The Thiol-Disulfide Homeostasis and Coenzyme Q 10 in Conjunction with Vitamin E Effect on Retinopathy Prematurity

Department of Ophthalmology, Faculty of Medicine, Afyonkarahisar Health Science University, Afyonkarahisar 03320, Turkey Department of Clinical Biochemistry, University of Health Sciences, Bursa Yuksek Ihtisas Training and Research Hospital, Bursa 16290, Turkey Newborn Clinic, University of Health Sciences, Bursa Yuksek Ihtisas Training and Research Hospital, Bursa 16290, Turkey Department of Clinical Biochemistry, Yildirim Beyazit University, 06760, Ankara, Turkey


INTRODUCTION
Retinopathy Of Prematurity (ROP) is a proliferative vasculopathy that usually develops in infants with low birth weight and low gestational age because of vascular dysfunction of the retina.Numerous factors have been proposed as aetiology of this disease, which causes structural impairment in immature retinal vessels and may result in blindness.The most significant factors are oxidative stress caused by oxygen (O 2 ) and O 2 metabolites [1,2] and associated inflammation [3].Studies demonstrating that Hb and Hb products, as well as haemogram parameters, are responsible for O 2 transport can be effective on ROP in infants, as they support the role of oxidative stress in ROP development [4].
Even full-term newborns may be exposed to oxidative stress as a result of exposure to high O 2 content in the natural environment compared with that in the mother's womb.Oxidative stress represents the imbalance between the production of Reactive Oxygen Species (ROS) and protective antioxidants [5].Oxidative stress and oxidative damage due to insufficient antioxidative response mechanisms in preterm infants are much higher than those in normal infants.In addition, because oxygen in the retina is supplied from the choroid, hyperoxygenation is observed in the inner and outer retina due to the lack of autoregulation between the choroid and the retina as well as inadequate antioxidant defence mechanism, particularly in premature infants.Hyperoxygenation causes oxidative stress in the retina, which plays an important role in ROP development [6 -8].Expression of Vascular Endothelial Growth Factor (VEGF), which causes neovascularisation in the retina, is regulated by signals associated with hypoxia [9].Oxidative stress also plays a role in pathological processes, including the regulation of VEGF expression in the retina [10].
Neonatal Intermittent Hypoxia (IH) is a recurrent, shortterm (<3 min) decrease in oxygen saturation [11].IH episodes are typically a consequence of an immature respiratory control system, and may result in the disruption of the antioxidant system, particularly in premature infants, leading to increased retinal neovascularisation.IH-induced mitochondrial ROS are toxic, causing lipid peroxidation and damaging cell membranes [11].
Coenzyme Q10 (CoQ10) is a potent antioxidant that plays a vital role in energy creation, electron transport and the mitochondrial electron transport chain.CoQ10 dissolves in fat and is often found in high-energy consuming tissues, such as the brain, eyes, liver and kidneys.The antioxidant is a naturally-occurring compound, primarily playing a bioenergenetic role in the cell [12].CoQ10 has been suggested to inhibit the developmental deficits caused by IH-induced oxidative stress [11].
Thiols are functional sulfhydryl groups that are contained within proteins and other molecules, such as glutathione and homocysteine.Thiol groups are converted to disulphide bonds under oxidative conditions, such as IH and the resulting bonds are degraded to thiols to create thiol-disulphide homeostasis.Thiol-disulphide homeostasis plays an important role in the maintenance of many physiological processes necessary for organisms, such as antioxidant defence and protein chemical structure stabilisation.A method developed in recent years makes it possible to measure thiol-disulphide homeostasis in the blood [13,14].
Recently.Unal et al. suggested that the shift in the thioldisulfide equilibrium towards disulphides in the first week can be attributed to the subjection of very low-birth-weighted preterms to oxidative stress [15].
This study was performed to determine whether one drop of topical administration of Coqun®, a potent antioxidant consisting of CoQ10 0.1% (w/v) and vitamin E TPGS (d-alphatocopheryl polyethylene glycol 1000 succinate) 0.5% (w/v) dissolved in aqueous saline isotonic solution [16], twice a day has any effect on the thiol-disulphide homeostasis in the ROP disease course.

