DOI: 10.

1159/000507026
Received: 11/29/2019
Accepted: 3/2/2020
Published(online): 3/9/2020
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Caffeine uptake into the vitreous after peroral coffee consumption
Leisser C. Stimpfl T. Ruiss M. Pilwachs C. Hienert J. Fisus A. Burgmüller W. Findl O.
Kronschläger M.
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Ophthalmic Research
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 Caffeine uptake into the vitreous after peroral coffee consumption

Christoph Leissera, Thomas Stimpflb, Manuel Ruissa, Caroline Pilwachsa, Julius Hienerta, Andreea

Fisusa, Wilhelm Burgmüllera, Oliver Findla, Martin Kronschlägera

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Vienna Institute for Research in Ocular Surgery, Department of Ophthalmology, Hanusch Hospital

Vienna, Austria

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Clinical Department of Laboratory Medicine, Medical University of Vienna, Austria

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Running head: Caffeine uptake into the vitreous

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Corresponding author:
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Oliver Findl, MD, MBA

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Hanusch Hospital

Heinrich Collin Strasse 30

A-1140 Vienna, Austria

+43 1 91021 84611

Fax: +43 1 91021 84619

oliver@findl.at

Keywords: caffeine, vitreous, epiretinal membrane, vitrectomy with membrane peeling, gas
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chromatography–mass spectrometry, blood serum concentrations

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Abstract

Introduction: Caffeine and its metabolites have antioxidant activity, scavenging reactive oxygen

species. Aim of our study was to measure caffeine concentrations in vitreous samples after peroral

caffeine intake.

Methods: This prospective study included patients scheduled for 23G pars plana vitrectomy with

membrane peeling due to epiretinal membranes. The study was performed in two parts: In the first

part patients were recruited into three different groups: group A consisted of habitual coffee

drinkers, that agreed to drink coffee containing 180mg caffeine one hour before surgery (n=10),

group B consisted of habitual coffee drinkers, that were not offered coffee before surgery (n=5), and

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group C consisted of non-habitual coffee drinkers, forming the control group (n=5). In the second

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part (group D) patients (habitual coffee drinkers) agreed to give additional blood serum samples for
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measurement of caffeine concentration. Harvested samples of vitreous (group A to D), epiretinal
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membranes (group A to C), and blood serum samples (group D) were examined for concentrations of
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caffeine with gas chromatography–mass spectrometry.

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Results: Samples of 40 eyes of 40 patients were harvested. The concentrations of caffeine in the
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vitreous samples were in group A of 1998 ng/ml +- 967 ng/ml and in group B of 1108 ng/ml +-874
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ng/ml. In group C caffeine concentrations were below 176 ng/ml in all vitreous samples. Both, groups
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A and B, had significantly higher concentrations of caffeine in the vitreous samples than group C

(p<0.002, p<0.01, Mann-Whitney-U test). Caffeine concentrations in epiretinal membranes were

below the limits of detection. Correlation of caffeine concentrations between blood serum samples

and vitreous samples in group D was high, with significantly higher caffeine concentrations in the

blood serum.

Conclusion: Coffee consumption leads to significant caffeine levels in the vitreous compared to

patients in the control group, and caffeine concentrations in the vitreous showed a high correlation

to blood serum concentrations of caffeine after peroral coffee consumption.

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Introduction

Caffeine (1,3,7-trimethylxanthine), a competitive adenosine receptor antagonist at physiological

concentrations [1,2] is present in various beverages such as coffee, green and black tea, coca cola,

and energy drinks. Adenosine receptors are present in the brain, blood vessels, kidneys, heart, the

gastrointestinal tract and respiratory system [2]. When binding to its receptors (the subtypes A2A and

A2B are located on the cerebrovascular smooth muscles), adenosine induces vasodilation [3-5].

