Keratoconus ( KC ) is a noninflammatory corneal ectasis which normally affects both eyes and with an incidence of about 1 per 2,000 in the general population ( Rabinowitz, 1998 ) , KC is going a important clinical job worldwide ( Zadnik et al, 1996 ) . The authoritative histopathological characteristics of KC include stromal cutting, Fe deposition in the epithelial cellar membrane and interruptions in the Bowman ‘s bed ( Rabinowitz, 1998 ) . KC is a heterogeneous disease, with several indicants of familial factors lending to the pathogenesis of stray KC such as duplicate surveies, bilateralism of the disease, familial collection and formal familial analyses ( Rabinowitz, 2003 ) . However, the function of environmental factors such as oculus friction and difficult contact lens wear may besides lend to the patterned advance of the disease in genetically susceptible persons ( Rabinowitz, 1998 ) . Although really small is known about the mechanisms taking to ectasia in KC, the current hypothesis is that the cutting of the cornea is due to abnormalcy in the collagen cross-linking and subsequent stromal thinning which leads to bulge of the cornea ( Li et al, 2007 ) . Other research lab surveies have besides indicated the degree alterations of transforming growing factor-I?2 and antioxidant enzymes such as matrix metalloproteinases, cathepsin V/L2 and tissue inhibitor of matrix metalloproteinases ( TIMPs ) in the eyes of KC patients ( Maier et al, 2007 ; Kenney et Al, 2005 ) . Another strong hypothesis of the development of KC was put frontward ( Wilson et al, 1996 ) who pointed out the function of the interleukin-1 system and other programmed cell death modulating systems which contributes to loss of keratocytes and finally stromal cutting. However, all of these suggested hypotheses need to be more clearly defined ( Rabinowitz, 1998 ) .
Paraoxonase 1 ( PON1 ) is an enzyme made up of 354 aminic acids with a entire molecular weight of 43 kDa ( Primo-Parma et Al, 1996 ; Mackness et Al, 1996 ) . PON1, which is associated with high-density lipoprotein ( HDL ) , catalyses the dislocation of phospholipid and cholesteryl-ester lipid peroxides in both low-density lipoprotein ( LDL ) and HDL, therefore doing it an of import hazard factor of artherosclerosis ( Mackness et al, 2004 ) . However, it was the ability of PON1 to protect the nervous system against organophosphate neurotoxicity that was foremost discovered ( Durrington et al, 2001 ) . The human PON1 cryptography sequence, located on the long arm of the human chromosome 7 ( q21.22 ) has two common polymorphism sites ; a Met ( M ) / Leu ( L ) permutation site at place 55 and a Gln ( Q ) / Arg ( R ) site at place 192, with the latter being more of import with respects to PON1 activity and affinity to certain substrates ( Primo-Parma et Al, 1996 ; Aviram, 2004 ) . Hence, the finding of the PON1 position of an person must non merely take into history the polymorphism nowadays but besides the degree of PON1 activity in that person in order to find the plasma PON1192 alloform responsible for the activity nowadays. This can be done utilizing a two-substrate enzymic check affecting two PON1 substrates ( normally paraoxon and diazoxon ) . Abnormalities between PON1 functional position and genotyping at place 192 can therefore bespeak mutant at other points in the PON1 cistron ( Richter et al, 2004 ) . As there are many factors which modulates the PON1 activity such as physiological factors ( eg. exercising ) , pathological factors ( eg. viral/bacterial infection, redness, diabetes ) , diet, alcohol ingestion and certain drugs ( eg. lipid-lowering lipid-lowering medicines ) , therefore is it of import to see these factors when finding of PON1 activity ( Aviram, 2004 ) . PON1 has been shown to hold antioxidant belongingss against oxidative emphasis ( Senti et al, 2003 ) , while oxidative emphasis has been associated with KC ( Kenney et al, 2005 ) . Therefore, PON1 may demo a protective function in the development of KC.
