·Clinical
Research· ·Current Issue· ·Achieve· ·Search Articles· ·Online Submission· ·About IJO·
Increased melatonin levels in aqueous humor of patients with
proliferative retinopathy in type 2 diabetes mellitus
Erdinc Aydin1,
Semsettin Sahin2
1Izmir Katip Celebi University, Faculty of Medicine, Ophthalmology, Izmir 35620, Turkey
2Gaziosmanpasa University Faculty of Medicine, Biochemistry, Tokat 60250, Turkey
Correspondence to: Erdinc Aydin. Izmir Katip Celebi Universitesi, Ataturk Egitim ve
Arastirma Hastanesi, Goz Klinigi, Karabaglar 35630, Izmir, Turkey.
erdincaydin@yahoo.com
Received: 2014-07-17
Accepted: 2015-10-13
Abstract
AIM: To report the association between melatonin levels
in aqueous humor and serum, and diabetic retinopathy (DR) grade in type 2
diabetic patients.
METHODS: Aqueous humor and plasma samples from 26 patients
with DR (in nonproliferative and proliferative stages) and 14 control subjects
were collected during cataract surgery after 6 p.m. Melatonin concentrations
were determined using an enzyme-linked immunosorbent assay (ELISA).
RESULTS: Melatonin
levels were significantly higher in the aqueous humor of patients with
proliferative diabetic
retinopathy (PDR) [18.57±2.67 pg/mL (range
15.20-23.06) vs 13.63±2.71
pg/mL (range 10.20-20.20), P=0.0001],
but not in those with nonproliferative retinopathy (NPDR) [13.79±2.56
pg/mL (range 9.80-20.10) vs 13.63±2.71
pg/mL (range 10.20-20.20), P=0.961]
compared to controls. There was
decrement in the plasma melatonin level of patients with PDR, but no
significant differences between the plasma melatonin levels of the study groups [5.37±1.74
pg/mL (range 2.85-8.65) vs 6.11±1.90
pg/mL (range 3.13-9.41), P=0.293],
or between control and DR groups [NPDR
6.11±1.90 pg/mL (range 3.13-9.41) vs
control 6.15±1.91
pg/mL (range 2.18-9.86);
PDR (5.37±1.74 pg/mL (range 2.85-8.65) vs control
6.15±1.91
pg/mL (range 2.18-9.86), P=0.808, P=0.264].
CONCLUSION: Elevated
melatonin levels in aqueous humor in PDR may indicate the level to be associated with
DR severity.
KEYWORDS: aqueous humor; diabetes mellitus; diabetic
retinopathy; melatonin
DOI:10.18240/ijo.2016.05.15
Citation: Aydin E, Sahin S. Increased melatonin
levels in aqueous humor of patients with proliferative retinopathy in type 2
diabetes mellitus. Int J Ophthalmol
2016;9(5):721-724
INTRODUCTION
Diabetic
retinopathy (DR) is one of the three major causes for visual impairment in
Western countries. A recent assessment of DR in the United States showed a high
prevalence of 28.5% among those with diabetes over 40 years old[1]. Diabetes mellitus (DM) activates pro-inflammatory
pathogenetic pathways i.e., the
polyol, protein Kinase C, hexosamine, and renin angiotensin system (RAS)
pathways, as well as advanced glycation end products formation. Molecular
changes result in blood flow alterations, formation of reactive oxygen species (ROS) and oxidative stress, induction
of inflammatory signaling systems and inflow of leukocytes, and ultimately
altered gene transcription, in turn promoting the biomolecular DR
characteristics. Formation of ROS is
increased in DM and hyperglycemia, and is directly related to vascular
dysfunction and other complications in diabetes [2]. The primary source of ROS is
considered to be overproduction of superoxide anions by the mitochondrial
electron transport chain (ETC)[3]. This increase in superoxide
formation leads to oxidative damage of mitochondrial and cellular lipids,
proteins, and nucleic acids.
Melatonin
(N-acetyl-5-methoxytryptamine) is a neurohormone found in all mammals,
including humans, and is mainly synthesized in pinealocytes. Its synthesis also
occurs in other tissues, such as bone marrow, gut, gastrointestinal tract,
lymphocytes, and in various parts of the eye including retina, ciliary body and
lachrymal gland[4-6]. In the eye, melatonin may
contribute to the regulation of retinomotor movements, rod outer segment disc
shedding, dopamine synthesis and release, and intraocular pressure, and as an
effective antioxidant and free radical scavenger[7-9]. It also protects the photoreceptor outer segment and other
ocular tissues from oxidative damage[10-13].
