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The eyes are the primary visual organ of an organism. Being part of the specific sensory system for vision, the eye converts visual light into a pattern of graded action potentials which can then be interpreted by the brain as an image (Widmaier et al., 2008).
The eye is a complex organ with highly differentiated tissues, each having specific functions in order to achieve vision. The outer portion of the eye is made up of three layers; the sclera which forms a tough, protective capsule, the choroid which is a darkly pigmented layer to absorb rays of lights at the back of the eye and the retina which is made up of specific photo-sensitive receptor cells to detect the light rays entering the eye. Within these layers are two fluid-filled chambers separated by the lens of the eye. To allow the entry of rays of lights into the eye, the anterior surface of the sclera specialised into the cornea while the choroid forms the iris (the coloured-structure of the eye), the ciliary muscle (which controls the shape of the lens for focusing) and the zonular fibers (which holds the lens in place). The anatomy of a human eye can be seen in Figure 1.1.
Vision is acquired by focusing rays of light onto a single point on the retina, the fovea centralis through the refractive actions of some structures in the eye. In order to achieve a clear vision, light from the environment enters the eye through the cornea, and is focused by the lens onto the fovea centralis. As this region of the retina has the highest visual acuity, failure of the eye to focus light onto fovea centralis results in blurred images and such conditions are known as refractive errors (Benjamin, 2006).
According to the World Health Organisation (WHO), refractive errors are very common eye disorders in which the eye cannot clearly focus images from the surrounding, resulting in blurred vision and in severe cases, visual impairment. It was estimated that about 153 million people worldwide suffers from visual impairments due to uncorrected refractive errors (World Health Organization, 2009). Two of the most common form of refractive errors present in healthy individuals are myopia and astigmatism.
Myopia, or commonly known as near-sightedness, is a condition in which the rays of light entering are focused in front of the retina. Such a condition may occur due to the eye having an elongated axial length (which is the straight length from the cornea to the fovea centralis) or an increase in refractive (focusing) power in one or more refractive components of the eye, such as the cornea or the lens (Borish, 1970).
Due to the numerous forms of myopia reported, many classification methods have been suggested. However, a review of the methods of classification by Grosvenor (1987) proposed seven broad headings for classifying myopia, which were rate of myopia progression, anatomical features of myopia, degree of myopia, physiological and pathological myopia, hereditary and environmentally induced myopia, theory of myopia development and age of myopia onset. For this study, the degree of myopia was used whereby subjects were grouped based on corrective power required which were high (more than -6.00 D), moderate (between -3.00 to -6.00 D), low (between -0.25 to -3.00 D) and normal (between -0.25 to 0.00) (Cline et al., 1997).
Astigmatism, on the other hand, is the failure of the cornea to focus light on a single point on the retina due to the difference in refractive powers of different radial planes of the cornea. This thus results in two centers of foci which cause distortion of the image seen (Shapiro, 1984). If the angle between the plane with the highest refractive power and that with the lowest is perpendicular are one another, the astigmatism is considered regular. Otherwise, it would be considered an irregular astigmatism (Duke-Elders et al., 1970). Irregular astigmatism should be noted as it is a sign of possible corneal distortion and thinning (Szczotka-Flynn et al., 2006; Rabinotwiz, 1998).
According to the National Eye Institute, the cornea is the clear, dome-shaped, outermost layer that covers the front of the eye. It is made up of five layers; the outermost epithelium, the Bowman's layer, the stroma, the Descemet's membrane and the endothelium. Functions of the cornea include shielding the insides of the eye from harmful elements from the environment and controlling and focusing the rays of light entering the eye. Hence, any injury to the cornea may result in severe visual loss. As the cornea is constantly exposed to the harms of the environment, the cornea must be able to cope and respond swiftly to damages or any injuries caused by the environment before vision is affected. Failure of the cornea to do so may lead to the development of corneal diseases and disorders. (National Eye Institute, 2010).
Keratoconus (KC) is most common primary ectasia of the cornea, characterised by bilateral, asymmetric, localised thinning of the corneal which leads to protrusion and coning of the cornea (Krachmer et al., 1984). As the corneal thinning progresses, one begins to develop high refractive errors and irregular astigmatism which severely affect visual acuity (Jimenez et al., 2010). KC has been indicated to have an onset at puberty, which usually progresses until the fourth decade of life (Rabinowitz, 1998). However, cases of KC developing earlier (Rahmen et al., 2006) or later in life (Lim et al., 2003) and even at birth, though rarely (Smolin, 1987) have been reported.