OBJECTIVES OF THE STUDY
Although there have been studies of KC developing in babies and besides in persons every bit tardily as the age of 51 old ages, bulk of KC patients develop this status between the ages of 12 to 20 old ages ( Hall KCG, 1963 ) , which is around the oncoming of pubescence. It could be truly lay waste toing for one to develop such a status at that point in life, when 1 is still immature and motivated. In Malaysia, the prevalence of KC may look to be of less important with studies of approximately 4 in 1169 ( 0.3 % ) in a population of oculus patients in an urban country and besides 0.3 % among school-aged kids ( Reddy SC et Al, 2008 ; Goh PP et Al, 2005 ) . However, due to the hard nature of naming KC in its developmental phases, many instances frequently go undetected until after multiple ailments from the patient and thorough analysis of the patient ‘s vision sharp-sightedness trial consequences ( Benjamin WJ, 2006 ) . Therefore, 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 sightlessness and terrible ocular damage in kids in Malaysia ( Reddy SC, 2001 ) , therefore more attempt should be carried out to plan an effectual and accurate sensing assay utilizing the promotions of molecular medical specialty to supply early intervention to these persons before the status worsens. Therefore, this brings to the aims of this survey, which are as follow:
To find PON1 activity in KC patients and to compare with non-KC controls.
To find the position of PON1 in plasma samples of KC and non-KC patients.
To place forecasters of KC from the accumulated informations by utilizing univariate and logistic arrested development analysis.
MATERIALS AND METHODS
Preparations prior to informations and sample aggregation
In order to obtain blood samples and informations from participants, a two-day information and sample aggregation event was organised. The event was held on the 22 – 23th May 2010 at Ophir Eye Clinic and Surgery. Prior to the event, readyings were made such as boxing a 21G acerate leaf, a disposable syringe, intoxicant swab, Elastoplast, a 6mL EDTA ( purple-top ) tubing, a 6mL Li Lipo-Hepin ( green-top ) tubing and some sweet into a fictile bag for each participants
Data and sample aggregation
Each participant was foremost given a brief account of the survey which they were traveling to take portion in and were asked to make full in an informed consent signifier before finishing the “ Keratoconus in the Malayan Population: Pathophysiological and Familial Surveies ” questionnaire signifier prepared by Shalini Arjunan, Prof Mary Anne, Dr Rozaida and Dr Jenny. Next, the participants ‘ spectacle powers were examined utilizing an auto-refractometer. Blood samples ( 5mL ) were so collected in the Li Lipo-Hepin ( green-top ) tubings from the participants of the survey and stored at 4A°C if can non be processed instantly. Blood samples were processed within the twenty-four hours. After that, each participant was examined utilizing a keratometer and a Pentacam to obtain their K-readings and corneal topography severally. Then, each participant was required to undergo a ocular sharp-sightedness trial and a biomicroscopy trial utilizing Snellen ‘s chart and Haag-Streit Slit Lamp severally. Finally, each participant ‘s trial readings were reviewed by Dr. Jenny P. Deva, adviser Ophthalmologist and Refractive Surgeon at Tun Hussein Onn Eye Hospital and the diagnosing of each participant was confirmed.
Preparation of plasma samples
After aggregation, the blood samples were centrifuged at 500 xg for 5 proceedingss at room temperature ( 25A°C ) to divide the plasma. Plasma ( top bed ) was so be carefully aliquoted in 500AµL batches into a few microcentrifuge tubings ( 1.5mL ) utilizing Pasteur pipettes depending on the sum of plasma available. All micro-centrifuge tubings were labelled and stored at -70A°C until activity measuring was carried out.
Two-substrate enzyme analysis
This survey was carried out by finding the position of Paraoxonase 1 in the plasma samples of the participants based on the two-substrate enzyme analysis as described by Richter RJ et Al, 2004, with little alterations. The rate of hydrolysis of the two substrates, which were Paraoxon and Diazoxon were measured utilizing Lamda 25 UV/VIS Spectrophotometer running KINLAB version 2.85.00. After blending with the several substrates for a few seconds, the rate of paraoxon and diazoxon hydrolysis were monitored continuously for two proceedingss. The initial rates of each sample were so multiplied by the deliberate transition factor, which was 5611 and 67000 to acquire paraoxonase and diazoxonase activity severally in U/L unit.
