Full text of DOI:10.1007/s004170100357

Graefe’s Arch Clin Exp Ophthalmol
(2001) 239:743–746
C L I N I C A L I N V E S T I G AT I O N
DOI 10.1007/s004170100357
Jens Berlau
Peter Lorenz
Ria Beck
Josef Makovitzky
Ursula Schlötzer-Schrehardt
Hans-Jürgen Thiesen
Rudolf Guthoff
Analysis of aqueous humour proteins of eyes
with and without pseudoexfoliation syndrome
Abstract Pseudoexfoliation syn-
drome (PEX) has been suggested to
represent a blood-aqueous barrier
impairment leading to a higher pro-
tein content in aqueous humour of
larised light microscopy. Aqueous
protein concentration was not signif-
icantly increased in PEX eyes in
comparison with non-PEX eyes. Fur-
thermore, we could not detect any
Received: 26 March 2001
Revised: 13 July 2001
Accepted: 8 August 2001
Published online: 11 September 2001
© Springer-Verlag 2001
eyes with PEX. However, the nature characteristic difference in protein
of a prospective PEX protein has not band sizes of the two groups after
yet been described. We set out to re- SDS PAGE. However, we were able
evaluate protein content and examine to show the presence of amyloid ex-
J. Berlau ( ) · R. Beck · R. Guthoff
✉
protein composition for prospective
PEX protein candidates in aqueous
clusively in aqueous humour of PEX
patients. Conclusion: our results do
Department of Ophthalmology,
University of Rostock, Rostock, Germany
e-mail: berlau@mampf.ieu.uni-jena.de
humour of eyes with PEX syndrome. not confirm a generally higher pro-
Aqueous humour of 52 patients with tein concentration in pseudoexfolia-
P. Lorenz · H.-J. Thiesen
Institute of Immunology,
University of Rostock, Rostock, German
PEX and 38 without PEX signs was
tion syndrome eyes. This does not
sampled during cataract or glaucoma necessarily contradict a blood-aque-
surgery. Total aqueous protein con-
centration in the samples was analy-
sed in 43 PEX specimens and 32
non-PEX specimens according to
Bradford. Aqueous protein composi- catable to pseudoexfoliation syn-
tion of all samples was determined
by sodium dodecylsulphate poly-
acrylamide gel electrophoresis (SDS findings support the theory that the
PAGE) and silver staining. Screening pseudoexfoliation syndrome is asso-
for amyloids was performed in nine
PEX samples and six non-PEX sam- protein.
ples by Congo Red staining and po-
ous barrier impairment but illustrates
the variance in protein concentration
between and within the two groups.
No characteristic protein band allo-
J. Makovitzky
Department of Gynaecology,
University of Rostock, Rostock, Germany
U. Schlötzer-Schrehardt
Department of Ophthalmology,
University of Erlangen-Nürnberg,
Erlangen, Germany
drome proteins could be detected in
any of the samples. However, our
Present address:
J. Berlau, Institute for Nutritional Science,
Department of Nutritional Toxicology,
Friedrich-Schiller-University of Jena,
Dornburger Straße 25, 07743 Jena, Germany
Fax: +49-3641-949672
ciated with an amyloid of a serum
the posterior surface of the cornea [12]. A simultaneous
mechanism of local production of the material by anteri-
Introduction
Pseudoexfoliation syndrome (PEX) is a disorder charac- or segment tissues and transport via the aqueous humour
terised by a widespread production and deposition of a has been suggested [20]. There is strong evidence that
distinctive fibrillar material in the eye and in various or- PEX represents an ocular manifestation of a systemic
gans such as skin, heart, lungs, liver, kidney and menin- disorder and that PEX material may be synthesised out-
ges [26]. In the eye, PEX is characterised by the pres- side the eye before it becomes evident in the anterior
ence of this material on the pupillary border, the anterior segment [27].
lens capsule, both surfaces of the iris, ciliary body, zon-
One of the most important clinical features of PEX is
ules, in the anterior chamber angle, and occasionally on the frequent association with open-angle glaucoma. In

744
of 10 µl of each sample was streaked on glass slides and air-dried.