Caffeine, in contrast, was reported to induce a vasoconstrictory response [6], to decrease ocular

blood flow [7,8], and to increase vascular resistance of retrobulbar vessels [9]. Nevertheless, when

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comparing habitual and non-habitual coffee drinkers, significant reductions of ocular blood flow after

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caffeine intake could only be detected in non-habitual coffee drinkers [10]. An explanation for this
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fact could be the up-regulation of adenosine receptors induced by chronic intake of caffeine [11,12].
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Almost all of perorally administered caffeine is absorbed in the gastrointestinal tract [13-15] within
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45 minutes [15] and peak plasma levels of caffeine in blood are detected 15 to 120 minutes after
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peroral consumption [14,16]. Its chemical properties (weak hydrophilic and weak lipophilic) allow
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caffeine distribution into all body fluids, and to pass the blood-brain barrier and all biological
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membranes [17-19]. Caffeine is metabolized in the liver by hepatic enzymes belonging to the

cytochrome P-450 family (mainly CYP1A2) to paraxanthine, theobromine, theophylline, 1-

methylxanthine, and 1-methyluric acid [20]. The half-life of caffeine has been reported to be 4–5 h

with modest intake of coffee and increases after higher levels of intake [21].

Major interest in caffeine and its metabolites is raised by their antioxidant activity due to scavenging

reactive oxygen species [22-25]. Experimental studies also indicated neuroprotective effects of

caffeine [26-28], therefore, knowledge about concentrations of caffeine in the vitreous will provide

an important basis for future investigations of neuroprotective effects of caffeine in the eye. Analysis
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of concentrations of several drugs in the vitreous, in case blood cannot be sampled, is a well-known

procedure in forensic toxicology, therefore, case reports of caffeine concentrations in the vitreous

are already published [29-30], but to our knowledge this study is the first to measure caffeine

concentrations on vitreous samples obtained during surgery. Recently, uptake of caffeine into the

lens capsule/epithelium after peroral caffee consumption was reported, with a dose dependent

manner [31].

Aim of our study was to measure caffeine concentrations in vitreous samples and surgically excised

epiretinal membranes (ERM) after peroral caffeine intake.

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Materials and Methods
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This prospective study included patients scheduled for pars plana vitrectomy with membrane peeling
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between September 2018 and November 2019 at the ophthalmic department of the Hanusch
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Hospital in Vienna, Austria. Inclusion criteria were age above 18 years, presence of an ERM with
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indication for surgery (defined as significant loss of vision and metamorphopsia due to the ERM), and

written informed consent for study participation. The study was performed in two parts: in the first

part caffeine concentrations were measured in vitreous and ERM samples in patients with and

without coffee consumption (group A, B, C), and in the second part caffeine concentrations in blood

serum and vitreous were examined (group D). Group A consisted of habitual coffee drinkers, that

agreed to drink coffee containing 180 mg caffeine one hour before surgery (n=10), group B and D

consisted of habitual coffee drinkers, that were not offered coffee before surgery (group B: n=5,

group D: n=20), and group C consisted of non-habitual coffee drinkers, forming the control group

(n=5). For groups A, B, and D no washout phases were defined, as the aims of the study were to
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examine average caffeine concentrations among habitual and non-habitual coffee drinkers and to

measure possible differences in caffeine concentrations among habitual coffee drinkers when

offered coffee one hour before surgery or not (groups A, B, and C), and to compare caffeine

concentrations between blood serum and vitreous (group D). For all study participants time between

last coffee intake and inclusion into the study was documented. All research and measurements

followed the tenets of The Declaration of Helsinki and were approved by the local ethics committee

of the city of Vienna. Clinical trials registration: NCT03646682. Written informed consent was

obtained from all patients in the study.

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Surgery was performed in a standardized fashion, with 23G pars plana vitrectomy and membrane
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peeling by an experienced vitreoretinal surgeon. Directly after placing the trocars and insertion of the
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irrigation, a sample of the vitreous was obtained with the cutter for measurement of caffeine

concentration in the sample. Special care was taken, that irrigation and the cutter were filled with air
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before harvesting the samples, to assure vitreous samples not to be diluted by balanced salt solution
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(BSS). After having harvested the vitreous sample, irrigation was turned to BSS, as usual for
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vitrectomy, and insufflated air in the vitreous cavity was removed. To visualize the ERM and internal
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limiting membrane (ILM), chromovitrectomy with a trypan blue and brilliant blue G-based dye
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(MembraneBlue-Dual, D.O.R.C., Dutch Ophthalmic Research Center, Rotterdam, Netherlands) was

performed. The ERM and ILM were peeled using an endgripping forceps, typically temporal to the

macula. Excised ERM were harvested for measurement of the caffeine concentrations (groups A to

C). All patients received non-steroidal anti-inflammatory eye drops and steroidal eye drops during

the first month after surgery. Combined phacoemulsification with implantation of an intraocular lens

and 23G pars plana vitrectomy with membrane peeling was performed only in case of coexisting

vision affecting cataract.

