
OF
473
VITREOUS
contaminated samples had a nondialyzable nitrogen content which was considerably higher than those which were uncontaminated. However, the hexosamine and the hexuronic acid concentrations were not affected by the tissue contaminatjon, indicating that the tissues surrounding the vitreous body contain very little of these monosaccharides. The nitrogen values of the aqueous humor are subject to the same errors as those of the vitreous body, but to a much lesser degree. TABLE Hydroxyproline
-
IX
and Collagen Content of the Vitreous of Different Animal Species
-
-
Number of eyes pooled
AI&la1
Body
Collagen
Hydroxyproline 1LiquidC
Geld
Liquid
Gel
ng./lOO
Squid II Frog Hen I Hen II Hen IIP Hen 111’~ Rooster I Rooster IIn Rooster IID Rooster III Turkey Ia Great horned Barred Rabbit 2 4 4 6 12 Guinea Sheep
Occipital Occipital Occipital Occipital Occipital Occipital Occipital Occipital Occipital Occipital Occipital Anterior Posterior Occipital
owl
owl weeks old weeks old weeks old months old months old pig
Steer Owl
monkey
-
Occipital Occipital Occipital Occipital Anterior Posterior Occipital Anterior Posterior Anterior Posterior Occipital
a “Clean” samples. b “Contaminated” samples. c Hydroxyproline analysis values body. d Hydroxyproline analysis values portion of the vitreous body.
60 72 20 30 20
-
0.06 0.02
100 60 2 -
4
-
12 12 20 20 8 7 50
-
14 3
-
0.33 -
2.0 2.7 2.2 3.3 3.1 2.7 2.8 3.2 2.8 2.2 2.0 0.9 1.2
0.04
45 50
?ng./lOO ml.
ml.
0.03
0.33
0.30 0.45 0.15
-
-
1.9 1.4 2.2 1.4 12.8 1.0 1.8 1.1 0.9 1.9 0.9 -
-
-
obtained
on the
liquid
obtained
on the high-speed
portion
2.46
of the sediment
18.2 20.1 16.4 24.6 23.1 20.1 20.9 23.9 20.9 16.4 14.9 6.7 9.0 14.2 10.4 16.4 10.4 95.5 7.5 13.4 8.2 6.7 14.2 6.7 -
vitreous of the gel
474
BALAZS,
LAURENT,
LAURENT,
Hydroxyproline
DEROCHE
AND
BUNNEY
Content
The hydroxyproline content of the sediment of the vitreous body, collected by high-speed centrifugation, is shown in Table IX. The collagen concentrations were calculated from the hydroxyproline content, assuming that the collagen of warm-blooded animals contains 13.5 % hydroxyproline (16). It was further assumed that the collagen of the frog contains 11.0 % hydroxyproline and that of the squid 9.0%. Only traces of hydroxyproline are present in the liquid vitreous body of the squid and in the liquid part of the avian vitreous body. The liquid vitreous of the owl monkey contained only 0.33 mg./lOO ml. hydroxyproline, one-third of the value found in the vitreous gel of the sheep, which contains the least collagen of all gels. In every animal studied, the hydroxyproline content was higher in the anterior part, next to the zonular region, than in the posterior segment. The difference was most striking in the rabbit, where it was 13 times higher in the anterior part than in the posterior. The least impressive difference was found in the sheep. Sialic Acid Content The sialic acid content of frogs (Table II), owls (Table V), rabbits (Table VI), sheep, steers, and owl monkeys (Table VIII) was determined. The highest values were found in steers and frogs. In owls, rabbits, and owl monkeys, the concentration was considerably less. DISCUSSION
It has been shown that hyaluronic acid, collagen, and water-soluble proteins, the main macromolecular components of the vitreous body of the cattle eye, are unevenly distributed in the gel (5). It has also been shown that part of the water-soluble proteins are glycoproteins (4, 18). Since it has been assumed by many authors that hyaluronic acid, glycoproteins, and collagen, together or individually, are responsible for the gel character of the vitreous body, it was of special interest to carry out a comparative biochemical study on these substances and correlate the results with the physical state of the vitreous body. Due to the small volume of vitreous body obtained from most animal eyes, it was not possible to isolate and characterize the macromolecules in the same way as in cattle. Instead, the concentration of certain macromolecular components such as nondialyzable hexosamine, hexuronic acid, nitrogen, and hydroxyproline was determined. The occipital sample of the vitreous body was used for these studies, since it can best be separated from the eye without massive contamination by neighboring tissues and with good reproducibility (5). This preparation,
COMPARATIVE
BIOCHEMISTRY
OF
VITREOUS
475
however, excludes a small anterior part around the zonular region of the vitreous body. Since the macromolecules are unevenly distributed in the vitreous body, the results are representative only of the sample used and not necessarily of the total vitreous body. In a few cases, when the volume of the vitreous body permitted, distribution studies were also made. Hyaluronic
Acid
Since hyaluronic acid is composed of equivalent amounts of N-acetylglucosamine and glucuronic acid, the vitreous body was analyzed for these two monosaccharides. Based on the results obtained on the bovine vitreous body, it was assumed that direct determination of the hexosamine in the hydrolyzates of dialyzed vitreous bodies would give correct values (6). Hexosamine, however, has not been separated chromatographically into glucosamine and galactosamine fractions. The carbazole method for the determination of hexuronic acid gave reliable results when pure hyaluronic acid or dialyzed bovine vitreous body was used (3). Substances which might interfere with the carbazole reaction were not observed in the dialyzed vitreous bodies during these studies. The nondialyzable hexosamine and hexuronic acid content of thevitreous body may be interpreted as an indication of the presence of mucopolysaccharides and glycoproteins. If one assumes that the dominant hexuronic acid-containing macromolecule in the vitreous body is hyaluronic acid, as in cattle, then one can estimate the hyaluronic acid content from the hexuronic acid analysis values. After subtracting the hyaluronic acid hexosamine from the total hexosamine values, one can further speculate on the glycoprotein content of the vitreous body. With the exception of the frog and the pigeon, hexuronic acid was found in the vitreous body of all of the animals investigated. The concentration varied considerably in the different species, with the highest in the vitreous body of the steer and the lowest in that of fowls. In squid, owls, sheep, steers, and owl monkeys, the hexosamine-hexuronic acid molar ratio is close to one. In chickens, pigeons, turkeys, guinea pigs, and calves, the hexosamine content is higher than the hexuronic acid, which indicates that a large part of the hexosamine belongs to proteins. In rabbits, considerable variation was found in the hexuronic acid concentration, while the hexosamine was more constant. The hexuronic acid concentration in the aqueous humor of mammals is very low. The concentration of hexosamine is somewhat higher, but always well below the level found in the vitreous body. The aqueous humor of birds, however, contains the same amount of hexosamine and hexuronic acid as the vitreous body. Abelsdorff and Wessely (19) reported that the aqueous humor of various
476
BALAZS,
LAURENT,
LAURENT,
DEROCHE
AND
BUNNEY