METHODS
Twenty-eight infants with a mean birth weight of 1,021 ± 330 g and a mean gestational age of 27.5 ± 2.0 weeks who followed up in the intensive care unit of our hospital between August 2018 and September 2018 for ROP were included in this study.This was a prospective observational case-control study; approval was obtained from the Ethics Committee of HSU Bursa Yuksek Ihtisas Training and Research Hospital.Infants were included in the study after obtaining the consent of their parents.This was a joint study by the newborn clinic, ophthalmology clinic and biochemistry clinic, and the principles of the Declaration of Helsinki were complied.
A Coqun® drop was administered twice a day to the eye's lower fornix between weeks 31 and 34 when ROP development was first observed in the infant's retinas.Fundus examinations were performed weekly following the initiation of Coqun®2x1 treatment.Thirty minutes before the examination, three drops of 0.5% tropicamide (tropamid, Bilim İlaç, Turkey) and 1% phenylephrine (Mydfrin, Alcon, USA) topical ophthalmic solutions were administered to the infants.After appropriate pupillary dilation, topical anaesthesia was performed using proparacaine hydrochloride (Alcaine®, Alcon, USA).Eyelids were opened using eye speculum, and all central and peripheral retinal areas were scanned by indirect ophthalmoscope and +28 dioptric lens vascular indentation.All images were recorded by Archimedes VGA imaging system (Pronova, Ankara, Turkey), and all examinations and treatments were performed by the same ophthalmologist.
Localisation (zone), amount of involvement (clock dial) and vascular examination findings (plus-preplus) were noted based on the severity of retinopathy and retinopathy between avascular areas according to the International ROP Classification criteria (ICROP): Stage 1 if there is a demarcation line, stage 2 if there is elevation or a 'ridge', stage 3 if there is extraretinal fibrovascular proliferation, stage 4 if there is extrafoveal/fovea-containing partial retinal detachment and stage 5 if there is total retinal detachment [17].These parameters were used to determine the infants' ROP stages.Aggressive posterior ROP (APROP, type II or rush-type ROP with a more posterior location, rapid progression instead of the conventional stage 1-5 progression and poor prognosis despite early treatment) diagnosis was made in the presence of these findings [18].Nutritional characteristics of infants were examined in three groups: those being fed breast milk, those fed a mix of breast milk and infant formula and those fed infant formulae only.Treatment of the infants was based on the Early Treatment for ROP (ETROP) criteria and BEAT ROP study data [19,20].
The dose of intravitreal bevacizumab (IVB) (Altuzan® 100 mg/dl, Roche, Switzerland) was 0.16-0.32mg (very low-low) for all infants [21].It was administered under local anaesthesia and sterile operating room conditions with 0.5% propargain hydrochloride drop (Alcaine®, Alcon, USA) using a 32-G needle through the limbus's periphery (0.5-1 mm) under topical anaesthesia.Laser photocoagulation (LPC) was performed in a sterile operating room, under sedation or general anaesthesia, using a laser device (Iridex; Oculight SL, Mountainview, CA, USA) on avascular regions for patients in group 2 and above who required this procedure.Coqun® drops were administered twice a day to ROP infants with or without anti-VEGF treatment with the same regimen.
Blood samples were obtained from all infants before and two weeks after Coqun® treatment.Centrifuged blood samples were stored at −80°C until analysis.The albumin level was studied using an Olympus AU 2700 autoanalyser.FRAP was measured using an adapted ELISA method to determine the total antioxidant level [22].Serum IMA levels were measured using a colorimetric assay method previously described by Bar-Or et al. [13].Thiol-disulphide homeostasis levels were measured using the automated direct measurement method used by Erel et al. [14].
The Statistical Package for Social Sciences version 21.0 software for Windows (IBM, Chicago, IL, USA) was used for data analysis.A paired-sample t-test was used for comparisons.Descriptive statistics were expressed as frequency and percentage.Normally distributed quantitative data were expressed as mean ± standard deviation.Non-normally distributed quantitative data were expressed as mean (range).P < 0.05 was considered statistically significant.

