Glaucoma is a major cause of blindness worldwide and is

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1 Glaucoma Elevation of Serum Asymmetrical and Symmetrical Dimethylarginine in Patients with Advanced Glaucoma Shahrbanou Javadiyan, 1,2 Kathryn P. Burdon, 1,2 Malcolm J. Whiting, 3 Sotoodeh Abhary, 1 Tania Straga, 1 Alex W. Hewitt, 1,4 Richard A. Mills, 1 and Jamie E. Craig 1 PURPOSE. Asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) are the dimethylated isomeric derivatives of the amino acid L-arginine. ADMA is an endogenous inhibitor of nitric oxide synthase (NOS), while SDMA is a competitive inhibitor of cellular uptake of L-arginine, the substrate for NOS. As such, these metabolites are associated with endothelial dysfunction. As the nitric oxide pathway and endothelial dysfunction have been implicated in glaucoma, the aim of this study was to investigate serum ADMA, SDMA, and L-arginine levels in individuals with advanced glaucoma compared with normal controls. In addition, we have investigated genetic variation in the DDAH1 and DDAH2 genes, encoding the enzymes responsible for degradation of ADMA, for association with ADMA level in glaucoma patients and controls. METHODS. Two hundred eleven patients with advanced glaucoma and 295 normal controls were recruited. Liquid chromatography tandem mass spectrometry was used to measure the serum ADMA, SDMA, and L-arginine levels of participants. Single nucleotide polymorphisms in the DDAH1 and DDAH2 genes reportedly associated with ADMA level were genotyped in all individuals. RESULTS. A significant increase in both serum ADMA and SDMA concentration was detected in advanced glaucoma cases compared with controls (P ). No significant change was detected in serum L-arginine concentration. No association of polymorphisms in DDAH1 and DDAH2 with either ADMA level or glaucoma was detected. CONCLUSIONS. The serum levels of two dimethylarginines, ADMA and SDMA, are associated with advanced glaucoma. These data further implicate the nitric-oxide pathway in glaucoma pathogenesis. (Invest Ophthalmol Vis Sci. 2012;53: ) DOI: /iovs From the 1 Department of Ophthalmology, Flinders University, Flinders Medical Centre, Adelaide, Australia; the 3 Chemical Pathology Directorate, SA Pathology, Flinders Medical Centre, Adelaide, Australia; and the 4 Centre for Eye Research Australia, University of Melbourne, Melbourne, Australia. 2 These authors contributed equally to the study. Supported by the RANZCO Eye Foundation and the Ophthalmic Research Institute of Australia. Salary for KPB and JEC supported by the National Health and Medical Research Council of Australia Fellowship schemes. Submitted for publication August 15, 2011; revised December 21, 2011 and January 24, 2012; accepted January 31, Disclosure: S. Javadiyan, None; K.P. Burdon, None; M.J. Whiting, None;S. Abhary, None;T. Straga, None;A.W. Hewitt, None; R.A. Mills, None;J.E. Craig, None Corresponding author: Kathryn P. Burdon, Department of Ophthalmology, Flinders University, GPO Box 2100, Adelaide, SA, 5001, Australia; kathryn.burdon@flinders.edu.au. Glaucoma is a major cause of blindness worldwide and is predicted to affect approximately 80 million people by It is a neurodegenerative disease characterized by the progressive death of retinal ganglion cells and subsequent optic nerve damage. 2 Elevated intraocular pressure (IOP) is an important risk factor, but mechanisms independent of IOP are also involved. 2 Apoptosis has been shown to be the mechanism of retinal ganglion cell loss in glaucoma; however, the precise triggers and regulatory factors in this disease are as yet unknown. 3 The protein arginine N-methyltransferase family of enzymes catalyze the methylation of L-arginine (L-Arg) to produce asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA). 4 SDMA and ADMA are physiological isomers. All three metabolites (ADMA, SDMA, and L-Arg) are involved in the nitric-oxide (NO) production pathway. Through competition of the binding site of L-Arg, ADMA is an endogenous inhibitor of nitric oxide synthase (NOS), the key endothelial enzyme that catalyses the conversion of L-arginine to L- citrulline and NO. SDMA does not directly inhibit NOS but appears to interfere with the cellular transport of arginine and some other amino acids. 