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Three standardized cups with 40-mL espresso (consisting of 60 mg of caffeine per cup), were given to

patients of group A one hour before surgery. Vitreous and ERM samples, harvested during surgery,

were transferred to sealed glass tubes and stored at 4°C prior to analysis. ERM samples were

analyzed using a previous published procedure [31] with a limit of detection of 0.5 ng caffeine per

ERM sample. To 0.1 ml of vitreous sample 10 microliters of isotope-labelled internal standard

solution (caffeine 13C3; 25 microgramms/ml methanol) and 0.7 ml of buffer solution (pH 7.4) were

added and transferred to an Extrelute®NT1-extraction column (Merck, Darmstadt, Germany). The

column was then extracted with 4 ml of dichloromethane, evaporated to dryness, and dissolved in

0.05 ml of ethyl acetate. One microliter was injected into a gas chromatography–mass spectrometry

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(GC–MS/MS) system, which consisted of a 7890B gas chromatograph coupled with a 7000C triple

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quad mass spectrometer (Agilent, Santa Clara, CA, USA). An autosampler AS 7693 was used for
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splitless injections onto a HP-5ms Ultra Inert capillary column (30 m, 0.25 mm internal diameter (ID),
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film thickness 0.5 micrometers; Agilent). The injector temperature was set to 280°C, the carrier gas
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was helium at a flow rate of 1.6 ml/min. The oven temperature was set to 160°C, held at this
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temperature for 2 minutes, heated at a rate of 30°C/min to 230°C, held for 2 minutes, followed by a
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rate of 20°C/min to 290°C, and held for 5.7 minutes. The transfer line temperature was set to 300°C.
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After electron impact (EI)-ionization the mass spectrometer was operated in scan-mode with m/z
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194.0 as quantifier and m/z 109.0 as qualifier for caffeine and m/z 197.0 as quantifier and m/z 111.0

as qualifier for the internal standard caffeine-13C3. The whole procedure was validated according to

the European Medicines Agency (EMA) Bioanalytical Method Validation Guideline achieving a LLOQ

of 150 ng/ml for caffeine (calibration curve and LLOQ: figure 1 and 2). The reference standard for

caffeine in this study was purchased from Fluka-Honeywell International, Inc. (Morristown, NJ, USA),

caffeine-13C3 was purchased from Sigma-Aldrich (St. Louis, Missouri, USA).

Statistical analysis was performed in a descriptive fashion for mean values, standard deviation, and
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range. All data were tested for normal distribution using the Shapiro-Wilk test. In case of a normal
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distribution, mean and standard deviation was calculated and otherwise median, interquartile range

(IQR) and range. T-test was used for normal distributed data and otherwise the Mann Whitney U test

was performed using the software tool BiAS (epsilon Verlag, Germany). A p<0.05 was regarded to

indicate significant differences between groups. For missing data, observations were excluded from

analysis. For caffeine concentrations below 150ng/ml, the limit of detection for vitreous samples,

statistical analysis was performed with the value 150ng/ml.

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Results

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Forty eyes of 40 patients could be recruited for this prospective study, among them 26 males and 14
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females, 23 right eyes and 17 left eyes. All patients had ERM, 27 with idiopathic ERM, 3 with ERM
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and vitreomacular traction, 3 with ERM and lamellar macular hole, 2 with diabetic ERM, 2 with
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secondary ERM after prior vitrectomy due to rhegmatogenous retinal detachment, one patient with
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ERM and myopic foveoschisis, one patient with secondary ERM after retinal vein occlusion, and one
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patient with ERM and coexisting full thickness macular hole. In 18 cases phaco-vitrectomy with
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membrane peeling was performed, due to coexisting vision affecting cataract. Mean age of the
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patients at the time of inclusion into the study was 70.6 +/-8.4 years (range: 48 years to 87 years).

The study was performed in two parts: in the first part caffeine concentrations were measured in

vitreous and ERM samples in patients with and without coffee consumption (group A, B, C), and in

the second part caffeine concentrations in blood serum and vitreous were examined (group D).

All vitreous samples in group A showed high concentrations of caffeine. Unfortunately, the first two

samples did not provide enough material for precise quantification of caffeine and therefore, had to
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be excluded from statistical analysis.