The aim of our current
study was to investigate the association between melatonin levels in aqueous
humor and serum, and DR grade in type 2 diabetic patients.
SUBJECTS AND METHODS
Subjects All
procedures were conducted in accordance with the Declaration of Helsinki, and
informed consent was obtained from all patients after approval from the
Institutional Review Board. The subjects were 40 patients (a total of 40 eyes)
who had undergone cataract surgery in the night-time (6 p.m.
and later). DR was documented
during a standard fundus examination in all patients, who underwent fluorescein
angiography (FA) if necessary to evaluate the presence of retinal neovascularization. No
patients who had hypertension, cardiac arrhythmia, and concomitant drugs affecting the autonomic nervous
system, including α and β adrenergic receptor blockers and other sympatholytic agents,
antidepressants, anorectics, cigarette smoking, sleep disorders, clinically
significant macular edema, glaucoma, were
recruited in the study. The patients with non proliferative retinopathy had
mild or no retinopathy without macular edema. The characteristics of patients
with nonproliferative diabetic retinopathy (NPDR), proliferative diabetic
retinopathy (PDR) and non diabetic subjects (controls) are summarized in Table
1. In literature, the
severity of diabetic retinopathy was graded according to the modified Early
Treatment Diabetic Retinopathy Study (ETDRS) retinopathy severity scale [14]
and International
Clinical Diabetic Retinopathy disease severity scale[15].
Table 1 Clinical and laboratory characteristics of the diabetic and control
groups
|
Parameters |
NPDR (n=13) |
PDR (n=13) |
Control (n=14) |
P |
|
Age (a) |
67.5±6.5 |
63.1±9.9 |
66.6±8.9 |
10.564 |
|
Sex (M/F) |
5/8 |
5/8 |
3/11 |
10.556 |
|
DM (a) |
12.21±3.98 |
18.0±6.56 |
|
20.009 |
|
HbA1C (%) |
6.9±0.6 |
7.4±1.1 |
|
20.980 |
|
Fakia/pseudofakia (fellow eye) |
7/6 |
5/8 |
7/7 |
10.834 |
|
MLTN (aqueous humor) |
13.79±2.56 |
18.57±2.67 |
13.63±2.71 |
10.0001 |
|
MLTN (plasma) |
6.11±1.90 |
5.37±1.74 |
6.15±1.91 |
10.447 |
1Kruskal Wallis test analysis; 2Mann-Whitney U test analysis. MLTN: Melatonin.
All of the patients
with type 2 DM were evaluated by careful biomicroscopic examination using a
fundus non contact lens (78 D).
Fundus findings were confirmed preoperatively by standardized fundus color
photography and FA, which was performed with a Topcon TRC-50IA fundus camera,
an image-net system (Tokyo Optical Co.
Ltd., Japan), and a preset lens with a slit-lamp.
Sample
Collection and Measurement
After
obtaining patient consent, the aqueous humor fluid (0.1-0.2
mL) was collected into sterile tubes during cataract surgery and then was
rapidly frozen to -40℃. Aqueous humor and
plasma samples were used for measurement of melatonin activity.
Melatonin
levels were determined with a commercially supplied ELISA kit (RE 54021; IBL,
Hamburg, Germany). The serum samples have to be extracted in advance. For
column preparation, 1 mL methanol was added to the columns and passed through
the column by centrifugation for 1min at 200 g,
and then 1 mL double distilled water was added to the columns in the same
procedures.
For
serum extraction, 0.5 mL standard, control or plasma samples were added to the
columns and centrifuged for 1min at 200 g.
After washing the columns with 1 mL, 10%
methanol and then centrifuging for 1min at 500 g, the extracts were eluted with
1 mL methanol by centrifugation for 1min at 200 g. The methanol was then
evaporated to dryness, using the evaporator centrifuge and the dried extracts
were reconstituted in 150 µL double distilled water. After this, 50 µL
extracted standard, control or serum samples were transferred into the
appropriate wells of the microtitre plate. To these wells, 50 µL melatonin
biotin and 50 µL melatonin antiserum were added. Following sealing with
adhesive foil, the plate was carefully shaken and incubated for 20h at 4℃.