Screening for amyloid was performed by 0.1% Congo Red stain-
ing and polarised light microscopy according to Romhányi [24].
addition, the incidence of cataract is high in eyes with
PEX [30,31]. Patients with PEX are at an approximately
ten fold increased risk of developing a rather aggressive
type of secondary open-angle glaucoma and a secondary
angle-closure glaucoma [23]. Furthermore, PEX is pres-
ently the most common cause of open-angle glaucomas
world-wide [22].
Despite its clinical significance, the exact composi-
tion of the abnormally produced material remains un-
known. Studies performed on the nature of PEX material
revealed an association with amyloid [17,19], but other
authors have shown the principal basement membrane
components heparan and chondroitin sulphate proteogly-
can, and entactin/nidogen [25], and particularly the elas-
tic fibre components elastin, amyloid P, vitronectin, and
fibrillin-1 to be integral constituents of PEX material
[10, 28,29].
Results
Total aqueous protein concentrations were not signifi-
cantly higher in PEX eyes (0.23±0.19 mg/ml, range
0.008–1.001 mg/ml) than in the non-PEX group
(0.26±0.23 mg/ml, range 0.038–0.875 mg/ml) (P>0.5,
Student’s t-test). Within the PEX group the aqueous hu-
mour of the 43 cataract patients showed a lower mean
protein concentration (0.237 mg/ml) than that of the
eight patients with secondary open-angle glaucoma
It has also been demonstrated that an impairment of
the blood-aqueous barrier is frequently associated with
PEX [5,8] leading to an altered composition of aqueous
humour. Increased aqueous protein concentrations were
reported as compared to age-matched controls [5, 6, 7,
13] as well as changes in the levels of alpha1-lipoprotein
and caeruloplasmin [2], transferrin [4] and fibronectin
[32]. Electrophoretic analyses of aqueous proteins dem-
onstrated either a prominent band at 12.5 kDa [5] or at
16.3 kDa [20] in aqueous humour of PEX patients.
It was the aim of this study to re-evaluate the protein
content and to examine the protein composition for the
possible presence of PEX-specific proteins in aqueous
humour of eyes with PEX syndrome. In addition, we
screened aqueous humour of patients with and without
PEX syndrome for the presence of amyloid proteins.
Materials and methods
Fig. 1 SDS-PAGE of aqueous humour of eight patients with PEX
(lanes 1–8) and eight patients without PEX (lanes 9–16). Note the
12.5 kDa band (arrow) visible in all specimens. (M marker)
Materials
Aqueous humour was obtained during intraocular surgery from 52
eyes of 52 patients who attended the University Eye Clinics in
Rostock and Erlangen (eight with secondary open-angle glauco-
ma, one with primary open-angle glaucoma, 43 with cataract, age
range 67–90 years, mean 77.1 years, male/female ratio 1:1.18)
with the characteristic clinical signs of PEX and from 38 eyes of
38 patients (all with cataract, two with an additional primary
open-angle glaucoma, age range 52–82 years, mean 70.4 years,
male/female ratio 1:1.53) without any clinical signs of PEX.
Methods
Total protein concentrations were determined for 43 of the 52
aqueous humour samples of PEX patients and 32 of the 38 sam-
ples of control patients using a Bradford assay (BioRad, Germa-
ny). Aqueous protein composition of 10 µl of the same samples
was determined by 15% sodium dodecylsulphate polyacrylamide
gel electrophoresis (SDS PAGE) and silver staining according to
standard protocols. The remaining nine PEX specimens and six Fig. 2 Positive amyloid reaction in aqueous humour of an eye
non-PEX specimens were used for amyloid screening. A volume with PEX (Congo red, ×200)

745
trol eyes. This finding does not necessarily contradict a
blood-aqueous barrier impairment but rather illustrates
the variance in protein concentrations between and with-
in the two groups. We conclude that PEX is not necessar-
ily characterised by increased protein concentrations in
aqueous humour. Aqueous protein concentrations appear
to be subject to high interindividual variations depending
on patient and development of the disease rather than be-
ing clearly related to the PEX process. Since the aqueous
protein concentrations may depend on the stage of dis-
ease progression, a possible explanation for these differ-
ences might be the fact that the majority of aqueous sam-
ples analysed in this study was obtained from patients
with PEX syndrome without glaucoma.