Clinical Features of KC
At a cellular level, KC has often been characterised by three classical histopathological signs; thinning of the corneal stroma, breakage in the Bowman's layer, and iron deposition within the basal layer of the corneal epithelium (Rabinowitz, 1998). However, a more recent report in the histopathology of KC indicated other changes such as thickening of nerve fibres, irregular and broken basement membrane and apoptotic cells in the epithelium (Sherwin et al., 2004).
As cellular pathogenesis progresses, typical signs and symptoms starts to appear depending on the severity of KC present. At subclinical stages, KC is present as forme fruste KC which does not produce any noticeable symptoms. Thus, forme fruste KC often remains undetectable by both patients and examiners unless specific ocular examinations have been carried out (Arntz et al., 2003).
As the disease progresses, patients begins to suffer from significant vision acuity deterioration which cannot be corrected with spectacles and increasing irregular astigmatism (Krachmer et al., 1984). Once KC has reach moderate to severe levels, obvious physical signs can be detected. Slit lamp findings commonly presented in KC patients are stromal thinning, Vogt's striae (which are fine lines parallel to the axis of the cone found in the deep stroma and Descemet's membrane), Fleischer's ring (accumulation of iron deposits surrounding the cone) and corneal scarring on the epithelial or subepithelial layer (McMahon, 2006), as shown in Figure 1.1.
However, the most important tool for diagnosing KC in recent times would have to be corneal topography. Advancements in diagnosing KC with corneal topography have proved to be valuable in documenting the different patterns of the anterior corneal surface in KC patients, which may provide new information of the possible pathogenesis pathway of this disorder (Rabinowitz at al., 1989). In fact, the use of corneal topography is important especially in the detection and documentation of corneal irregularities in the early stages of KC, such as in forme fruste KC as it was reported that large amounts of corneal deformation were detected in the cornea of several KC suspects despite presenting normal keratometry and refractive data, in addition to normal slit lamp findings (Maguire et al., 1989). Through corneal topography, both KC and forme fruste KC can be detected by an "asymmetric bow tie with skewed radial axis" shape (Figure 1.2) seen in the corneal topograph (Rabinowitz, 2007).
In recent years, tremendous effort have also been made to ease detection of KC using corneal topography, especially during the forme fruste stage through the development of several index-based classification methods as reported by Maeda & Klyce (1994), Smolek & Klyce (1997), Schwiegerling & Grevenkamp (1996), Rabinowitz & Rasheed (1999), McMahon et al.(2006) and Mahmoud et al.(2008). To date, there are two optical instruments with built-in software based on these indices which can detect and monitor the progression of KC; the Pentacam (Emre et al., 2007) and the Ocular Response Analyzer (Fontes et al., 2010). As more accurate indices are being developed as time passes, researchers comes closer and closer to deriving the perfect formula to detecting the earliest stages of KC which can address the subjective nature of diagnosing KC.
Despite the heavy dependence of current KC diagnostic methods on computer-assisted optical measuring instruments, the strong genetic link found in KC have opened a new door for researchers to develop new diagnostic methods based on molecular genetic tests. However, most are still in development stage and far from ready for clinical use (Rabinowitz, 2007).
In literature, the most common prevalence of KC was approximate to be about 1 in 2000 in the general population (Krachmer et al., 1984). However, this value may vary considerably between different populations with reports of as low as 4 per 100,000 (Duke-Elders & Leigh, 1965) to as high as 600 per 100,000 (Hofstetter, 1959). However, it was found that most reports of KC prevalence were based on western populations and reports on prevalence of KC in Asian population remain scarce (Mahadevan et al. 2009). Several studies have indicated higher incidences of KC among Asian-Indians originating from Northern Pakistan when compared to Caucasians from the same area (Pearson et al., 2000; Georgiou et al., 2004). There have also been reports of increased KC expression among the Mediterranean and Middle Eastern populations, which could have been influenced by the hot, dry climate present there (Tanabe et al., 1985; Tabbara, 1999; De Cock, 1994) while populations made up of high percentages of Chinese showed lower prevalence (Shi et al., 2007).