Activity analysis utilizing SPSS
The paraoxonase and diazoxonase activities of each sample together with other informations from the questionnaire done by Shalini Arjunan and genotype informations done by Yvonne Yong were so pooled together into SPSS version 17.0 for farther analysis. Trials which were carried out include age and sex demographic distribution, independent t-test, Pearson ‘s Correlation, spread secret plan, Kruskal-Wallis trial, one-way ANOVA, etc.
RESULTS AND DISCUSSION
For this survey, a population of 66 participants were successfully identified, consisting of 9 keratoconus patients ( 13.6 % ) , 2 forme fruste keratoconus patients ( 3.0 % ) , 9 high myopic patients ( 13.6 % ) , 11 moderate myopic patients ( 16.7 % ) and 35 normal controls ( 53.0 % ) . This population, dwelling of 27 Malays ( 40.9 % ) , 9 Chinese ( 13.6 % ) and 30 Indians ( 45.5 % ) had an age scope of 7 to 68 old ages old. There were somewhat more males than females in this population, that is 35 ( 53 % ) to 31 ( 47 % ) participants. There were undistinguished difference of ages among both genders and besides the cultural groups.
Exploratory informations analysis on this sample population revealed that the distribution of the paraoxonase activity and age of the participants were non distributed usually, giving a p-value of 0.019 and 0.025 severally when utilizing the Shapiro-Wilk trial. Merely diazoxonase activity gave a undistinguished p-value ( 0.446 ) , therefore bespeaking a normal distribution. It was of import to transport out such trials foremost to find whether a parametric or nonparametric trial should be used for analysis.
The activity of Paraoxonase 1 was successfully determined by mensurating the rate of hydrolysis of paraoxon and diazoxon substrate, harmonizing to the method used in Richter RJ et Al ( 2004 ) , with some minor accommodations. With these informations, the phenotype of each participant was successfully identified by plotting a spread graph of diazoxonase activity against paraoxonase activity, to distinguish the persons with functionally homozygous for PON1192Q, heterozygotes for PON1Q/R192Q/R and homozygous for PON1192R. These phenotype information was subsequently confirmed with informations from my co-worker, Yvonne who carried out Paraoxonase 1 genotyping for polymorphism 192QR by limitation enzyme digestion. Both informations from Paraoxonase 1 activity finding and Paraoxonase 1 genotyping were found to be coincident and accurate.
When these participants were divided harmonizing to familial relation to the KC patients, 18 were first degree relations to the KC patients, 3 were 2nd degree relations and 20 had no blood dealingss to the patients, while 16 were omitted as they did non suit the standards for normal healthy controls. Based on such classs, it was found that KC patients and relations may hold a important difference in paraoxonase activity when groups together against the normal controls with a p-value of 0.057. However, this value is non important plenty to reason a possible correlativity and may necessitate more KC to make a important degree. This was non the instance for diazoxonase activity when compared in similar mode, where the activity in KC patients and comparative were non significantly different from that of normal controls.
The most important consequences of this survey nevertheless, were obtained when KC patients, including forme fruste KC patients were compared against the other participants, labelled non-KC. When categorised as such, the paraoxonase activity of KC patients were significantly difference than that of the non-KC participants ( p-value = 0.022 ) . This indicates that paraoxonase activity may hold a function in the development of KC patients. However, different cultural groups besides showed important difference in paraoxonase activity when tested, viz. between Malays and Indians and this may lend as an implicit in factor since many of the KC patients where Indians. Hence, comparing surveies between KC/non-KC position and cultural groups must be carried out to find any relation between the two variables.
The paraoxonase and diazoxonase activity of each participant were successfully and accurately measured. Based on genotype informations from my co-worker Yvonne, the phenotype informations obtained from the diazoxonase – paraoxonase spread secret plan was rather dependable. There were important happening which relates paraoxonase activities with KC/non-KC position, but it may be due to an underlying factor such as cultural group. Further statistical analysis and re-definition will be required to obtain more important informations.