Each well was then washed three times with assay buffer and added with 150 µL
enzyme conjugate. The plate was sealed again and incubated for 120 min at room
temperature on an orbital shaker at 500 rpm. After incubation and washing three
times with assay buffer, each well was then added with 200 µL freshly prepared
paranitrophenyl phosphate (PNPP) substrate solution. The plate was further
incubated for 30min at room temperature on an orbital shaker. The substrate
reaction was stopped by adding 50 µL of PNPP stop solution into the wells. The
absorbance values were measured by a spectrophotometer. Finally, 50 µL of PNPP
stop solution were added to each well, and the optical density was measured
with a photometer at 405 nm (reference wavelength: 600-650 nm)[16].
Statistical
Analysis For
statistical analysis, variables were determined to have abnormal distribution
by the Shapiro-Wilk Tests, and Kruskal-Wallis Tests were used in the
comparisons of groups. For the comparison of differences within groups the Mann-Whitney
U test was used. For categorical variable (gender),
Chi-square tests were used to find out differences between groups. A
P<0.05 was defined as
statistically significant. Statistical analyses were performed using the
Statistical Package for Social Science's software
(SPSS version 15, SPSS Inc., Chicago, IL, USA).
RESULTS
The demographic and
clinical data of the diabetic patients and controls are summarized in Table 1.
No statistically significant differences were noted among the groups with
regard to age and sex distribution (P=0.564, P=0.556, respectively).
There were significant differences between the DM duration (P=0.009), but no significant differences
in HbA1c levels of NPDR and PDR groups (P=0.980).
Aqueous humor and
plasma melatonin concentrations, shown as mean±standard deviation (range,
pg/mL), for the NPDR group were 13.79±2.56 pg/mL (range 9.80-20.10), and
6.11±1.90 pg/mL (range 3.13-9.41), respectively, and for the PDR group were
18.57±2.67 pg/mL (range 15.20-23.06) and 5.37±1.74 pg/mL (range 2.85-8.65),
respectively. The aqueous humor and plasma melatonin concentrations for the
control group were 13.63±2.71 pg/mL (range 10.20-20.20) and 6.15±1.91 pg/mL
(range 2.18-9.86), respectively
(Table 1). When the study groups were compared with the control group,
melatonin levels were found to be significantly increased in the aqueous humor
of the patients with PDR (P=0.0001),
but no remarkable increased in the aqueous humor of the patients with NPDR (P=0.981). There was decrement in the
plasma melatonin level of patients with PDR, but no significant differences
between the plasma melatonin levels of the study groups (P=0.293), or between control
and study groups (NPDR vs control ;PDR
vs control, respectively) (P=0.808;
P=0.264).
The significant correlations
were detected between the duration of DM and melatonin levels in the aqueous
humor of diabetic patients, between the levels of HbA1c and melatonin levels in
the aqueous humor of diabetic patients (Spearman’s
rho: 0.572, P=0.002:
Spearman’s rho: 0.438; P=0.025).
DISCUSSION
Oxidative stress is
implicated in the etiology of many ocular diseases such as glaucoma, retinal
degeneration, ocular inflammation, cataracts, and diabetic complications[13,17]. Diabetes has been shown to be a
state of increased free radical production[18]. Various mechanisms contribute to the formation of free
radicals in diabetes, and may include not only increased non-enzymatic and
auto-oxidative glycosylation, but also metabolic stress resulting from changes
in energy metabolism, levels of inflammatory mediators, and the status of
antioxidant defense[19]. Moreover, elevated levels of
various reactive oxygen and nitrogen species have been identified in diseased
ocular structures. These reactants damage the structure and deplete the eye of
natural defense systems, such as antioxidants, glutathione, and the antioxidant
enzyme, superoxide dismutase (SOD). Oxidative damage in the eye leads to the
apoptotic degeneration of retinal neurons and fluid accumulation.