owls, unlike other birds, has a high relative viscosity and that the aqueous humor close to the cornea is the most viscous. Barany et al. (20) found that hyaluronidase prepared from testes, from bacteria, and from snake or bee venom decreases the viscosity of the owl aqueous humor. Our finding that the viscosity and the hexuronic acid content were considerably higher in the sample of the aqueous humor taken from the region of the cornea than in the sample taken from the central part of the aqueous humor supports the assumption of Barany et al. that hyaluronic acid is unevenly distributed in the aqueous humor of the owl. Most probably hyaluronic acid is present only as a viscous layer (gel) on the interior surface of the cornea. These observations may be of some importance in connection with the theory that the aqueous flow is regulated by the mucopolysaccharides formed or collected on the surface of the trabecular meshworks (21).6 Water-Soluble
Proteins
The nitrogen content of the soluble protein fraction of the dialyzed and centrifuged vitreous body is obtained by subtracting the hexosamine nitrogen from the total nitrogen value (4). The soluble protein concentration, calculated from the nitrogen values, is low in all species, indicating that the vitreous body of all animals is a tissue compartment well separated from the blood vessel system. In several cases, the actual protein content of the vitreous body is probably considerably lower than the values reported, because in small eyes it is seldom possible to prepare the samples free from blood and tissue contamination. Since the hexosamine-hexuronic acid molar ratio is higher than one, it is most probable that some of the hexosamine is part of the glycoproteins, which may represent a considerable portion of the proteins (4). It is interesting that the owl monkey had the lowest protein nitrogen concentration, which, if correlated with the sialic acid content, suggests that approximately half of the proteins could be glycoproteins. Several authors have studied the hexosamine and nitrogen content of the vitreous body of sheep, rabbits, and guinea pigs. Bernbridge and Pirie (22) found that the hexosamine and nitrogen concentration in the rabbit vitreous body decreases during the first weeks of postnatal life. Meyer et al. (9), Boruchoff and Woodin (23), and Cristiansson (24) reported hexosamine analyses on sheep, rabbits, and guinea pigs which are comparable to ours. These authors used hexosamine analysis values to calculate the hyaluronic 6 Recently, L. Berggren showed in frozen owl eyes (Striz aluco) that a material which stains metachromatically with cationic dyes is present in the cornea1 part of the wall of the anterior chamber. This material is absent in the regions next to the lens and the iris. (Personal communication.)
COMPARATIVE
BIOCHEMISTRY
OF
VITREOUS
477
acid concentration in the vitreous body. One of the authors (E. A. B.) reported earlier (5) that hyaluronic acid is unevenly distributed in the adult cattle vitreous body. This observation was based on hexosamine and hexuronic acid determinations and on the quantitative preparation of hyaluronic acid. It was also found that the concentration of hyaluronic acid depends on the a,ge of the animal. Schweer et al. (25,26) later reported that the hexosamine content is unevenly distributed in the cattle vitreous body and that its concentration is dependent on the age of the animal, which is in agreement with these findings. Collagen The insoluble collagen fraction was obtained from the vitreous body after high-speed centrifugation. Collagen is the only protein which contains a large amount of hydroxyproline, and therefore, hydroxyproline is a reliable measure of the collagen concentration (16). It was interesting to note that collagen was present in all vitreous gels but was absent or very low (owl monkey) when the vitreous body was in liquid state. It is present in all animals in concentrations of the same magnitude, being somewhat higher in birds. The results indicate that collagen is a necessary component of the vitreous body if it is in gel state. Distribution
qf Macromolecules in the Vitreous Body
It ha,s been reported (5) that hyaluronic acid, collagen, and glycoproteins are unevenly distributed in the bovine vitreous body. Hyaluronic acid and glycoproteins are present in highest concentration in the gel next to the retina (3, 4, S), and the collagen is highest in the anterior segment next to the zonular region (5). Some of the distribution experiments were repeated on the vitreous body of owls, sheep, rabbits, and monkeys. Hydroxyproline, hexosamine, hexuronic acid, and nitrogen analysis values indicat,e an uneven distribution of macromolecular components similar to that found in the bovine vitreous body. One can therefore state that an uneven distribution of hyaluronic acid, soluble proteins, and collagen in the vitreous body is a general phenomenon in all animals thus far investigated. Ascorbic Acid The gel character of the vitreous body does not seem to depend on a high ascorbic acid content. In the vitreous body of rabbits of various ages, there was a large variation in the ascorbic acid content which was independent of age. In embryos the concentration was higher than in the mother animal, which corresponds to the finding of Kinsey et al. (27) in rabbit aqueous humor and Balazs et al. (6) in cattle vitreous body.
478
BALAZS, LAURENT,
LAURENT,
DEROCHE AND BUNNEY
SUMMARY
A comparative biochemical study was made on the vitreous body of the following animals: squid, frogs, tortoises, chickens, turkeys, pigeons, owls, rabbits, guinea pigs, sheep, cattle, and owl monkeys. It was found that: 1. The vitreous body of the squid is a liquid and has a low viscosity. The vitreous body of the owl monkey is a viscous fluid. In the chicken, turkey, and pigeon, the posterior part of the vitreous body is a liquid; the rest is a gel. In the other animals the vitreous body is in gel sta.te. 2. The total ascorbic acid content is high (in the order of 10 mg./lOO ml.) in adult mammals and tortoises, and it is ten times lower in the other animals investigated. The amount of oxidized ascorbic acid in mammals is about 10% of the total ascorbic acid content. 3. Only when the vitreous body is in gel state does it contain collagen which can be sedimented by high-speed centrifugation. 4. Hexuronic acid is present in the vitreous body of all species,but with a large variation in concentration. The highest values were found in squid and in some mammals (cattle, owl monkeys, sheep). Its concentration is much less in birds, rabbits, and guinea pigs. In frogs no hexuronic acid could be determined. 5. In most animals the hexosamine concentration was higher than the hexuronic acid, indicating that hexosamine-containing compounds other than hyaluronic acid, such as glycoproteins, are present. The sialic acid content supports these findings. 6. The aqueous humor of birds contains hexosamine and hexuronic acid in about the same concentrations as the vitreous body. The aqueous humor of the owl showed an uneven distribution of hexosamine and hexuronic acid. A viscous layer, presumably hyaluronic acid, was found behind the cornea. The hyaluronic acid concentration of the aqueous humor of the mammals investigated was 100 times less than that of the vitreous body. 7. In all animals the soluble protein concentration of the vitreous body is very low (about 1% of the serum concentration), indicating that there is a distinct barrier between the vitreous body and the blood vessel system. 8. Hyaluronic acid, soluble proteins, and collagen were unevenly distributed in the vitreous body of all animals investigated. REFERENCES 1. BALAZS, E. 2. VARGA, L., 3. BALAZS, E. 4. SUNDBLAD, Abstr. p. 5. BALAZS, E.