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Mean concentration of caffeine in the vitreous samples in group A (habitual coffee drinkers, that

received coffee containing 180 mg caffeine one hour before surgery) was 1998 ng/ml +- 967 ng/ml

(range: 967 ng/ml to 3517 ng/ml), in group B (habitual coffee drinkers, that were not offered coffee

on the day of surgery) 1108 ng/ml +-874 ng/ml (range: 205 ng/ml to 2360 ng/ml), and in group C

(non-habitual coffee drinkers) caffeine concentrations were below 150 ng/ml in all vitreous samples,

except one with a caffeine concentration of 176 ng/ml. There were no significant differences in

caffeine concentrations measured in the vitreous samples between group A and B (p=0.122996, t-

test), but both groups A and B, had significantly higher concentrations of caffeine in the vitreous

samples than group C (group A versus group C: p<0.002, Mann-Whitney-U test; group B versus group

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C: p<0.01, Mann-Whitney-U test). Concentrations of caffeine in eyes with prior vitrectomy or phaco-

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vitrectomy were within the range of the others in their specific groups (prior vitrectomy: one patient
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in group A with 1210 ng/ml and one patient in group B with 1340 ng/ml; phaco-vitrectomy: 2
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patients in group A with 3517 ng/ml and 1684 ng/ml, one patient in group B with 345 ng/ml, and 3
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patients in group C all with <150 ng/ml).

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All ERM samples in the groups A, B, and C had caffeine concentrations below 0.5 ng per sample,
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being under the limit of detection.

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Median caffeine concentrations in group D were 964.5 ng/ml (IQR 343.4 ng/ml to 1897.1 ng/ml;

range: 151.3 ng/ml to 6186 ng/ml) in the blood serum samples, and 762.5 ng/ml (IQR 371.7 ng/ml to

1310.8 ng/ml; range: 147.7 ng/ml to 5394 ng/ml) in the vitreous samples. Correlation between

corresponding blood serum samples and vitreous samples was high (r=0.9944, Pearson; rho=0.9895,

Spearman), nevertheless blood serum concentrations of caffeine were significantly higher than

concentrations of caffeine in the vitreous (p=0.0001, Wilcoxon matched pairs test).

Median time interval between last caffeine intake at home and inclusion into the study was 16.5
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hours, IQR 10.3 hours to 24 hours in group A (range: 5 hours to 24 hours), 24 hours, IQR 12 hours to
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24 hours in group B (range: 8 hours to 24 hours), 20.5 hours, IQR 18.3 hours to 25 hours in group D

(range: 4 hours to 45 hours), and in group C one patient had the last caffeine intake 2 days and 2

patients 2 weeks before inclusion into the study, while the other patients did not drink beverages

containing caffeine at all.

Interindividual variations in measured concentrations of caffeine between habitual coffee drinkers

that received coffee containing 180 mg of caffeine (group A) or not (groups B and D) are shown in

figure 3.

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Discussion
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All vitreous samples, except those in the control group (group C), showed high concentrations of

caffeine, with significantly higher concentrations of caffeine in groups A and B compared to group C,
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and a tendency to higher caffeine concentrations among habitual coffee drinkers offered coffee one

hour before surgery (group A) compared to habitual coffee drinkers that did not drink coffee at least
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8 hours before surgery (group B). Furthermore, concentrations of caffeine in the blood serum and

vitreous showed high correlation, with significantly higher concentrations of caffeine in the blood

serum.

Concentration of caffeine in ERM was below the limits of detection, an outcome that could be

explained either by the fact, that ERM samples are small and the total amount of caffeine in the ERM

samples was too low for detection, or caffeine was washed out of ERM in the surgical time interval

between start of surgery and ERM peeling.

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Major interest in caffeine and its metabolites is raised by their antioxidant activity due to scavenging

reactive oxygen species [22-25] and potential neuroprotective effects of caffeine [26-28].

Furthermore, caffeine absorbs a minor fraction of the UV-A component of daylight and its

metabolites xanthine, hypoxanthine, and uric acid are capable of quenching triplet riboflavin, an

excited state of riboflavin originating from oxidation by light absorption [32,33]. High concentrations

of caffeine in the vitreous body, as shown by the results of the present study, can be hypothesized to

have protective effects directly due to the antioxidant activity of caffeine and its metabolites, and in

a second way by the absorption of UV-A daylight in the vitreous body. As the vitreous body is in

direct neighborhood to the retina, only separated by the ILM, and caffeine is capable of passing all

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biological membranes [17-19] the above mentioned effects can further be hypothesized to have

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beneficial effects on the retinal tissue, too – nevertheless, this needs to be proven in further studies
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in the future.
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Besides direct effects of caffeine, pharmacologic blockade of A2A receptors (as induced by caffeine)
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leads to reduced neuroinflammation mediated by microglia [34] and experimental studies indicated
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caffeine to reduce microglial activity, increase proinflammatory cytokines, and decrease loss of
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retinal ganglion cells in rat models of glaucoma and ischemia-reperfusion [26-28].

