ACTA OPHTHALMOLOGICA 1958

ANALYSIS OF VITREOUS AND AQUEOUS HUMOUR OF ENUCLEATED EYES

BY PAPER ELECTROPHORESIS

BY

Henrik Forsius (Helsinki)

Normally the vitreous body is a gel in a high state of turgescence. According to Duke-Elder, the vitreous contains only 0.0652 per cent total protein, divided into mucoprotein, residual protein, albumin and globulin.

On filtered vitreous, Hesselvik (1939) differentiated by electrophoresis globulins, albumins, and hyalomucoids. The albumin was a somewhat faster moving component than the serum albumin. Brunish, Rowen, Rodman and Irwine (1954) distinguished albumin, alpha and beta-globulin in vitreous, but no gamma-globulin, and noted that albumin/globulin quotient was lower than in serum. Rao, Kulkarni, Cooper and Radhakrishnan (1955) found large amounts of gamma-globulin, some albumin, and a small, faster moving com- ponent.

The vitreous is readily transformed to sol. if slightly traumatized or treated with hyaluronic acid. The liquefied vitreous of which the ophthalmologists have great experience in connection with cataract extractions, is due, according to Duke-Elder, to changed metabolism owing to low-grade toxins or to slight inflammatory processes. Supply of foreign inflammatory products from in- flammations of uvea and retina causes accumulation of exudate, invasion of leukocytes, etc.

On measuring the albumin content of the vitreous, Franceschetti and Wieland (1928) obtained 0.213 per cent in a case of glaucoma.

Foreign albuminous substances are readily absorbed by the vitreous. Even if an eye is preserved at a temperature of 0 degrees for a day only, the protein will increase to the double (Brunish, Rowen, Rodman and Irwine, 1954).

Among previous investigations by paper electrophoresis, that of Stemmerman (1952) may be mentioned. In a case of melano chorioideae with yellowish liquefied vitreous, he obtained a blood-serum-like proteinogram. In the same year d'Erno, in a case of tuberculous uveitis, obtaine& hyalomucoid 21.9 per cent, albumin 9.6 per cent, and globulin 13.2 per cent.

Cagianut and Wunderly (1953) used paper electrophoresis in four cases: one each of melanoma and hydrophthalmus, and two of glaucoma. After concen-

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txatioii of the vitreous which was clear in all cases, the almumin content was noted: 61-77.8 per cent. The alphal-globulin and alphaz-globulin fractions separated well, and appeared in about equal amounts.

Usiiig paper electrophoresis, Vilstrup and Kornerup (1955) analysed vitreous from an eye enucleated owing to processes in the anterior chamber. The firm vitreous was washed and dissolved with hyaluronidase. The albumin/globulin quotient was low (43-46/57-54).

Aqueous humour. The normal aqueous humour contains less proteins than the vitreous. According to Duke-Elder it contains 0.0201 per cent proteins, consisting of albumin and globulin. In disease, the protein content in the aqueous increases rapidly. Kornfeld (1941), for instance, showed that the albumin content in senile cataract with cortical breakdown was somewhat raised, whereas, in compensated glaucoma, it stayed within normal boundaries (5-16 ing O h ) .

In acute and secondary glaucoma, the protein content increases tenfold and over, as revealed by Franceschetti and Wieland (1928), the highest protein value, 2.37 1 per cent, being noted in hydrophthalmus congenitus.

Verrey (1955) mentions values up to 6 per cent total protein in ulcus corneae with hypopyon, and Paycha (1955) 1.0-1.1 per cent in iritis and detachment of the retina. The albumin content of the aqueous is too low to be directly determined by paper electrophoresis. By an own method Niedermeier con- centrated normal aqueous humour and found 0.01 g/l albumin and 0.003 g/l globulin.

Esser, Heinzler and Pau (1954) also collected aqueous humour obtained from different eyes and concentrated the albumin content by ultra-filtration. By paper electrophoresis they obtained 0.6 per cent of a protein that moved somewhat faster than albumin, 64.9 per cent albumin, 4.8 per cent alphal- globulin, 7.5 per cent alphaz-globulin, 18.5 per cent beta-globulin and 4.1 per cent gamma-globulin. The corresponding albumin content in serum was lower, 59.0 pel cent, and the gamma-globulin in serum was higher, 16.5 per cent. The distribution of the globulin-fractions differed if the sample was taken post mortem. the gamma-globulin content of aqueous then being higher and the alpha-gl obulin content lower than in the aqueous in vivo.

Several paper electrophoresis investigations on pathologically changed aqueous humour have been made. Witmer (1952) reported the results of his examinations of 8 cases of acute iritis and 13 cases of chronic uveitis and, in addition, 15 cases of chronic uveitis with only slight clinical irritation symptoms (see Table 1) .

Wunderly and Cagianut (1952) studied pathological aqueous by paper electro- phoresis in 12 cases of various diseases of the anterior part of the eye. The albumin content was often lower than in serum, and the alpha-globulin content was higher. In chronic inflammations, the alpha-globulin in the aqueous in-

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Table 1. Proteinograms of aqueous humour according to various authors.