A., Am. J. Ophthalmol. 38, No. I, Part II, 21-28 (1954). AND BALAZS, E. A., Am. J. Ophthalmol. 38, No. 1, Part II, 29-36 (1954). A., in preparation. L., AND BALAZS, E. A., Intern. Congr. Biochem. 4th Congr. Vienna 1968. 20. A., Acta XV11 Cont. Ophthalmol., 1964, II, 1019-24 (1955).
COMPARATIVE
BIOCHEMISTRY
OF
VITREOUS
479
6. BALAZS, E. A., LAURENT, T. C., AND LAURENT, U. B. G., .I. Biol. Chem. 234,422430 (1959) . 7. BALAZS, E. A., Am. J. Ophthalmol. 42, Part I, 137 (1956). 8. JOHNSON, S. W., Biochem. J. 30, 1430-7 (1936). 9. MEYER, K., SMYTH, E. M., AND GALLARDO, E., Am. J. Ophthalmol. 21, 1083-90 (1!#38). 10. KINSEY, V. E., Am. J. Ophthalmol. 30, 1262-6 (1947). 11. ROE, J. H., MILLS, M. B., OESTERLING, M. J., AND DAMRON, C. M., J. BioZ. Chem. 174, 201-8 (1948). 12. BLIX, G., Acta Chem. &and. 2, 467-73 (1948). 13. DISC:HE, Z., J. BioZ. Chem. 167, 189-98 (1947). 14. MA, T. S., AND ZUAZAGA, G., Znd. Eng. Chem., Anal. Ed. 14,280-2 (1942). 15. WERNER, I., AND ODIN, L., Acta Sot. Med. Upsaliensis 67,23041 (1952). 16. NEUMAN, R. E., AND LOGAN, M. A., J. BioZ. Chem. 166,549-56 (1950). 17. MARTIN, C. J., AND AXELROD, A. E., Proc. Sot. Exptl. BioZ. Med. 83,461-2 (1953). 18. DISCIHE, Z., DANILCZENKO, A., AND ZELMENIS, G., The neutral heteropolysaccharides in connective tissue. Ciba Foundation Symposium, Chem. and BioZ. Mucopolysaccharides, 1968, pp. 11636. 19. ABELSDORFF, G., AND WESSELY, K., Arch. Augenheilk. 64,65-125 (1909). 20. BARANY, E. H., BERGGREN, L., AND VRABEC, F., Brit. J. OphthaZmoZ. 41, 25-30 (1957). 21. BARANY, E. H., in “Glaucoma.” Josiah Macy Jr. Foundation, New York, 1955, pp. 123-221. 22. BEMBRIDGE, B. A., AND PIRIE, A., Brit. J. OphthaZmoZ. 36, 784-9 (1951). 23. BORUCHOFF, S. A., AND WOODIN, A. M., Brit. J. OphthaZmoZ. 40, 113-18 (1956). 24. CRISTIANSSON, J., Acta OphthaZmoZ. 36,336-59 (1957). 25. SCHWEER, G., AND S~DHOF, H., Arch. Ophthalmol. 167, 85-91 (1955). 26. SCHWEER, G., AND KARELL, E., Arch. Ophthalmol. 167, 422-8 (1956). 27. KINSEY, V. E., JACKSON, B., AND TERRY, T. C., Arch. OphthaZmoZ. 34, 415-17 (1945).
OF
BIOCHEMISTRY
AND
BIOPHYSICS
Studies on the Structure VIII. Endre From
Retina and Ear
the
81, 464-479
(1959)
of the Vitreous
Comparative
Body.
Biochemistry’
A. Balazs, Torvard C. Laurent, Ulla B. G. Laurent, Marcella H. DeRoche and Doris M. Burmey Foundation, Infirmary
Department and Harvard Received
of Ophthalmology of the Massachusetts Medical School, Boston, Massachusetts September
Eye
24, 1958
INTRODUCTION
The biochemical composition of the vitreous body of cattle has been studied in this laboratory with special regard to the ascorbic acid, carbohydrates, and proteins contained, and the results have been reported in earlier papers of this series (l-6). In order to obtain a broader concept of the biological role of the vitreous body, a comparative study was carried out on its chemical composition. The concentration of ascorbic acid and of certain components of macromolecules was determined in the eyes of animals of various speciesrepresenting the zoological classesof Cephalopoda, Amphibia, Reptilia, Aves, and Mammalia. A short communication on this subject was published earlier (7). Similar studies made on t’he vitreous body of fishes will be reported in a separate paper. The ascorbic acid content of the vitreous body of horses, oxen, sheep, pigs and guinea pigs was determined by Johnson (8). The hexosamine concentration of the vitreous body and the aqueous humor was studied by Meyer et al. in oxen, sheep, pigs, dogs, rabbits, cats, and humans (9). MATERIALS The following animals were used: squid (Loligo pealii), frog (Rana catesbeiana), tortoise (Pseudemys scripta elegans), chicken (Gallus gallus), turkey (Meleagris gallopavo), pigeon (Columba livia), great horned owl (Bubo virginianus virginianus), barred owl (Striz varia varia), guinea pig (Cavia porcellus), rabbit (Lepus cuniculus), sheep (Ovis Aries), and cattle (Bos Taurus). A primate belonging to the family of i This investigation was supported National Institute of Neurological and by Fight-for-Sight Grant-in-Aid Blindness, Inc., New York, N. Y. Paper No. 67, Retina Foundation.