4 The result of the inhibition of NOS by ADMA is the reduction of NO bioavailability. NOS itself exists in three isoforms: endothelial (enos), neuronal (nnos), and inducible (inos). 5 Each isoform is produced by distinct cell types. In particular, enos is produced by endothelial cells. NO is an essential metabolite and acts as an antioxidant. It is involved in a variety of processes maintaining the proper function of vascular endothelium. 6 It is also involved in the inhibition of apoptosis of endothelial cells under shear stress 7 via the S-nitrosylation and cyclic GMP dependent pathway. 8 The anti-apoptotic properties of NO have also been reported in primary neurons. 9 It has been suggested that the increased level of enos may have neuroprotective effects by enhancing vasodilation. 10 Dimethylaminohydrolase (DDAH) is the enzyme responsible for degradation of ADMA to its metabolites dimethylamine and citrulline. 4 Two isoforms, DDAHI and DDAHII (encoded by the DDAH1 and DDAH2 genes, respectively), have been identified. DDAHI is predominant in tissues with nnos, while DDAHII is more dominant in tissues with enos expression. 11 It has been shown that overexpression of DDAH1 in mice causes a reduction in ADMA level and an increase in sensitivity of the vasculature to NO. 12 A physiological role for NO in the regulation of IOP has been suggested, and the use of NO-releasing compounds to treat glaucoma has also been considered. 13 Considering the involvement of ADMA and SDMA in the L-arginine nitric-oxide pathway and multifaceted roles of NO in apoptosis and vasodilatation, we hypothesize a potential association of serum ADMA, SDMA, and L-Arg levels with glaucoma. An association of ADMA levels with diabetes mellitus, diabetic retinopathy, nephropathy, and cardiovascular disease has been previously reported Here, we present data from patients with and Investigative Ophthalmology & Visual Science, April 2012, Vol. 53, No Copyright 2012 The Association for Research in Vision and Ophthalmology, Inc.

2 1924 Javadiyan et al. IOVS, April 2012, Vol. 53, No. 4 without advanced glaucoma. In addition, we previously found a strong association between single nucleotide polymorphisms (SNPs) in the DDAH1 and DDAH2 genes and serum ADMA levels in patients with type 2 diabetes mellitus. 18 Thus, we investigated these SNPs for association with both glaucoma and with ADMA level in glaucoma patients. METHODS Participant Recruitment Cases were recruited from the Ophthalmology clinic of Flinders Medical Centre, Adelaide, Australia. Controls consisted of Flinders Medical Centre volunteers, spouses of patients, and residents of assisted living facilities (nursing homes) in Adelaide. Approval was obtained from the Human Research Ethics Committee of Flinders University and Flinders Medical Centre, and the study adhered to the tenets of the Declaration of Helsinki. In total, 506 individuals consisting of 211 cases and 295 controls of Caucasian ethnicity were recruited. Advanced glaucoma was defined as visual field loss related to glaucoma with at least two out of the four central squares having a Pattern Standard Deviation (PSD) <0.5% on a Humphrey 24-2 field, or a mean deviation of less than -22 db, or in the absence of field testing, loss of central acuity related to glaucoma in the worst eye. Evidence of glaucomatous optic disc changes in both eyes was an additional entry criterion. Patients with ocular pathology (other than open angle glaucoma or cataract) were excluded. Information on worst recorded IOP and age of glaucoma diagnosis was obtained from the case notes. All patients were under the care of ophthalmologists and on treatment for glaucoma; however, IOP readings prior to initiating treatment were obtained where possible. Control participants were examined and included if they had no evidence of glaucoma, normal IOP, and no family history of glaucoma. Measurement of Serum ADMA, SDMA, and L- Arginine Level Serum was obtained from peripheral whole blood collected in EDTA for each participant. Whole blood was centrifuged at 2700g. The serum was then stored at -808C in 1-mL aliquots until ready for use. Measurement of serum ADMA, SDMA, and L-Arg concentrations were obtained using liquid chromatography tandem mass spectrometry as previously reported. 