Author Disease I-.* Albumin I Prt;r

Wunderly, Healthy Steiger and persons Bohringer Aqueous

Serum

19.4 10.4 9.2 62.6

18.3 13.8 8.8 59.1

creases and the gamma-globulin decreases; in polynuclear reactions the gamma- globulin increases rapidly.

Chinaglia and Franco (1953) found that the albumin-globulin quotient was lower in aqueous than in serum, whereas the alpha-globulin level rose in iritis, iridocyclitis and keratoiritis. Lens, Iris, retina. On examination of the albumin content of the lens, Stem-

mermann (1952) observed two peaks in the proteinogram: one between the alphal- globulin and alphag-globulin, and another a t the beta-globulin. The first peak may be divided into three subfractions (Fransois, Wieme, Rabaey and Neetens, 1953).

The albuminous substances in the tissue of the iris and retina are also com- posed of globulins; the most important point in the proteinogram of the tissue of iris is the beta-globulin, while the retinal tissue contains mainly gamma- globulin (Munich and Oswald, 1955).

The author’s investigations. The material comprises 44 eyes, enucleated at the Ophthalmic Clinic of the University of Helsinki. The reason for enucleation was traumatic uveitis in 28 cases, corneal ulcers in 7 cases, chronic uveitis in 2 cases, choroid melanoma in one case, and absolute glaucoma in 7 cases.

57 1 37*

Methods: The eyes were collected as soon as possible after enucleation. The aqueous humour was emptied by puncture; the bulb was washed and opened by incision along the equator, and the vitreous thus obtained. If the vitreous was firm, the part that could be sucked up by pipette after slight compression with the pipette was collected. The lowest protein level at which proteinograms may be read lies slightly below 1/2 per cent. For that reason, in solitary cases, when no Tyndall phenomenon was seen in the anterior chamber, or the protein content of the vitreous was too low, the aqueous was concentrated by evapora- tion in vacuum.

Electrophoresis, mainly according to Grassman, Hannig and Knedel, was perfornied. The bands were stained with amino-black 10 B, photometrically read, and assessed by planimeter. My modification has been previously described in detail (Forsius, 1954).

Seruin proteinograms were taken in most cases parallely with the analyses of the aqueous and vitreous. The total protein in the eye liquids was determined by Kjeldahl's micro-method.

As blood is often present in the enucleated bulbs and the cell-proteins of the blood, in consequence thereof, often dominate the picture, proteinograms were made of different blood components to render analysis of the proteinograms of eye liquids possible: Blood plasma proteinograms were made of heparinized blood after centrifugalization. Secondly, total blood drawn by venous puncture was heniolyzed by deep freezing with carbonic-acid snow, the cell membranes were eliminated by centrifuge, and the proteinogram made. For determination of the proteinogram of erythrocytes, the cells were separated from heparinized blood, washed three times in saline solution, and centrifuged after each washing. The erythrocytes were further hemolyzed with carbonic-acid snow and centri- fugalizetl. Proteinograms were made in a similar way of a concentration of leukocytes according to Wasastjerna's method; the ratio of leukocytes and erythrocytes obtained was 1 : 3.

RESULTS

Traumatic uveitis (Table 2). 28 eyes were examined. The table shows the distribution of proteins in the vitreous and aqueous in per cent. The vitreous was liquefied in all instances. No eyes enucleated immediately after trauma are included in the material. The earliest enucleation was performed three weeks after injury.

In 18 cases the period elapsed between trauma and enucleation was 3 weeks to 8 months, and in 6 cases 2-18 years. No considerable difference was seen between the two groups. In the group covering a longer period of time, the albumin content was somewhat higher both in aqueous and vitreous. Three

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3

c) c

573

c ... 8 9 0 , - e,

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cases of hemophthalmus were compared. In the first case, enucleation had been performed 13 weeks after trauma, and the proteinogram resembled that of whole blood. In the other two cases, enucleation had been performed 2 and 15 years after trauma, and the proteinogram revealed a preponderance of albumin, in spite of the vitreous being highly sanguinous in both cases.

Furthermore, cases in which the perforation had occurred 1) through cornea and lens, 2) through cornea and sclera, probably injuring the ciliary body, and 3) through sclera, were compared.

The albumin content of the vitreous and the aqueous was highest in group 3. Corneal ulcer (Table 3). In corneal ulcers, the gamma and beta-globulin

occurred in the proteinogram in about equal percentages both in aqueous and vitreous humour. As expected, the protein content of the vitreous was low, 0.44 per cent.

a

Graph I . Proteinograms on various parts of blood and on aqueous humour.

a) serum proteinogram. b) traumatic uveitis without hypopyon but with high protein content. c) aqueous humour in glaucoma absolutum. Protein content 1,9 O/o.

d) traumatic uveitis with hypopyon. e) whole blood hemolyzed by deep-freezing. f ) sanguinous aqueous humour. g) isolated erythrocytes hemolyzed by deep-freezing. h) suspension of leucocytes hemolyzed by deep-freezing.