in part by a research grant (B-1146) from the Diseases and Blindness, Public Health Service, G-180(C6) of the National Council to Combat
464
COMPARATIVE
BIOCHEMISTRY
OF
VITREOUS
465
Cebidae, owl monkey or Dourocoulis (Aotus trivirgatus), was also used. The age of the animals, if determinable, is reported in the tables. The eyes, collected immediately after the death of the animal, were dissected for analysis within 1 hr. The one exception, the squid, was packed in ice for 24 hr. prior to preparation of the vitreous body. The ascorbic acid content of eye tissues depends on the blood level and, therefore, on the diet of the animal (10). Animals kept and fed in our laboratory before they were sacrificed received a controlled diet: tortoises received lettuce; owls, cattle liver and mice; guinea pigs, Purina guinea pig chow2 containing 0.033% ascorbic acid; rabbits, Purina rabbit chow2 containing no ascorbic acid; and owl monkeys received fresh fruit rich in ascorbic acid. Thedietsof steers, sheep, pigeons, chickens, turkeys, squid, and frogs could not be controlled. Throughout these investigations the vitreous body samples used were pooled from several eyes. Unless otherwise stated, the so-called “occipital” sample (5) of the vitreous body, which was collected from the posterior section of the eyeball after it had been divided by an equatorial cut behind the lens, was used. In some experiments the gel was subdivided into “anterior,” “posterior,” “central,” and “cortical” samples, as described earlier (5). In birds, the posterior part of the vitreous body was partially liquid. This portion was collected and analyzed separately. In many cases it was difficult to completely free the retina from the vitreous body; samples containing retinal tissue are classified as “contaminated” and those free from retina are termed “clean , . ” The vitreous gels were centrifuged for 2 hr. at 105,000 X g immediately after preparation. This treatment resulted in a water-clear liquid supernatant and a sediment, both of which were used for analysis. In some cases the aqueous humor was also collected for analyses by puncturing the anterior chamber through the cornea with a needle and applying slight suction with a syringe. The samples were dialyzed for 24 hr. against a pH 7.0 M/150 phosphate buffer containing 0.1 M sodium chloride, and the hexosamine, hexuronic acid, nitrogen, and sialic acid contents were determined. Ascorbic acid was determined on undialyzed samples. The sediment after centrifugation was used for hydroxyproline determination. METHODS
Ascorbic acid was determined according to Roe et al. (11) using 2,4-dinitrophenylhydrasine. This method permits the determination of both reduced and oxidized (dehydroascorbic acid and diketogulonic acid) ascorbic acid. Hexosamine was determined by a modification of the method of Elson and Morgan (12) after hydrolysis of the vitreous body and the .aqueous humor in 6 N hydrochloric acid for 16 hr. at 96”. It has been shown previously that hexosamine determinations made on the total vitreous hydrolyzates of cattle are not subject to error because of interfering substances (6). Hexuronic acid was determined according to the carbazole method (13) using glucuronic acid as standard. Nitrogen was determined on centrifuged and dialyzed vitreous body samples by the micro-Kjeldahl method (14). Sialic acid was determined by Bial’s test (15). Hydroxyproline was determined on the sediments of the high-speed centrifugate * Ralston
Purina
Co.,
St. Louis
2, MO.
466
BALAZS,
LAURENT,
LAURENT,
DEROCHE
AND
BUNNEY
(16, 17). In warm-blooded animals collagen was calculated from the hydroxyproline content using the multiplication factor 7.46 (16). The viscosity of the vitreous body of some animals and the viscosity of the aqueous humor of owls was studied on the supernatant after high-speed centrifugation (105,000 X g) and was measured at 37” in an Ostwald capillary viscometer. The results are expressed as specific viscosity (q., = qr - 1). RESULTS
State of the Vitreous Body and the Aqueous Humor Only two animals were found to have an entirely liquid vitreous body, viz., the squid and the owl monkey.3 In hens, turkeys, and pigeons the vitreous body behind the lens is a solid gel, while the part next to the retina is liquid. In frogs, tortoises and owls, and in all mammals except the owl monkey, the entire vitreous body is a gel. The vitreous body of the owl monkey is a viscous liquid with a specific viscosity of 8.20. After 1 hr. of centrifugation at 105,000 X g, the specific viscosity dropped to 6.86. When the centrifuged sample was dialyzed against 0.15 N NaCl, the viscosity dropped further to 5.41. After treatment for 20 min. at 37” with testis hyaluronidase (57 units U.S.P. (TR)/~IL),~ the specific viscosity dropped to 0.09. The specific viscosity of the liquid vitreous of the squid was 0.06. There was some difference between the viscosity of the liquid and the gel part of the vitreous body of birds. The liquid and the gel of pooled samples of the vitreous body of turkeys were centrifuged at, 105,000 X g for 2 hr. and subsequently dialyzed against 0.14 N NaCl. The specific viscosity of the liquid vitreous body was 0.02 and that of the liquid supernatant of the vitreous gel was 0.06. The aqueous humor of all the animals investigated was a liquid of low specific viscosity (
reported that another (Galago crassicaudatus analyses were made Inc.,
Philadelphia,
primate, agisym[Luck, C. Pa.
COMPARATIVE
BIOCHEMISTRY
TABLE Ascorbic
Acid
Content
of the Vitreous Number of eyes pooled
Stat’
OF
467
VITREOUS
I Body
in Different
of,$~,,vltreo”s
Animal
Total
ascorbic
Species acid
mg./lOO ml.
Squida
50
Tortoise Chicken (I-yr.old hens) Chicken (13-15 days old) Pigeon Great horned owl Guinea pig Rabbitb Rabbit embryos (26 days old) Sheep Steer
78 20
a Det,erminations death. b Determinations to 2 years.
Liquid Gel Gel Liquid Gel
20
0.3 0.3 0.3
2.2 0.5 15.0 6.8 f 3.4 2.8
50 61
Gel Gel
16.5 13.7
24 hr.
after
were
made
on ten
groups
death.
Animals
of animals
were
ranging
ml.
0.3
Gel Gel Gel Gel Gel
made
ascorbic
acid
mg./lOO
1.3 6.0 0.7 0.7 1.2
24 2 12 2-20 16
were
Oxidized
0.1 0.4 1.1 0.5-1.2 1.0 3.2 1.2
kept
on ice
in age from
after 5 days
in all birds and in squid. Higher values (in the order of 10 mg./lOO ml.) were found, essentially, in all mammals and in tortoises. The amount of oxidized ascorbic acid is usually low. In mammals it constitutes about 10 % of the total ascorbic acid; in the other animals investigated, the values obtained were unreliable because of low concentration. The ascorbic acid concentration of the aqueous humor of the great horned owl was determined and was found to be 0.7 mg./lOO ml. The total ascorbic acid concentration of the aqueous humor of the owl monkey was 5.7 mg./lOO ml. and that of the oxidized ascorbic acid was 1.1 mg./lOO ml. Ascorbic acid determinations on the rabbit vitreous body were carried out on ten groups of animals of different ages (from 5 days to 2 years old). No age dependence was found. However, there was a large variation in the analysis values (Table I) despite the fact that the animals were kept on an ascorbic acid-free diet. The ascorbic acid concentration of the vitreous body of 26-day-old rabbit embryos6 was lower (2.8 mg./lOO ml.) than the corresponding value in the mother (4.2 mg./lOO ml.). 6 The
gestation
period
of rabbits
is approximately
31 days.