The exact composition of PEX material and the
pathogenesis of PEX are still unclear. So far, no PEX-
specific proteins could be identified with certainty in the
aqueous humour, although two electrophoretic studies
have suggested proteins of a molecular weight of 12.5
kDa and 14.4/16.3 kDa as possible candidates [6, 21].
Fig. 3 Positive amyloid reaction in aqueous humour of an eye
with PEX (Congo red, polarised light, ×200)
(0.380 mg/ml). However, this difference was not signifi- The results of this study confirmed a protein band of
cant (P>0.05, Student’s t-test). SDS-PAGE did not reveal 12.5 kDa, which was, however, present in all samples in-
any significant band differences between both groups dependent of PEX. However, this band had been only
(Fig.1).
observed in 56% of PEX eyes in the study by Küchle et
Amyloid exhibiting typical green birefringence was al. [6]. A band of 14.4 kDa only occurred in two non-
demonstrated in the presence of Congo Red under cross- PEX aqueous humour samples, whereas a 16.3 kDA
polarised light in all nine examined aqueous humour band was found in all samples. Thus, our results do not
samples of PEX patients (Fig. 2, Fig. 3). The six non- confirm the presence of one or several specific proteins
PEX samples were negative for amyloid.
in the aqueous humour of PEX eyes. However, the possi-
bility of the presence of proteins which are not solubili-
sed by standard methods or not detectable with one-
dimensional SDS-PAGE electrophoresis cannot be ruled
out.
Discussion
PEX has been suggested to represent the intraocular
Although some cases with PEX and primary amyloi-
manifestation of a systemic disorder, characterised by dosis have been described [11], the relationship of PEX
grey dandruff-like material deposited in the anterior part material with amyloid has always been a matter of much
of the eye. Many clinical studies have demonstrated an debate. Whereas a few studies suggested PEX material
impaired blood-aqueous barrier function in eyes with to be a type of amyloid [17, 19], most studies examining
PEX [5, 7, 8, 9, 15] which may lead to protein leakage the nature of PEX material by Congo Red staining or im-
and increased total aqueous protein concentration. Iris munohistochemistry with specific antibodies have yield-
fluorescein angiographic and electron microscopic stud- ed negative reactions for amyloid [3, 14, 18]. Thus, the
ies [1, 25] could show that the origin of the increased amyloid theory lacked any conclusive evidence. In the
aqueous proteins is mainly the iris vasculature [1, 6, 8, present study, clear evidence for the presence of amyloid
16].
proteins in the aqueous humour of PEX eyes but not of
The results of our study do not confirm a significant control eyes was provided by Congo Red staining.
mean increase in total aqueous protein concentration in Therefore, these findings indicate an association of PEX
eyes with PEX. This finding is in accordance with with amyloid formation and further substantiate the amy-
Ringvold and co-workers [21] who described increased loid theory on the pathogenesis of PEX syndrome. How-
protein concentrations only in lens capsule specimens, ever, the exact type of amyloid in the aqueous humour
but not in aqueous humour samples of PEX eyes. How- and the deposition of amyloid in anterior segment tissues
ever, our findings contradict the results of Küchle and of PEX eyes remain to be elucidated in future studies.
co-workers [6] who found significantly higher protein
concentrations in aqueous humour of patients with PEX
with and without glaucoma. However, the protein con-
centrations measured in this study showed considerable
interindividual variations both in PEX eyes and in con-

746
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