In Malaysia, literature on KC were limited or none existence. In a study on the causes of childhood blindness in Malaysia carried out students from six schools for the blind found that out of the 332 children identified, only eight (2.4%) suffered from KC (Reddy & Tan, 2001) while in the district of Gombak, only a mere 0.3% of school-aged children there were diagnosed with KC (Goh et al., 2005). A study similar study carried out under an urban setting found that 0.3% of eye patients of the UMMC were KC (Reddy et al., 2008). However, these reports do not represent the actual figure fairly and the prevalence in Malaysia may be much higher than those reported.
Despite heavy efforts put into research of the possible aetiology and pathogenesis of KC over the last few decades, results have remained inconclusive and poorly understood. There have been several indications of an underlying genetic factor, and also links to possible biochemical and biomechanical mechanisms (Jimenez et al., 2010).
KC is a heterogenous disease, with several indications of genetic factors contributing to the pathogenesis of isolated KC such as bilaterality of the disease, twin studies, familial aggregation and formal genetic analyses (Rabinowitz, 2003). Out of the 19 pairs of monozygotic twins affected by KC have been reported in literature to date, most reported of KC prevalence in both twins, but at different levels of severity. This suggested the strong genetic factor over the development of KC, but also indicates the role of environmental factors (Parker et al., 1996; Weed et al., 2006; Bechara & Waring, 1996). Early familial studies indicated familial relations were present in 6-8% of KC subjects (Hammerstein, 1974). However, the invention of corneal topography had more or less confirmed the role of familial relations in KC development with findings of up to 50% of KC subjects having at least one affected family member (Gonzalez & McDonnell, 1992). This association was further strengthen by recent findings that relatives of KC patients had a risk of up to 67 times higher to develop KC compared to those who did not (Wang et al., 2000). Few genetic analyses have identified possible mutations present in KC patients, however results remain inconclusive and conflicting (Jimenez et al., 2010).
Theories on a biochemical mechanism have been suggested by some researchers, most of which supports the hypothesis that loss of corneal structural components eventually leads to KC development. Although very little is known about the mechanisms leading to ectasia in KC, a current hypothesis is that the thinning of the cornea is due to abnormality in the collagen cross-linking and subsequent stromal thinning which leads to protrusion of the cornea (Li et al., 2007). Other laboratory studies have also indicated increased levels of proteases and decrease levels of protease inhibitors such as Î±2-macroglobulin and Î±1-antiprotease in the eyes of KC patients (Fukuchi et al., 1994; Sawaguchi et al., 1989; Sawaguchi et al., 1994). Another strong hypothesis for the development of KC pointed out the role of the interleukin-1 system and other apoptosis regulating systems which contributes to loss of keratocytes and eventually stromal thinning (Wilson et al., 1996).
From a biomechanical aspect of the development of KC, most studies have pointed towards the role of oxidative damage to corneal tissue in KC eyes. Both common risk factors of KC, atopy and mechanical trauma cause by eye rubbing have been implicated to cause oxidative damage to the cornea (Kenney & Brown, 2003). The role of atopy in the development of KC have been unclear as initial reports of association between KC and atopy was most probably because atopic individuals have a tendency for eye rubbing due to the severe itch they suffer (Bawazeer et al., 2000). However, there have been studies which found topographic differences and faster KC development in atopic individuals compared to normal individuals (Kaya et al., 2007; Hargrave et al., 2003). Hence, most investigation is needed to confirm such associations. Mechanical trauma caused by eye rubbing have also been implicated numerous times as a significant risk factor of KC due to the high percentage of KC patients with positive history of eye rubbing, ranging between 66% (Coperman, 1965) to 73% (Karseras & Ruben, 1976). However, a causal relationship is still hard to confirm as evidence for eye rubbing being a risk factor is circumstantial, based only on clinical observation and subjective response (Koenig & Smith.,1993). McMonnies (2007) argued though, that it was still reasonable to conclude eye rubbing as the cause of not all but some forms of KC, because eye rubbing is not a necessary condition for KC development despite its circumstantial nature.