468
BALAZS,
LAURENT,
LAURENT,
DEROCHE
AND
BUNNEY
Hexosamine and Hexuronic Acid Content of the Vitreous Body and the Aqueous Humor Determinations of nondialyzable hexosamine and hexuronic acid in the vitreous body of different animal species are shown in Tables II, III, V, VI, VII, and VIII. For comparison, some determinations of the aqueous humor are listed in Tables IV, V, VI, and VIII. The vitreous body of the squid contained approximately equivalent amounts of hexuronic acid and hexosamine (Table II). The frog vitreous body contained about one-third of the hexosamine concentration of the squid, but there was no detectable amount of hexuronic acid (<0.05 mg./lOO ml.). In the vitreous body of birds the hexosamine and the hexuronic acid concentration is low, with the hexosamine usually higher than the hexuronic acid (Table III). The only exception was the owl vitreous body, where the hexuronic acid content was about equivalent to the hexosamine (Table V). In fowls, the values are not influenced by the age or the sex of the animal. Both the liquid and the gel contained about the same amounts of hexosamine and hexuronic acid. The aqueous humor of birds contained hexosamine and hexuronic acid in the same concentrations as the vitreous body (Tables IV and V). In owls, however, the anterior part of the aqueous humor contained 20 times more hexosamine and hexuronic acid than the posterior part. The hexosamine and the hexuronic acid concentrations of the total aqueous humor, collected with a syringe, were in the same magnitude as those of the vitreous body. The results of determinations carried out on some mammals are given in Tables VI, VII, and VIII. The rabbit vitreous body contained hexosamine and hexuronic acid in the same low concentrations as the bird (Table VI), with the hexosamine somewhat higher than the hexuronic acid value. The concentrations of hexosamine and hexuronic acid are not appreciably affected by age (Table VII). The values in the guinea pig were similar to those found in the rabbit TABLE Analysis
of the Vitreous
II
The volume of the vitreous body collected 0.6 ml. and from each frog eye 0.1-0.2 ml. Number of eyes pooled
Animal
Hexosamine mg./lOO ml.
Squid Squid Frog
I II
50 60 72
15.2 13.6 4.6
of Squid
Body from
and
each squid
Hexuronic
acid
mg./lOO ml.
11.8 16.0 <0.05
Frogs eye was approximately
Nitrogen mg./1OOml.
13.8 12.2 12.5
Sialic
acid
ntg./lOO ml.
2.8
COMPARATIVE
BIOCHEMISTRY
TABLE
OF
469
VITREOUS
III
Analysis of the Vitreous Body of Birds Hens I and II were 2- to 4-year-old animals. Hens III-A were animals with active ovarial function, and Hens III-B were animals with inactive ovarii. Turkeys I were 22-week-old males. The ages group II are unknown. Roosters I, II, and III are animals between The volume of the liquid portion of the vitreous body collected eye was approximately 0.9 ml., and the volume of the gel was 1.3 Number of eyes pooled
Animal
State
ofb~~yvitreous
Hemsamine mg./lOO
Hen
I
20
Hen
II
Hen
III--A
20
Hen
III-B
20
Rooster
I
Rooster
IIQ \
Rooster Rooster
IF III
50
Chicken days Turkey
(15-17 old) Ia
50 20
56 Turkey
Ib
Turkey
II
1
Pigeon
5 “Clean” samples. b “Contaminated”
44 24
Gel Liquid Gel Liquid Gel Liquid Gel Liquid Gel Liquid Gel Liquid Gel Gel Liquid Gel Liquid Gel Liquid Gel Liquid Gel Liquid Gel Liquid
1.7 2.7 3.4 7.8 1.0 1.1 4.6 5.0 2.5 2.1 3.4 0.8 0.5 0.7 1.1 1.5 2.8 1.7 3.8 3.8 2.6 1.7 3.0
ml.
2- to 3-year-old of the same age of the turkeys in 1 and 3 years old. from each turkey ml.
Hexuronic
ng./lOO
acid
ml.
Nitrogen ng.
/lOO ml.
16.9 16.3 33.4 6.5
0.7 1.2 1.0 1.5 1.3 1.6
6.0 9.1 17.7 10.1 6.0 6.1 21.3 23.8
22.4 7.2 5.3 8.7 42.2
samples.
(Table VIII). In the owl monkey the concentrations of these two monosaccharides was much higher. In calves the hexosamine concentration was considerably higher than the hexuronic acid. In adult steers the concentrations of hexosamine and hexuronic acid were the highest of all animals investigated. The hexosamine and the hexuronic acid concentrations were compared in anterior and posterior, as well as in cortical and central, samples of the vitreous body of owls, rabbits, sheep, and owl monkeys (Tables V and
470
BALAZS,
LAURENT,
LAURENT,
DEROCHE
Analysis
of the Aqueous
TABLE Analyses
were
made
on pooled
Allima
samples
mg./lOO
n The
I II 1.
volume
of the
of
Humor of 4569
Hexuronic ml.
humor
the Aqueous
7
Number of eyes pooled
Species
Nitrogen
ml.
mg./lOO ml.
1.0 2.0 0.8 collected
Humor
-
acid
nzg./lOO
TABLE
of
Anal@s
Birds
eyes.
4.6 2.5
aqueous
BUNNEY
IV
Hexosamine
Rooster Rooster Turkey
AND
each
turkey
eye was 0.2 ml.
V and
-
from
13.2 57.0 36.6
the Vitreous
Body
of Owls
-
Analysis made on
-
rng./lOO
Barred
horned
Before
After
ml.
1.3 Total Total Anterior Posterior Total
owl
viscosity
hyaluronidase treatment
-Great owl
Specific
2.0-J 1.8 19.4
2.6 1.8 12.4
0.7
0.7
2.9 5.4
0.84
0.05
0.80
0.02
2.93 0.12
2.3
1.1
1.3 2.5 1.8
1.7 9.2
1.2
1.7
0.45
0.11
0.13
0.02
a value
of 21.5
.
V itreous
Great owl
Barred
horned
owl -
0 Hexosamine mg./lOO ml.
hod:
Central Cortical Occipita Occipita Occipita. determination
1.2 2.1 2.1 1.0
-
made
on the undialyzed
0.6
-
sample
gave
VIII). In owls, sheep, and owl monkeys the anterior or the central sample showed a significantly lower concentration than the posterior and cortical samples. This difference was much less in rabbits. The distribution of hexosamine and hexuronic acid was studied in the vitreous body of the owl monkey in the following way: The eye was frozen in Dry Ice-acetone immediately after the death of the animal. The sclera,
COMPARATIVE
BIOCHEMISTRY
OF
TABLE Analysis
of the Vitreous Age of animal
Sample
Body
months
Vitreous body Occipital Vitreous body Anterior Posterior Aqueous humor
1 12
VI
and the Aqueous
Number of eyes pooled
471
VITREOUS
HeXOSamine ?&I00
ml.
Humor
of Rabbits
Hexu&nic
Nitrogen
mg./lOO ml.
mg./lOO
Sialic acid ml.
mg./loo
20 10
2.3 2.2
1.7 1.9
16.5 8.3
0.8 0.6
8
2.9 2.4 0.3 0.4
2.1 2.3 <0.3 <0.3
1.3 0.7 4.5 6.0
0.9 0.8
?nl.
12
6 12 TABLE Analysis
of the Vitreous
Age
Number of eyes pooled
2 weeks 4 weeks 7 weeks 8 weeks 6 months 1 year 2 years
12 12 4 12 20 10 4
Body
VII of Rabbits
Volume obtained from one eye ml.
0.15 0.3 0.5 0.6 0.8 1.1 1.3
of Different
Hexosamine mg./loo
2.7 1.8 2.4 2.1 2.3 2.4 2.5
Ages
Hexuronic acid ml.
mg./lOO ffll.