DISCUSSION
Despite a limited number of cases in this study, the significant decrease in serum FRAP levels, which is an indicator of total protective antioxidant level, may lead to increased oxidative stress and progression of this disease.
A decrease in serum FRAP levels indicates that these infants are under severe oxidative stress.The FRAP method, which is an iron (III) ion-reducing antioxidant power method, is suitable for determining hydrophilic and lipophilic antioxidants and measuring the sum of all antioxidants in the environment, excluding thiols [3].
CoQ10s have a high molecular weight and a lipophilic structure that is almost too strong to melt in an aqueous environment.Although there are studies suggesting that CoQ10 has poor bioavailability because it is not water-soluble, there are other studies that show its benefits many organs in the body [11].Its strong lipophilic structure limits its therapeutic value [23 -25].
Although there is no study investigating FRAP levels in the blood following topical CoQ10 treatment in newborns and preterm infants, one study reported serum CoQ10 levels in newborns and preterm infants [22].In this study, CoQ10 initially showed a very low level but increased in vivo in the serum with the presence of vitamin E, which is a potent antioxidant.In newborns exposed to oxidative stress without any external treatment, this increase was caused only by the nutrition and CoQ10 levels produced by the body itself.This increase occurred much slower in preterm infants with a disease, which was attributed to the delay in enteric feeding (i.e., a delay in intestinal maturation) [6].
CoQ10 inhibits haemolysis of erythrocytes by hydrogen peroxide in preterm infants by preventing membrane phospholipid peroxidation and thereby free radical formation.It also strengthens defence against oxidative aggression in preterm infants [2].
Oxidative stress plays a critical role in retinal injury and the prognosis of ROP, diabetic retinopathy and ischaemic oxidative retinopathies, such as age-related macular degeneration (AMD); therefore, CoQ10 has an important role in all of these oxidative retinopathies [3].In their recently published study, Beharry et al. demonstrated that CoQ10 and n-3 PUFA supplementation in IH elicited protective effects against ROP [11].CoQ10 reduced VEGF expression, maintained astrocytic integrity, reduced neovascularisation and normalised retinal layers.In addition, n-3 PUFAs reduced VEGF expression, promoted IGF-I secretion, reduced neovascularisation and normalised retinal layers [11].
Inhibition of ROS production by intravitreal luteolin injection, another potential antioxidant, has reduced VEGF gene transcription [26].Increasing antioxidant gene expression regulated by Nrf2 via intraperitoneal ebselen (a mimic of glutathione peroxidase) injection protected retinal Müller cells and vessels from oxidative stress-induced injury, and led to reduced retinal VEGF expression due to oxidative stress [27].
ROP is an ischaemic retinopathy.Combining CoQ10 and opoloxyethanyl-alpha-tocopheryl sebacate α-water-soluble αtocopherol (vitamin E) α-with a non-covalent bond at a ratio of 2:1 produces a strong hydrophilic substance.Clinical and laboratory studies have proven that it passes through the corneal epithelium into the eye [16].
In premature infants, IH can result in the disruption of the antioxidant system, leading to disruption of dopamine secretion and thereby neurological damages and increased retinal neovascularisation.The antioxidant effect caused by CoQ10 treatment prevents this neurological damage, protects both inner retina and visual cortex and reduces neurodegenerative damage [11].
Sato et al. showed that CoQ10 levels were low at birth, and that CoQ10 plasma levels rapidly increased in the neonatal period when administered in combination with another strong antioxidant, such as vitamin E. This increase was slower in preterm infants with much lower birth weight and gestational age, which was attributed to the late onset of enteric feeding [6].
No significant difference was found between the pre-Coqun and post-Coqun groups in terms of total thiol, native thiol, disulphide, native/total thiol ratio and disulphide/total thiol levels.Based on these results, Coqun® treatment (one drop twice a day) did not alter the systemic serum thioldisulphide homeostasis.The concentration of thiol-based antioxidants in plasma is lower than in cells: they are formed by serum albumin-SH (~80%) and low molecular weight thiols (i.e., cysteine, cysteinylglycine, gamma glutamylcysteine, glutathione and homocysteine).In addition to oxidative stress, many factors affect the plasma thiol/disulphide homeostasis, which may have affected the outcomes of ROP patients [28].
Most recent animal studies have shown that growth is significantly impaired in cases where IH is induced, whereas oral CoQ10 treatment can decrease ROS accumulation by stimulating antioxidant activity and n-3 PUFA treatment can decrease VEGF induced by IH, and therefore, may have beneficial effects on growth and growth-supporting factors [6].
ROP is ischaemic retinopathy.Studies show that CoQ10 can achieve T-fam protein expression in ischaemic retina to provide neuroprotection against mitochondrial changes mediated by oxidative stress in ischaemic retinal damage [14].Increased reactive oxygen species can activate the inflammatory response by triggering cytokines (inflammatory cytokines, such as IL-1β, TNF-α and IL-6), resulting in oedema development in endothelial cells, decreased blood flow and increased ischaemia, thereby accelerating neovascularisation [25].
Conflicting reports have been published regarding vitamin E supplementation and its use in ROP treatment or prevention.Two meta-analyses have reported beneficial effects, while another study has reported no effect [29 -31].Some limitations of this study include the limited number of patients and non-generalisability of the results, as this was a single-centre study.
Although the serum antioxidant levels decreased following the use of topical Coqun® 2 x 1 treatment, the clinical course of ROP in infants was much more stable.Even if serum antioxidant levels do not rise, these levels increase in the vitreous fluid, which may be associated with stabilisation in ROP level.Because the antioxidant levels in the infants' vitreous fluid cannot be checked, investigating these parameters in multi-centre clinical studies or animal models may provide more definitive results.However, the results of this study suggest that administration of CoQ10 not only topically but also orally may be beneficial in the clinical prognosis of infants with this condition.