Paraoxonase 1 (PON1; EC 18.104.22.168/ EC 22.214.171.124) is a calcium-dependent esterase which is physically associated to high-density lipoprotein (HDL) molecules. PON1 was initially discovered for its ability to hydrolyse the organophosphate paraoxon, hence the name given (Durrington et al., 2001). PON1 has two EC designations as it was initially thought that PON1 and arylesterase were two different enzymes. However, subsequent research demonstrated that PON1 was also responsible for the arylesterase activities (Sorensen et al., 1995).
The PON Family
The PON1 gene encoding for the enzyme, belongs to a super family of PON genes. Together with PON2 and PON3, the PON gene family is located on the long arm of human chromosome 7 at position q21.22 (Primo-Parmo et al., 1996). With evidence of high similarities between the PON genes at the amino acid and nucleotide levels, there are strong indications that these genes may have a common evolutionary precursor, with PON2 being the oldest member followed by PON3 and PON1 (Draganov & La Du, 2004). With the discovery of the protective effects of PON1 towards low-density lipoprotein (LDL) and HDL, there has been a tremendous rise in the number of studies on PON1 (Aviram, 2004). PON2 and PON3, although less studied, have also been shown to display antioxidant properties and possible antiatherogenic capacities similar to PON1 (Ng et al., 2004).
Biochemical Structure and Active Sites
PON1 is made up of 354 amino acids with a total molecular weight of 43 kDa (Primo-Parma et al., 1996; Mackness et al., 1996). PON1 messenger ribonucleic acid (mRNA) expression is limited to the liver in humans. When secreted into the blood circulation, PON1 exclusively binds to HDL (Mackness, 1989a, 1989b).
At a molecular level, the PON1 protein consists of six Î²-bladed propellers, with four Î²-strands making up each blade at shown in Figure. At the top of the propeller, three Î±-helices are responsible for the binding of PON1 to the HDL molecule. Calcium have also been shown to be important in PON1 activity, by maintaining the active site of PON1 through direct participation or maintaining appropriate conformation of the active site, and by facilitating the removal of diethyl phosphates from the active site (Harel et al., 2004). The Apolipoprotein-A1 (apoA1) in HDL molecules have also been implicated to stabilize and stimulate hydrolytic activities of PON1 (Rosenblat et al., 2006).
As PON1 has two active sites specific to different substrates, the biochemical functions of PON1 can be classified according to which active sites present contributed to the biochemical function. The calcium-dependent site of PON1 has been attributed to ability to hydrolyse organophosphates and related substrate, thus possibly providing protection against organophosphate neurotoxicity (Costa et al., 2005a). On the other hand, a Cys283 residue-dependent site contributes to PON1 ability to hydrolyse oxidised lipids. Thus, it has been establish for some time already of the role of PON1 in the development of atherosclerosis (Mackness & Mackness, 2004), mainly due to its protective effects towards LDL against oxidation and by stimulation of the HDL-mediated macrophage cholesterol efflux (Rosenblat et al., 2006).
In vitro, PON1 have been successfully shown to hydrolyse active metabolites of organophosphates such as paraoxon, chlorpyrifos oxon and diazoxon, and also nerve agents such as soman and sarin (Costa et al., 2003). Many studies have since been carried out to determine whether the level of PON1 activity in an individual may determine possible susceptibility to organophosphate exposure (Manthripragada, 2010; Chia et al.,2009; Costa et al., 2005), however no conclusive results have been achieved.
Atherogenesis is the condition in which the intima layers of arteries gradually thickens, leading to decreased elasticity, narrowing and reduced blood circulation throughout the body (Matsuura et al., 2006). As atherogenesis have been attributed to oxidative modification of LDL in the arterial walls (Mackness et al., 1999) , PON1 plays important anti-atherogenic roles by providing LDL protection against lipid oxidation by lipid hydroperoxides (Adachi & Tsuijimoto, 2006). As oxidised phospholipids have been indicated to activate adhesion of monocytes to endothelium, a key event in the pathogenesis of atherosclerosis, early removal of these oxidised lipid by-products may prevent the development of atherosclerosis (Heinecke & Lusis, 1998). The hydrolytic active sites of PON1 have also been shown to contribute to the stimulation of HDL-mediated macrophage cholesterol efflux (Rosenblat et al., 2006). Increased cholesterol efflux thus prevents deposition of cholesterol in the arterial wall, a process which contributes to the acceleration of atherosclerosis development (Esparragon et al., 2006). Thus, the role of PON1 is clearly protective against atherosclerosis through its antioxidant effect towards lipid oxidation of both LDL and HDL, in addition to its stimulatory effect towards HDL-mediated cholesterol efflux.