0.6 1.6 1.0 1.4 1.6 1.6
Nitrogen mg./loo
ml.
6.0 3.4 2.7 3.8
and retina were removed from the still-frozen vitreous body. Then the liquid which dripped from the surface of the thawing vitreous body was collected in four 0.3-0.7-ml. fractions. The hexosamine concentration in these fractions, representing concentric portions starting from the retina toward the center and the lens, was as follows: 27.8, 26.1, 18.7, 11.8 mg./lOO ml., respectively. The hexuronic acid concentration in the same sampleswas 36.1, 35.7, 24.8, 16.4 mg./lOO ml., respectively. This indicates that the concentration decreasestoward the center and the lens as in all other mammals. The aqueous humor of all of the mammals investigated had a very low hexosamine content. In steers the hexosamine content of the aqueous humor was nearly 100 times lower than that of the vitreous body. This difference was not so great in calves, owl monkeys, and sheep. As a rule, the hexuronic acid concentration was lower than the hexosamine in the aqueous humor. choroid,
472
BALAZS,
LAURENT,
LAURENT,
DEROCHE
AND
BUNNEY
TABLE VIII and the Aqueous Humor of Mammals A volume of 0.2 ml. vitreous body was collected from each guinea pig. From 4 to 5 ml. vitreous b%ly and 0.8 ml. aqueous humor were collected from each sheep eye. From each owl monkey eye were collected 2.0 ml. vitreous body and 0.4 ml. aqueous humor. Analysis
of the Vitreous
Body
-
Animal
Sample
Number of eyes pooled
w./~OO
Guinea pig Sheep
Steer (1-3 yr. old) Calf (2~preeks old) Owl monkey I
Owl monkey II
Vitreous body Vitreous body Posterior Anterior Aqueous humor Vitreous body Aqueous humor” Vitreous body Aqueous humoru Vitreous bodyc Cortical Central Aqueous humor Vitreous bodyc
7 50
14 8
2.5
14.6 4.4 1.7 29.6 0.3 9.6 0.5 19.ld 10.9 6.0 0.5 18.7
Iexuronic acid
Nitrogen
W./O0
W&/pJ
1.9
14.6
14.4 3.3 0.9 29.9 0.1 4.0 0.1 21.7 13.7 8.5
11.1 10.1 47.1 7.9
Sialic acid
mg;.,oo
2.8 1.6 1.1 3.4
10.0 7.lb
4.2
1.5
a Before analysis the aqueous humor was concentrated 7X by dialysis against polyvinylpyrrolidone (Plasdone type ND-K-30, General Aniline and Film Co., N. Y.). The values are calculated for the unconcentrated aqueous humor. b The nitrogen content before dialysis was 20.0 mg./lOO ml. c The vitreous body was withdrawn with a syringe using a 22-gage needle. d The hexosamine determination gave a value of 27.0 mg./lOO ml. in the undialyeed sample. I s!.
,‘7
Nitrogen Content of the Vitreous Body
.The:&trogen content of the dialyzed vitreous body of different, animals was also, determined, and the results are tabulated in Tables II, III, V, VI,
VII, an$,VIII. Concentrations in the order of 10 mg./lOO ml. were found in all animals. This is only about 1% of the nitrogen concentration of dialyzed serum. The nondialyzable nitrogen values, however, showed considerable variation within each animal species,especially in birds. This is due to the difficulties encountered in the separation of the vitreous body from adhering tissues. Even the slightest retinal contamination increases the nitrogen value consideral$y. In two experiments the vitreous body of roosters (Rooster Prepn. II) and of turkeys (Turkey Prepn. I) was divided into two parts: one contaminated and one macroscopically uncontaminated. The
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contaminated samples had a nondialyzable nitrogen content which was considerably higher than those which were uncontaminated. However, the hexosamine and the hexuronic acid concentrations were not affected by the tissue contaminatjon, indicating that the tissues surrounding the vitreous body contain very little of these monosaccharides. The nitrogen values of the aqueous humor are subject to the same errors as those of the vitreous body, but to a much lesser degree. TABLE Hydroxyproline
-
IX
and Collagen Content of the Vitreous of Different Animal Species
-
-
Number of eyes pooled
AI&la1
Body
Collagen
Hydroxyproline 1LiquidC
Geld
Liquid
Gel
ng./lOO
Squid II Frog Hen I Hen II Hen IIP Hen 111’~ Rooster I Rooster IIn Rooster IID Rooster III Turkey Ia Great horned Barred Rabbit 2 4 4 6 12 Guinea Sheep
Occipital Occipital Occipital Occipital Occipital Occipital Occipital Occipital Occipital Occipital Occipital Anterior Posterior Occipital
owl
owl weeks old weeks old weeks old months old months old pig
Steer Owl
monkey
-
Occipital Occipital Occipital Occipital Anterior Posterior Occipital Anterior Posterior Anterior Posterior Occipital
a “Clean” samples. b “Contaminated” samples. c Hydroxyproline analysis values body. d Hydroxyproline analysis values portion of the vitreous body.
60 72 20 30 20
-
0.06 0.02
100 60 2 -
4
-
12 12 20 20 8 7 50
-
14 3
-
0.33 -
2.0 2.7 2.2 3.3 3.1 2.7 2.8 3.2 2.8 2.2 2.0 0.9 1.2
0.04
45 50
?ng./lOO ml.
ml.