CONCLUSION
Twenty-eight premature infants with a mean gestational age of 27.6 ± 1.99 (24-32) weeks, a mean birth weight of 1012 ± 318 (520-1750) g and a mean duration of care in the incubator of 64 ± 28  days were included in the study.
Three of the infants were breastfed, seven were fed infant formula and 18 were fed both.All infants were in ROP stages 2-3; none had stage 4A and above ROP.Two infants had necrotising enterocolitis and six had APROP findings.One infant was O 2 dependent.
Coqun® treatment (one drop twice a day) was initiated in the newborn unit at gestational weeks 31 and 34, when the initial ROP stages were detected in the infants.Blood antioxidant levels were measured when treatment was initiated and two weeks after initiation.Table 1 shows the serum antioxidant levels of the infants before and two weeks after the Coqun® treatment.
Fundus examinations were performed every week following the initiation of Coqun® treatment.None of the infants progressed in their ROP stage.A total of eight infants (six with APROP and two with stage 3 ROP) had to receive a low dose of IVB injection (0.312 mg) at a mean age of 36.7 weeks.
After two weeks of treatment, the ROP stage regressed in two infants with stage 2 plus (+) ROP.No progression was seen in the other infants; however, eight infants with APROP and stage 3 plus (+) ROP had to receive a low dose of IVB.No infant was treated with LPC.

HUMAN AND ANIMAL RIGHTS
No Animals were used in this research.All human research procedures followed were in accordance with the ethical standards of the committee responsible for human experimentation (institutional and national), and with the Helsinki Declaration of 1975, as revised in 2013.
been approved by the Ethics Committee of HSU Bursa Yuksek Ihtisas Training and Research Hospital.

Table 1 . Serum antioxidant levels in infants before and 2 weeks after the initiation of Coqun® treatment.
FRAP: Ferric-reducing ability of plasma; IMA: Ischemic modified albumin; ABSU: Absorbance unit.