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Regular caffeine intake could be associated with a small degree of decreased risk of Parkinson

disease [35-37] and Alzheimer dementia [37,38]. Furthermore, moderate coffee consumption is

inversely associated with risk of cardiovascular disease, with the lowest risk at 3-5 cups a day [39],

and coffee consumption in general decreases the incidence of major causes of death, such as heart

disease, type 2 diabetes, and several types of cancer [36,40-43]. Caffeine has an established role in

the treatment of acute migraine [44], postlumbar puncture headache [45], and neonatal apnea [46].
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There is evidence, that caffeine has the potential of inhibiting cataract development [47-57], and

peroral or topical caffeine intake achieves significant concentrations of caffeine in the lens [31,52].

Caffeine is capable of preventing oxidative stress in the lens, a factor that leads to loss of lens

transparency [58], by actively scavenging reactive oxygen species [22-25]. Ultraviolet B (UV-B)

radiation is a major cause of oxidative stress in the lens, as it is absorbed by proteins and fibers and

damages tissue by photochemical formation of reactive oxygen species [59,60].

Effects of caffeine on intraocular pressure (IOP) are controversially discussed, while caffeine can

cause a temporary increase in IOP of 1 – 3 mmHg [61-64] for about 2 hours, in general there is a

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positive association between increased IOP and daily coffee drinking only among patients with

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primary open angle glaucoma (POAG) [65]. Daily caffeine intake was not associated with an increased
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risk of developing POAG, too, except in subjects with a family history of glaucoma [66]. Nevertheless,
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there is a 1.66-fold increased risk of developing exfoliation glaucoma among subjects consuming
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more than 3 cups of coffee per day [67].

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Caffeine consumption showed no association to the incidence of early age-related macular

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degeneration during a 5-year follow-up [68], and in the Coimbra Eye Study subjects without age
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related macular edema had a significantly higher consumption of caffeine, fibres, beta-carotene,

vitamin C and E [69]. Heavy coffee drinking with intake of up to 20 cups per day should be avoided,

as it has the potential to cause central ring scotomas, sparing the central 1-2° [70].

Concluding our results, coffee consumption leads to significant caffeine levels in the vitreous

compared to patients in the control group, and caffeine concentrations in the vitreous showed a high

correlation to blood serum concentrations of caffeine after peroral coffee consumption.

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Acknowledgements

The authors would like to express their thanks to the Adele Rabensteiner Foundation for financially

supporting the study.

Statement of Ethics

All research and measurements followed the tenets of The Declaration of Helsinki and were

approved by the local ethics committee of the city of Vienna. Written informed consent was

obtained from all patients in the study.

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Disclosure Statement

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O. Findl is a scientific advisor for Carl Zeiss Meditec AG, but has no personal interest in the products
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mentioned. All authors declare, that there are no conflicts of interest.
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Funding Sources
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The study was supported by a grant from the Adele Rabensteiner Foundation, Austria. The Adele
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Rabensteiner Foundation did not influence outcomes of the study or manuscript preparation in any
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way.
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Author Contribution
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C. Leisser1-4, T. Stimpfl1,2,5, M. Ruiss2,5, C. Pilwachs2,5, J. Hienert2,5, A. Fisus2,5, W. Burgmüller2,5, O.

Findl1,5, M. Kronschläger1,5 (1conception of the study, 2data acquisition, 3statistical analysis,

4
preparation of the manuscript, 5critical review of the manuscript)
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Legends of Figures

Figure 1: Calibration curve and quality controls for caffeine (ng/mL).

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Figure 2: Ion chromatogram of lower limit of quantification for caffeine.

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Figure 3: Median concentrations of caffeine measured in the samples between habitual coffee
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drinkers, that were offered a cup of coffee containing 180 mg of caffeine one hour before surgery
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(group A) or not (groups B and D). The outcomes show interindividual variability in the measured
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concentrations of caffeine in vitreous (V) and blood serum samples (S).

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