0.03
0.33
0.30 0.45 0.15
-
-
1.9 1.4 2.2 1.4 12.8 1.0 1.8 1.1 0.9 1.9 0.9 -
-
-
obtained
on the
liquid
obtained
on the high-speed
portion
2.46
of the sediment
18.2 20.1 16.4 24.6 23.1 20.1 20.9 23.9 20.9 16.4 14.9 6.7 9.0 14.2 10.4 16.4 10.4 95.5 7.5 13.4 8.2 6.7 14.2 6.7 -
vitreous of the gel
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BALAZS,
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LAURENT,
Hydroxyproline
DEROCHE
AND
BUNNEY
Content
The hydroxyproline content of the sediment of the vitreous body, collected by high-speed centrifugation, is shown in Table IX. The collagen concentrations were calculated from the hydroxyproline content, assuming that the collagen of warm-blooded animals contains 13.5 % hydroxyproline (16). It was further assumed that the collagen of the frog contains 11.0 % hydroxyproline and that of the squid 9.0%. Only traces of hydroxyproline are present in the liquid vitreous body of the squid and in the liquid part of the avian vitreous body. The liquid vitreous of the owl monkey contained only 0.33 mg./lOO ml. hydroxyproline, one-third of the value found in the vitreous gel of the sheep, which contains the least collagen of all gels. In every animal studied, the hydroxyproline content was higher in the anterior part, next to the zonular region, than in the posterior segment. The difference was most striking in the rabbit, where it was 13 times higher in the anterior part than in the posterior. The least impressive difference was found in the sheep. Sialic Acid Content The sialic acid content of frogs (Table II), owls (Table V), rabbits (Table VI), sheep, steers, and owl monkeys (Table VIII) was determined. The highest values were found in steers and frogs. In owls, rabbits, and owl monkeys, the concentration was considerably less. DISCUSSION
It has been shown that hyaluronic acid, collagen, and water-soluble proteins, the main macromolecular components of the vitreous body of the cattle eye, are unevenly distributed in the gel (5). It has also been shown that part of the water-soluble proteins are glycoproteins (4, 18). Since it has been assumed by many authors that hyaluronic acid, glycoproteins, and collagen, together or individually, are responsible for the gel character of the vitreous body, it was of special interest to carry out a comparative biochemical study on these substances and correlate the results with the physical state of the vitreous body. Due to the small volume of vitreous body obtained from most animal eyes, it was not possible to isolate and characterize the macromolecules in the same way as in cattle. Instead, the concentration of certain macromolecular components such as nondialyzable hexosamine, hexuronic acid, nitrogen, and hydroxyproline was determined. The occipital sample of the vitreous body was used for these studies, since it can best be separated from the eye without massive contamination by neighboring tissues and with good reproducibility (5). This preparation,
COMPARATIVE
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475
however, excludes a small anterior part around the zonular region of the vitreous body. Since the macromolecules are unevenly distributed in the vitreous body, the results are representative only of the sample used and not necessarily of the total vitreous body. In a few cases, when the volume of the vitreous body permitted, distribution studies were also made. Hyaluronic
Acid
Since hyaluronic acid is composed of equivalent amounts of N-acetylglucosamine and glucuronic acid, the vitreous body was analyzed for these two monosaccharides. Based on the results obtained on the bovine vitreous body, it was assumed that direct determination of the hexosamine in the hydrolyzates of dialyzed vitreous bodies would give correct values (6). Hexosamine, however, has not been separated chromatographically into glucosamine and galactosamine fractions. The carbazole method for the determination of hexuronic acid gave reliable results when pure hyaluronic acid or dialyzed bovine vitreous body was used (3). Substances which might interfere with the carbazole reaction were not observed in the dialyzed vitreous bodies during these studies. The nondialyzable hexosamine and hexuronic acid content of thevitreous body may be interpreted as an indication of the presence of mucopolysaccharides and glycoproteins. If one assumes that the dominant hexuronic acid-containing macromolecule in the vitreous body is hyaluronic acid, as in cattle, then one can estimate the hyaluronic acid content from the hexuronic acid analysis values. After subtracting the hyaluronic acid hexosamine from the total hexosamine values, one can further speculate on the glycoprotein content of the vitreous body. With the exception of the frog and the pigeon, hexuronic acid was found in the vitreous body of all of the animals investigated. The concentration varied considerably in the different species, with the highest in the vitreous body of the steer and the lowest in that of fowls. In squid, owls, sheep, steers, and owl monkeys, the hexosamine-hexuronic acid molar ratio is close to one. In chickens, pigeons, turkeys, guinea pigs, and calves, the hexosamine content is higher than the hexuronic acid, which indicates that a large part of the hexosamine belongs to proteins. In rabbits, considerable variation was found in the hexuronic acid concentration, while the hexosamine was more constant. The hexuronic acid concentration in the aqueous humor of mammals is very low. The concentration of hexosamine is somewhat higher, but always well below the level found in the vitreous body. The aqueous humor of birds, however, contains the same amount of hexosamine and hexuronic acid as the vitreous body. Abelsdorff and Wessely (19) reported that the aqueous humor of various
476
BALAZS,
LAURENT,
LAURENT,
DEROCHE
AND
BUNNEY
owls, unlike other birds, has a high relative viscosity and that the aqueous humor close to the cornea is the most viscous. Barany et al. (20) found that hyaluronidase prepared from testes, from bacteria, and from snake or bee venom decreases the viscosity of the owl aqueous humor. Our finding that the viscosity and the hexuronic acid content were considerably higher in the sample of the aqueous humor taken from the region of the cornea than in the sample taken from the central part of the aqueous humor supports the assumption of Barany et al. that hyaluronic acid is unevenly distributed in the aqueous humor of the owl. Most probably hyaluronic acid is present only as a viscous layer (gel) on the interior surface of the cornea. These observations may be of some importance in connection with the theory that the aqueous flow is regulated by the mucopolysaccharides formed or collected on the surface of the trabecular meshworks (21).6 Water-Soluble
Proteins
The nitrogen content of the soluble protein fraction of the dialyzed and centrifuged vitreous body is obtained by subtracting the hexosamine nitrogen from the total nitrogen value (4). The soluble protein concentration, calculated from the nitrogen values, is low in all species, indicating that the vitreous body of all animals is a tissue compartment well separated from the blood vessel system. In several cases, the actual protein content of the vitreous body is probably considerably lower than the values reported, because in small eyes it is seldom possible to prepare the samples free from blood and tissue contamination. Since the hexosamine-hexuronic acid molar ratio is higher than one, it is most probable that some of the hexosamine is part of the glycoproteins, which may represent a considerable portion of the proteins (4). It is interesting that the owl monkey had the lowest protein nitrogen concentration, which, if correlated with the sialic acid content, suggests that approximately half of the proteins could be glycoproteins. Several authors have studied the hexosamine and nitrogen content of the vitreous body of sheep, rabbits, and guinea pigs. Bernbridge and Pirie (22) found that the hexosamine and nitrogen concentration in the rabbit vitreous body decreases during the first weeks of postnatal life. Meyer et al. (9), Boruchoff and Woodin (23), and Cristiansson (24) reported hexosamine analyses on sheep, rabbits, and guinea pigs which are comparable to ours. These authors used hexosamine analysis values to calculate the hyaluronic 6 Recently, L. Berggren showed in frozen owl eyes (Striz aluco) that a material which stains metachromatically with cationic dyes is present in the cornea1 part of the wall of the anterior chamber. This material is absent in the regions next to the lens and the iris. (Personal communication.)