Melatonin continuously regenerates
and offers a frontier anti-oxidative defense for both the anterior and
posterior eye against conditions such as photo-keratitis, cataract, glaucoma,
retinopathy of prematurity, and ischemia/reperfusion injury[10]. Immunocytochemical analysis of
ocular tissues obtained from various animals like chickens, rats, and humans
has shown that melatonin receptors (MT1, MT2) are distributed in the cornea,
choroid, sclera, photoreceptors, RGCs and retinal blood vessels[20-22]. Additionally, the
existence of melatonin receptors in the iris and ciliary processes has led to
the proposal that they are involved in aqueous humor secretion and the
circadian rhythm of intraocular pressure[23]. The existence of melatonin
biosynthetic pathway in the mammalian retina was initially supported by the
discovery of retinal hydroxyindole-O-methyl transferase (HIOMT) activity. The
gene encoding HIOMT is selectively expressed in retinal photoreceptor cells
that rhythmically secrete melatonin suggests that photoreceptors contain an
endogenous ‘‘clock’’ that regulates melatonin biosynthesis[24]. This has been confirmed in the mammalian retina, photoreceptors, either rods or
cones, contain circadian oscillators[25].
Melatonin treatment increased SOD
and glutathione S-transferase (GST) activities in plasma, erythrocyte lysate
and liver and kidney tissue[26-27]. Reiter et al[28] reported that melatonin led to a
dramatic decrease in free radical production. It
has been demonstrated in some studies that melatonin and anti-oxidant (SOD, GST)
concentrations are elevated in both the vitreous fluid and aqueous humor of
patients with active proliferative diabetic retinopathy[29-30].
Recent studies have demonstrated a close correlation between oxidative stress
and morphological changes in the trabecular meshwork, highlighting that anterior chamber involvement in patients with PDR may be
caused partly by redox-state imbalances[31-32]. In
some human studies, decreased plasma melatonin levels were reported in type 2 DM[33-34]. Hikichi et
al[35] demonstrated that serum melatonin
levels were significantly lower in the diabetic group than in the non-diabetic
group, and were lower in the PDR group than in the non-diabetic and NPDR
groups, but no significant difference was found between the non-diabetic and
NPDR groups. In our
study, the melatonin
levels of patients with PDR were found to be lower in plasma, but significantly
higher in aqueous humor compared to NPDR patients and controls. These findings
showed accordance with the results of plasma melatonin studies in Literature.
Furthermore, there was a significant correlation between the duration of DM and
aqueous humor melatonin levels of diabetic patients in our study. All patients in the PDR group of our study underwent panretinal photocoagulation. Retinal dysfunction induced not only
by advanced diabetic retinopathy but also by panretinal photocoagulation should be associated with diminished retinal
light perception. Bughi et al[36] reported that bilateral photocoagulation for PDR may be associated with loss of normal circadian cortisol variation.
Consequently,
we speculated that the advanced retinal dysfunction alters serum melatonin
secretion in the patients with PDR due to light perception loss. On the other hand, elevated melatonin
levels in aqueous humor may be linked to actively anti-oxidative,
anti-angiogenic processes and morphological changes of the trabecular meshwork
in type 2 diabetics. We propose that melatonin
level in aqueous humor may increase with the severity of DR, and that the levels of melatonin in
aqueous samples reflect the intraocular concentrations. Main limitations of this study were its small sample size and high standard
deviations of the mean concentrations of melatonin. Further studies with larger patient
cohorts are required, however, it looks like that melatonin may be promising
for the treatment of DR in the future.
ACKNOWLEDGEMENTS
Conflicts of Interest: Aydin E, None; Sahin S, None.
REFERENCES
[Top]
1 Zhang X, Saaddine JB,
Chou CF, Cotch MF, Cheng YJ, Geiss LS, Gregg EW, Albright AL, Klein BE, Klein
R. Prevalence of diabetic retinopathy in the United States, 2005-2008. JAMA 2010;304(6):649-656. [CrossRef] [PubMed] [PMC free article]
2 Giacco F, Brownlee M. Oxidative stress and diabetic
complications. Circ Res 2010;107(9):1058-1070.