Substrates of PON1
As stated earlier, PON1 has been associated with a wide range of substrates, ranging from active metabolites of organophosphates to oxidised lipids such as lipid peroxides. However, the physiological substrate still remains to be known with investigations still ongoing (Ng et al., 2005). Recent evidence have shown have identified a range of lactones including 5-hydroxyeicosatetraenoic acid lactone (Teiber et al., 2006) and homocysteine thiolactone (Kosaka et al., 2005) and also hydrogen peroxide (Aviram et al., 1998b; Karabina et al., 2005) as possible candidates for PON1 hydrolysis.
Variation in PON1 Activity
It has been known for some time that PON1 activity highly fluctuates, varying by 13- up to 40-folds among individuals in a given population (Davies et al., 1996; Richter & Furlong, 1999). Although the mechanism of PON1 activity expression is not well described, studies of PON1 gene expression have found that PON1 promoter polymorphisms, especially that of 192QR, 55LM and -108CT may account for up to 25% of variation in serum PON1 levels (Leviev & James, 2000). The rest of the variation may be accounted for by a great number of other factors, both environmental and biological (Costa et al., 2005).
PON1 Q192R Polymorphism
Although there at least 198 known single-nucleotide polymorphism (SNPs) in the human PON1 gene (La Du, 2003), most of the variation of PON1 activity are influenced by three common polymorphism sites; the 55LM, 192QR and -108CT polymorphism sites. The 55LM and 192QR polymorphism is located at Exons 3 and 6 respectively, while -108CT polymorphism is found in the regulatory regions of the gene (Durrington et al., 2001).
Among these, the 192QR polymorphism was found to be the most important with the wild-type Q allele associated with low paraoxonase (POase) activity (Mueller et al., 1983). The PON1 192R allele possibly improves the positioning of paraoxon on the active site, thereby producing a more effective hydrolysis and higher activity (Harel et al., 2004). However, the opposite is true for the hydrolysis of nerve agents and oxidised HDL or LDL with PON1 192Q allele being more efficient at metabolising those substrates.
Many studies have been carried out on the association of PON1 with diseases, however most have only examined the most common nucleotide polymorphisms present. However, such analysis does not provide the complete picture of the levels of plasma PON1 activity even if all polymorphisms present in PON1 gene were tested. This is due to the high variation of PON1 activity expressed and also the many modulating factors, both biologically and environmentally. Thus, a functional genotype analysis would provide much more information as measurements of plasma PON1 activity takes into account all polymorphisms and other factors which might have affected the expressed activity (Costa et al., 2005). Such an approach have been referred to as the determination of PON1 status, which takes into account the measurement of PON1 activity coupled with PCR genotying of codon 192 to identify any possible discrepancies due to possible undiscovered mutations in the PON1 gene (Richter & Furlong, 1999). For this study, the determination of PON1 status was carried out using a two-substrate method as described by Richter et al., 2004).
Free radicals are molecules which are characterised by the presence of one or more unpaired electrons. Being unpaired, there electrons are highly reactive as they continuously attempts to pair up with other molecular structures to achieve a more stable state. Thus, these free radical species can react with most cellular macromolecules, changing molecular structures and eventually modifying the cellular functions of the cell (Wu & Cederbaum, 2003). Due to its vital role in the respiratory chain of all living organisms, oxygen has commonly been associated with free radical formation, forming reactive oxygen species (ROS) such as superoxides (O2â€¢-), hydroxyls (â€¢OH), hydroperoxyls (HOOâ€¢) and alkoxyls (ROâ€¢) (Evans & Halliwell, 2001).
As free radicals are being formed constantly through metabolic functions, cells have adapted protective mechanisms to detoxify these free radicals before any damage is done to the cells (Wu & Cederbaum, 2003). These protective mechanisms are most likely to involve antioxidants, which are any substances which are able to significantly hold-up or restrain the oxidation of a substrate, when present in low amounts relative to the oxidised substrate (Halliwell & Gutteridge, 1989). Antioxidants may be present in two forms, whether enzymatic or non-enzymatic in nature and they act by three main mechanisms; preventing the production of free radicals, terminating the free radical chain reaction and repairing the damage caused by free radicals (Sies, 1993).