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Graph I I . Proteinograms on vitreous humour.

i) vitreous humour in a case of traumatic uveitis. k) vitreous humour in choroid melanoma. 1) proteinogram in a case of abscess in the vitreous humour.

m) proteinogram on subretinal fluid in a case of uncomplicated retinal detachment.

Chronic uueitis (Table 3). Only 2 cases of chronic uveitis were analysed. In one case the albumin content of the vitreous was too low to yield a readable graph. Gamma-globulin dominated the only proteinogram of the vitreous obtained.

Chorioid melanoma (Table 3) . The only case included was that of a woman aged 70 years. From the opened bulb, 0.5 can brownish fluid was obtained; after centrifugalization it was found to contain an abundance of erythrocytes and occasional leukocytes. The protein content was not measured but, judging from the strong colour of the proteinogram, it must have been of the same magnitude as that of serum. The gamma-globulin level was high, and the alpha-globulin level was low,

Absolute glaucoma (Table 3). Seven cases of primary absolute glaucoma were analysed. The protein content of the vitreous was low (0.94 O/O). The albumin content of the aqueous was measured in one case, and found to be 1.7 per cent.

The proteinogram of the vitreous which in these cases was jelly-like or mucous, and in only one case yellowish, resembled that of serum. The proteino- grams of aqueous humour and serum agreed in general, but individual varia- tions occurred.

If the proteinograms of vitreous are divided on the basis of their macro- scopical appearance, it is observed that when the vitreous changes due to in-

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flammation and blood, a relative decrease in albumin and alpha-globulin content occurs, while the gamma- and beta-globulins increase. This is seen from Table 4, in which the proteinograms are divided into two groups. The first group comprises with clear, mucous, or may be yellowish vitreous, and the second group cases with yellow, sanguinous, or purulent vitreous.

If the proteinograms of aqueous are divided on the basis of the clinical picture, i. e., according to the power of the Tyndall phenomenon, it is observed that if this phenomenon is weak, a proteinogram resembling that of serum is obtained, and, similarly, in the case of a moderate Tyndall phenomenon. When there is a strong Tyndall phenomenon without hypopyon, the proteinogram of aqueous humour may resemble that of serum but, when the cell content is high, and especially if leukocytes or erythrocytes are present, then the albumin level decreases and gamma-globulin and beta-globulin predominate (Table 4). In this connection von Sallmann’s observation may be mentioned, i. e., that the protein content and the Tyndall phenomenon do ot always agree, because the albumin does not appear equally distinctly in the form of a Tyndall pheno- menon as do other proteins.

No. of cases

Table 4. Eyes grouped according to strength of Tyndall phenomenon in anterior chamber.

Globulins - Albumin

Y 1 B l a z 1 a1

Globulins - No.

of cases Y B a2 a1

Table 5. Eyes grouped according to degree of inflammation.

Protein 010

Albumin

10 Clear, mucous, yellowish 13.7 11.2 5.3 2.5 67.3 2.219

9 Yellow, cloudy, sanguinous 24.4 20.3 6.8 1.4 47.1 8.712

51 I

FinaIly, on study of the separate components of the proteinogram, it may be noted that high protein levels are generally associated with high gamma- globulin levels, independently of the cell content in the eye liquid. This oc- curred. for instance, in the two cases in which the aqueous and the vitreous humour, respectively, coagulated in the test tube. The proteinogram of the sanguiiious eye liquids revealed an enermous increase in beta-globulin. The alpha-globulin content was low, especially in the vitreous, but approached, re1 ativcly, the corresponding serum percentages in proteinograms poor in proteins, mainly in glaucoma, and often in inflammations of the anterior chamber without increased cell content. The closer the clinical picture ap- proachcd normal conditions, the higher did the relative albumin content be- come, and the proteinogram resembled that of serum.

SUMMARY

The material includes 44 eyes enucleated owing to traumatic uveitis, corneal ulcers, chronic uveitis, chorioid melanoma and absolute glaucoma.

The ;iqueous and vitreous were examined by paper electrophoresis, sometimes after concentration in vacuum, and compared with serum. Proteinograms of hernolyAed whole blood, and of separated erythrocytes and leukocytes were made.

I f traumatic uveitis arose due to perforation of the sclera, the albumin con- tent was higher both in vitreous and aqueous, as compared with eyes per- forated through cornea or both cornea and sclera. If erythrocytes and leuko- cytes were present, the gamma-globulin and beta-globulin values were higher. In hyphemia or in the case of the vitreous being sanguinous, similar proteino- grams were obtained as in tests by paper electrophoresis of vitreous and in whtch the beta-globulin predominated completely. The erythrocytes did not predominate in the sanguinous vitreous if a number of years had elapsed since the injury.

The alpha2-globulin level was low, especially in the vitreous; this was noted also in subretinal fluid. If the protein content of the aqueous was low, the alphaz-proteins were often of the same magnitude as in serum, especially in glaucoma.

The protein content of the vitreous was measured in 21 cases, and found to be 4,74 in traumatic uveitis, 0.94 per cent in absolute glaucoma, and 0.44 per cent in corneal ulcer.

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