[CrossRef] [PubMed] [PMC free article]
3 Naudi A, Jove M, Ayala V, Cassanye A, Serrano J,
Gonzalo H, Boada J, Prat J, Portero-Otin M, Pamplona R. Cellular dysfunction in
diabetes as maladaptive response to mitochondrial oxidative stress. Exp Diabetes Res 2012;2012:696215. [CrossRef] [PubMed] [PMC free article]
4 Tosini G, Pozdeyev N, Sakamoto K, Iuvone PM. The
circadian clock system in mammalian retina. Bioessays
2008;30:624-633. [CrossRef]
[PubMed] [PMC free article]
5 Martin XD, Malina HZ, Brennan MC, Hendrickson PH,
Lichter PR. The ciliary body-the third organ found to synthesize indoleamines
in humans. Eur J Ophthalmol 1992;2(2):67-72. [PubMed]
6 Mhatre MC, van Jaarsveld AS, Reiter RJ. Melatonin in
the lacrimal gland: first demonstration and experimental manipulation. Biochem Biophy Res Commun 1988;153(3):1186-1192. [CrossRef]
7 Besharse JC, Dunis DA. Methoxyindoles and photoreceptor
metabolism: activation of rod shedding. Science
1983;219(4590):1341-1343. [CrossRef] [PubMed]
8 Sengupta A, Baba K, Mazzoni F, Pozdeyev NV, Strettoi E,
Iuvone PM, Tosini G. Localization of melatonin receptor 1 in mouse retina and
its role in the circadian regulation of the electroretinogram and dopamine
levels. PLoS One 2011;6(9):e24483. [CrossRef] [PubMed] [PMC free article]
9 Agorastos A, Huber CG. The role of melatonin in
glaucoma: implications concerning pathophysiological relevance and therapeutic
potential. J Pineal Res
2011;50(1):1-7. [CrossRef]
[PubMed]
10 Siu AW, Maldonado M, Sanchez-Hidalgo M, Tan DX, Reiter
RJ. Protective effects of melatonin in experimental free radical-related ocular
diseases. J Pineal Res
2006;40(2):101-109. [CrossRef] [PubMed]
11 Lundmark PO, Pandi-Perumal SR, Srinivasan V, Cardinali
DP. Role of melatonin in the eye and ocular dysfunctions. Vis Neurosci 2006;23(6):853-862. [CrossRef] [PubMed]
12 Rosen RB, Hu D,Chen M, McCormick SA, Walsh J,
RobertsJE. Effects of melatonin and its receptor antagonist on retinal pigment
epithelial cells against hydrogen peroxide damage. Mol Vis 2012;18:1640-1648. [PMC free article]
[PubMed]
13 Mirunalini S, Karthishwaran K,
Dhamodharan G, Mohan S. Melatonin attenuates lipid peroxidation and enhances
circulatory antioxidants during mammary carcinogenesis in rats. J Biochem Tech 2010;2(3):171-174.
14 Grading diabetic retinopathy from stereoscopic color
fundus photographs--an extension of the modified Airlie House classification.
ETDRS report number 10. Early Treatment Diabetic Retinopathy Study Research
Group. Ophthalmology 1991;98 (Suppl
5):786-806. [CrossRef]
15 Wilkinson CP, Ferris FL 3rd, Klein RE, Lee PP, Agardh
CD, Davis M, Dills D, Kampik A, Pararajasegaram R, Verdaguer JT; Global
Diabetic Retinopathy Project Group. Proposed international clinical diabetic
retinopathy and diabetic macular edema disease severity scales. Ophthalmology 2003;110(9):1677-1682. [CrossRef]
16 Khaleghipour S, Masjedi M, Ahade H, Enayate M, Pasha
G, Nadery F, Ahmadzade G. Morning and nocturnal serum melatonin
rhythm levels in patients with major depressive disorder: an analytical
cross-sectional study. Sao Paulo Med J
2012;130(3):167-172. [CrossRef]
17 Ohia SE, Opere CA, Leday AM. Pharmacological
consequences of oxidative stress in ocular tissues. Mutat Res 2005;579: 22-36. [CrossRef] [PubMed]
18 Yang H, Jin X, Kei Lam CW, Yan SK. Oxidative stress
and diabetes mellitus. Clin Chem Lab Med
2011;49(11):1773-1782. [CrossRef]
[PubMed]
19 Griesmacher A, Kindhauser M, Andert SE, et al. Enhanced serum levels of
thiobarbituric-acid-reactive substances in diabetes mellitus. Am J Med 1995;98(5):469-475. [CrossRef]
20 Scher J, Wankiewicz E, Brown GM, Fujieda H. MT(1)
melatonin receptor in the human retina: expression and localization. Invest Ophthalmol Vis Sci
2002;43(3):889-897. [PubMed]
21 Wiechmann AF, Rada JA. Melatonin receptor expression
in the cornea and sclera. Exp Eye Res
2003;77(2):219-225. [CrossRef]
22 Rada JA, Wiechmann AF. Melatonin receptors in chick
ocular tissues: implications for a role of melatonin in ocular growth
regulation. Invest Ophthalmol Vis Sci 2006;47(1):25-33. [CrossRef] [PubMed]
23 Alarma-Estrany P, Pintor J. Melatonin receptors in the
eye: location, second messengers and role in ocular physiology. Pharmacol Ther 2007;113(3):507-522. [CrossRef] [PubMed]
24 Cahill GM, Besharse JC. Circadian clock functions
localized in xenopus retinal photoreceptors. Neuron 1993;10(4):573-577 [CrossRef]