When there exist an imbalance between free radicals and antioxidants which fall in favour of free radicals, there is a potential of severe cellular damage caused by free radicals and this condition is known as oxidative stress (Sies, 1985). Many studies have shown that a state of oxidative stress can lead to tissue damage (Sies, 1997) and possibly play important roles in the aetiology of many diseases (Brownlee, 2001).
Oxidative Stress in the Human Cornea
As described earlier, the cornea protects the insides of the eye from environmental harms. However, by doing so the cornea itself is constantly exposed to harmful elements such as ultraviolet (UV) radiation, which have been reported widely as an environmental stress factor which contributes to the formation of free radicals in most cells and tissues (Wenk et al., 2001). As the cornea absorbs most of the UV radiation entering the eye, it is most likely that the cornea is especially predisposed to damages from ROS. Studies have reported of various antioxidant enzymes present in the cornea, such as aldehyde dehydrogenase (ALDH3), superoxide dismutase (SOD), catalase, glutathione peroxidise and glutathione reductase, all of which contribute to the removal of ROS in the cornea. (Abedinia et al., 1990; Rao et al.,1987).
Oxidative Stess in Keratoconus
Many studies have been carried out on the role of oxidative stress in KC cornea. A study on oxidative stress levels in human corneal diseases found accumulation of cytotoxic by-products from both lipid peroxidation and nitric oxide pathways of oxidative stress in KC corneas. From these findings, it was suggested that KC corneas do not process the ROS in a similar manner to healthy corneas, which could have contributed to the pathogenesis of KC (Buddi et al., 2002). Other studies have found decreased levels of important corneal antioxidant enzymes in KC cornea, such as ALDH3 (Gonghowiardjo et al., 1993), SOD (Behndig et al., 1998) and catalase (Kenney et al., 2005), an enzyme responsible for hydrogen peroxide hydrolysis. When taken together, the lack of these enzymes in KC corneas have significantly increased accumulation of malonaldehyde (MDA) from lipid peroxidation pathways and nitrotyrosine (NT) from nitric oxide pathways. Thus, all of these findings have led to a working hypothesis, the "Cascade Hypothesis" which states that KC corneas may have an underlying defect in their ability to detoxify ROS and thus suffers from oxidative damage that triggers a cascade of events which eventually lead to corneal thinning (Kenney & Brown, 2003).
Oxidative Stress and PON1
As stated earlier, PON1 have been showed to play an important antioxidant role in protection against atherosclerosis through its action of preventing lipid oxidation of both LDL and HDL molecules. PON1 have also been shown in various studies to be able to neutralise the toxic effects of lipid peroxides. As a recent study have found expression of PON1 mRNA human cataractous lens which was expected due of the widely accepted role of PON1 as an antioxidant enzyme (Hashim et al., 2009), there is a high possibility of PON1 having the same antioxidant role in the cornea. Thus, this study hypothesized that there would be decreased PON1 activity due to the presence of oxidative stress in KC cornea.
Justification of This Study
Although there have been reports of KC developing in infants and also in individuals as late as the age of 51 years, the majority of KC patients develop this condition between the ages of 12 to 20 years (Hall, 1963), which is around the onset of puberty. It is really devastating for one to develop such a condition at this point in life, when one is still young and motivated. In Malaysia, the prevalence of KC appears low with reports of about 4 in 1169 (0.3%) in a population of eye patients in an urban area and 0.3% among school-aged children (Reddy et al., 2008; Goh et al., 2005). However, due to the difficult nature of diagnosing KC in its different developmental stages, many cases often go undetected until after multiple complaints from the patient and after thorough analysis of the patient's vision acuity test results (Benjamin, 2006). Thus, the prevalence of KC in the general Malaysian population could be much higher than reported. As KC is reported as among the top five treatable causes of blindness and severe visual impairment in children in Malaysia (Reddy, 2001), more effort should be carried out to design effective and accurate detection assays using the advancement of molecular medicine to provide early treatment to these individuals before the condition worsen.
The objectives of this study are as follows:
To determine predictors of KC status and POase activity from pooled data
To determine PON1 activity and status in KC patients and to compare with non-KC controls.
To determine the strength of the association between KC and POase activity and identify possible confounders to this association, if any.