COMPARATIVE
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477
acid concentration in the vitreous body. One of the authors (E. A. B.) reported earlier (5) that hyaluronic acid is unevenly distributed in the adult cattle vitreous body. This observation was based on hexosamine and hexuronic acid determinations and on the quantitative preparation of hyaluronic acid. It was also found that the concentration of hyaluronic acid depends on the a,ge of the animal. Schweer et al. (25,26) later reported that the hexosamine content is unevenly distributed in the cattle vitreous body and that its concentration is dependent on the age of the animal, which is in agreement with these findings. Collagen The insoluble collagen fraction was obtained from the vitreous body after high-speed centrifugation. Collagen is the only protein which contains a large amount of hydroxyproline, and therefore, hydroxyproline is a reliable measure of the collagen concentration (16). It was interesting to note that collagen was present in all vitreous gels but was absent or very low (owl monkey) when the vitreous body was in liquid state. It is present in all animals in concentrations of the same magnitude, being somewhat higher in birds. The results indicate that collagen is a necessary component of the vitreous body if it is in gel state. Distribution
qf Macromolecules in the Vitreous Body
It ha,s been reported (5) that hyaluronic acid, collagen, and glycoproteins are unevenly distributed in the bovine vitreous body. Hyaluronic acid and glycoproteins are present in highest concentration in the gel next to the retina (3, 4, S), and the collagen is highest in the anterior segment next to the zonular region (5). Some of the distribution experiments were repeated on the vitreous body of owls, sheep, rabbits, and monkeys. Hydroxyproline, hexosamine, hexuronic acid, and nitrogen analysis values indicat,e an uneven distribution of macromolecular components similar to that found in the bovine vitreous body. One can therefore state that an uneven distribution of hyaluronic acid, soluble proteins, and collagen in the vitreous body is a general phenomenon in all animals thus far investigated. Ascorbic Acid The gel character of the vitreous body does not seem to depend on a high ascorbic acid content. In the vitreous body of rabbits of various ages, there was a large variation in the ascorbic acid content which was independent of age. In embryos the concentration was higher than in the mother animal, which corresponds to the finding of Kinsey et al. (27) in rabbit aqueous humor and Balazs et al. (6) in cattle vitreous body.
478
BALAZS, LAURENT,
LAURENT,
DEROCHE AND BUNNEY
SUMMARY
A comparative biochemical study was made on the vitreous body of the following animals: squid, frogs, tortoises, chickens, turkeys, pigeons, owls, rabbits, guinea pigs, sheep, cattle, and owl monkeys. It was found that: 1. The vitreous body of the squid is a liquid and has a low viscosity. The vitreous body of the owl monkey is a viscous fluid. In the chicken, turkey, and pigeon, the posterior part of the vitreous body is a liquid; the rest is a gel. In the other animals the vitreous body is in gel sta.te. 2. The total ascorbic acid content is high (in the order of 10 mg./lOO ml.) in adult mammals and tortoises, and it is ten times lower in the other animals investigated. The amount of oxidized ascorbic acid in mammals is about 10% of the total ascorbic acid content. 3. Only when the vitreous body is in gel state does it contain collagen which can be sedimented by high-speed centrifugation. 4. Hexuronic acid is present in the vitreous body of all species,but with a large variation in concentration. The highest values were found in squid and in some mammals (cattle, owl monkeys, sheep). Its concentration is much less in birds, rabbits, and guinea pigs. In frogs no hexuronic acid could be determined. 5. In most animals the hexosamine concentration was higher than the hexuronic acid, indicating that hexosamine-containing compounds other than hyaluronic acid, such as glycoproteins, are present. The sialic acid content supports these findings. 6. The aqueous humor of birds contains hexosamine and hexuronic acid in about the same concentrations as the vitreous body. The aqueous humor of the owl showed an uneven distribution of hexosamine and hexuronic acid. A viscous layer, presumably hyaluronic acid, was found behind the cornea. The hyaluronic acid concentration of the aqueous humor of the mammals investigated was 100 times less than that of the vitreous body. 7. In all animals the soluble protein concentration of the vitreous body is very low (about 1% of the serum concentration), indicating that there is a distinct barrier between the vitreous body and the blood vessel system. 8. Hyaluronic acid, soluble proteins, and collagen were unevenly distributed in the vitreous body of all animals investigated. REFERENCES 1. BALAZS, E. 2. VARGA, L., 3. BALAZS, E. 4. SUNDBLAD, Abstr. p. 5. BALAZS, E.
A., Am. J. Ophthalmol. 38, No. I, Part II, 21-28 (1954). AND BALAZS, E. A., Am. J. Ophthalmol. 38, No. 1, Part II, 29-36 (1954). A., in preparation. L., AND BALAZS, E. A., Intern. Congr. Biochem. 4th Congr. Vienna 1968. 20. A., Acta XV11 Cont. Ophthalmol., 1964, II, 1019-24 (1955).
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6. BALAZS, E. A., LAURENT, T. C., AND LAURENT, U. B. G., .I. Biol. Chem. 234,422430 (1959) . 7. BALAZS, E. A., Am. J. Ophthalmol. 42, Part I, 137 (1956). 8. JOHNSON, S. W., Biochem. J. 30, 1430-7 (1936). 9. MEYER, K., SMYTH, E. M., AND GALLARDO, E., Am. J. Ophthalmol. 21, 1083-90 (1!#38). 10. KINSEY, V. E., Am. J. Ophthalmol. 30, 1262-6 (1947). 11. ROE, J. H., MILLS, M. B., OESTERLING, M. J., AND DAMRON, C. M., J. BioZ. Chem. 174, 201-8 (1948). 12. BLIX, G., Acta Chem. &and. 2, 467-73 (1948). 13. DISC:HE, Z., J. BioZ. Chem. 167, 189-98 (1947). 14. MA, T. S., AND ZUAZAGA, G., Znd. Eng. Chem., Anal. Ed. 14,280-2 (1942). 15. WERNER, I., AND ODIN, L., Acta Sot. Med. Upsaliensis 67,23041 (1952). 16. NEUMAN, R. E., AND LOGAN, M. A., J. BioZ. Chem. 166,549-56 (1950). 17. MARTIN, C. J., AND AXELROD, A. E., Proc. Sot. Exptl. BioZ. Med. 83,461-2 (1953). 18. DISCIHE, Z., DANILCZENKO, A., AND ZELMENIS, G., The neutral heteropolysaccharides in connective tissue. Ciba Foundation Symposium, Chem. and BioZ. Mucopolysaccharides, 1968, pp. 11636. 19. ABELSDORFF, G., AND WESSELY, K., Arch. Augenheilk. 64,65-125 (1909). 20. BARANY, E. H., BERGGREN, L., AND VRABEC, F., Brit. J. OphthaZmoZ. 41, 25-30 (1957). 21. BARANY, E. H., in “Glaucoma.” Josiah Macy Jr. Foundation, New York, 1955, pp. 123-221. 22. BEMBRIDGE, B. A., AND PIRIE, A., Brit. J. OphthaZmoZ. 36, 784-9 (1951). 23. BORUCHOFF, S. A., AND WOODIN, A. M., Brit. J. OphthaZmoZ. 40, 113-18 (1956). 24. CRISTIANSSON, J., Acta OphthaZmoZ. 36,336-59 (1957). 25. SCHWEER, G., AND S~DHOF, H., Arch. Ophthalmol. 167, 85-91 (1955). 26. SCHWEER, G., AND KARELL, E., Arch. Ophthalmol. 167, 422-8 (1956). 27. KINSEY, V. E., JACKSON, B., AND TERRY, T. C., Arch. OphthaZmoZ. 34, 415-17 (1945).