25 Tosini G, Iuvone PM (Eds.).
Chapter 4, Role of Melatonin and Dopamine in the Regulation of Retinal
Circadian Rhythms. The Retina and
Circadian Rhythms Germany: Springer; 2014;49-68.
26 Anwar MM, Meki AR. Oxidative stress in
streptozotocin-induced diabetic rats: effects of garlic oil and melatonin. Comp Biochem Physiol A Mol Integr Physiol 2003;135(4):539-547.
[CrossRef]
27 Tan DX, Reiter RJ, Manchester LC, Yan MT, El-Sawi M,
Sainz RM, Mayo JC, Kohen R, Allegra M, Hardeland R. Chemical and physical
properties and potential mechanism: melatonin as a broad spectrum antioxidant
and free radical scavenger. Curr Top Med
Chem 2002;2(2):181-198. [CrossRef]
28 Reiter RJ, Tan DX, Mayo JC, Sainz RM, Lopez-Burillo S.
Melatonin, longevity and health in the aged: an assessment.Free Radic Res 2002;36(12):1323-1329. [CrossRef]
29 Chiquet C, Claustrat B, Thuret G, Brun J, Cooper HM,
Denis P. Melatonin concentrations in aqueous humor of glaucoma patients. Am J Ophthalmol 2006;142(2):325-327. [CrossRef] [PubMed]
30 Izzotti A, Bagnis A, Saccà SC. The role of oxidative
stress in glaucoma. Mutat Res
2006;612(2):105-114. [CrossRef]
[PubMed]
31 Saccà SC, Izzotti A, Rossi P, Traverso C. Glaucomatous
outflow pathway and oxidative stress. Exp
Eye Res 2007;84(3):389-399. [CrossRef] [PubMed]
32 Mancino R, Pierro DD, Varesi C, Cerulli A, Feraco A,
Cedrone C, Pinazo-Duran MD, Coletta M, Nucci C. Lipid peroxidation and total
antioxidant capacity in vitreous, aqueous humor, and blood samples from
patients with diabetic retinopathy Mol
Vis 2011;17:1298-1304. [PMC free article]
[PubMed]
33 Tutuncu NB, Batur MK, Yildirir A, Tutuncu T, Deger A,
Koray Z, Erbas B, Kabakci G, Aksoyek S, Erbas T. Melatonin levels decrease in
type 2 diabetic patients with cardiac autonomic neuropathy. J Pineal Res 2005;39(1):43-49. [CrossRef] [PubMed]
34 Peschke E, Frese T, Chankiewitz E, Peschke D, Preiss
U, Schneyer U, Spessert R, Mühlbauer E. Diabetic Goto Kakizaki rats as well as
type 2 diabetic patients show a decreased diurnal serum melatonin level and an
increased pancreatic melatonin-receptor status. J Pineal Res 2006;40(2):135-143. [CrossRef] [PubMed]
35 Hikichi T, Tateda N, Miura T. Alteration of melatonin
secretion in patients with type 2 diabetes and proliferative diabetic
retinopathy. Clin Ophthalmology
2011;5:655-660. [CrossRef]
[PubMed] [PMC free article]
36 Bughi S, Shaw S,
Bessman A. Laser damage to retinal ganglion cells: the effect on circadian
rhythms. J Diabetes Complication
2006;20(3):184-187. [CrossRef]
[PubMed]
[Top]