18,19 The method was validated by the inclusion of deuterated internal standards (Cambridge Isotope Laboratories, Andover, MA). The between-run coefficient of variation for ADMA and SDMA was calculated as 9.3% and 13.0% at the concentrations of 1.40 and 1.38 lm, respectively. Genotyping Peripheral whole blood was obtained from each participant. DNA was extracted using the QiaAmp Blood Maxi Kit (Qiagen, Valencia, CA) and diluted to 20 ng/ll for genotyping. SNP genotyping was performed using predesigned Taqman assays (Applied Biosystems, Carlsbad, CA) for SNPs rs and rs These were genotyped in all cases and controls according to the manufacturer s protocols on a Step-One Plus Real-Time PCR instrument (Applied Biosystems). Statistical Analysis Statistical analysis was performed using SPSS (Version 18.0, SPSS Inc., Chicago, IL). ADMA, SDMA, and L-Arg were log transformed to achieve an approximately normal distribution. The independent t-test was used to compare means, and linear regression was performed to incorporate adjustment for age and sex in the cohorts. Student s t-test and the v 2 test were used to detect differences in age and sex, respectively, between cases and control participants. Pearson correlation coefficient TABLE 1. Demographic and Clinical Characteristics of Cases and Controls* was used to detect correlation of sex and age with serum measures. Genetic associations were analyzed using v 2 tests and linear regression implemented in PLINK (Version 1.07). 20 Using the Genetic Power Calculator, 21 this study has 85% power to detect SNPs accounting for 5% of the variance of these traits. RESULTS Cases Controls P Value N Age (y) at recruitment Female (%) 47.4% 54.2% 0.12 Self-reported diabetes (%) 12.3% 11.2% 0.70 Age (y) at diagnosis Duration (y) of POAG Worst recorded IOP (mmhg) Worst recorded IOP >21 (%) 73.6% * Data are presented as mean standard deviation or as percentage (%) of the cohort. A total of 211 patients with advanced glaucoma and 295 control participants were recruited. Demographic characteristics of the cohort are displayed in Table 1. There was no significant difference in age, sex, or diabetes status between the case and control cohorts. Approximately 74% of the cases had a worst recorded IOP of greater than 21 mmhg. The average age of diagnosis of glaucoma was 61 years, with a mean duration of glaucoma of 16 years in this cohort. Age at study recruitment was positively correlated with log(adma) in both the normal control (R = 0.259; P = ) and advanced glaucoma groups (R = 0.159; P = 0.021). Log(SDMA) was also associated with age in the normal control (R = 0.396; P = ) as well as the advanced glaucoma group (R = 0.239; P = ). Log(L-Arg) was not significantly correlated with either age or sex. There was no correlation between worst recorded IOP and any of the analytes. Serum ADMA level in glaucoma cases (mean = 0.64 lm) was higher compared with controls (mean = 0.60 lm) with P value of (Fig. 1A). Similarly, SDMA level was also higher in glaucoma patients (mean = 0.71 lm) than normal controls (mean = 0.61 lm) with P value of (Fig. 1B). There was no statistically significant difference in L-Arg levels between glaucoma cases (mean = lm) and controls (mean = lm) as shown in Figure 1C. The associations with ADMA and SDMA remained significant after adjustment for age and sex (Fig. 1). The associations with ADMA and SDMA remained significant after excluding participants with diabetes mellitus and adjusting for age and sex (P = and P = , respectively). We investigated two SNPs previously reported to be associated with serum ADMA concentration in type 2 diabetes mellitus. Table 2 shows association results of these SNPs (rs in DDAH1 and rs in DDAH2) with serum ADMA in the glaucoma cases and controls separately and in the total cohort. Serum ADMA level was found to be associated with the DDAH1 SNP rs in the control group (Table 2) before adjustment for age and sex b (95% confidence interval [CI]) =-0.01( to ). No association was detected in the advanced glaucoma group. Analysis of all participants in this study revealed a borderline level of association b (95% CI) =-0.01(-0.02 to ) which did not remain statistically significant after adjusting for age and sex. No

3 IOVS, April 2012, Vol. 53, No. 4 Elevated Dimethylarginines in Advanced Glaucoma 1925 FIGURE 1. Comparison of untransformed level (lm) of mean ADMA (A), mean SDMA (B), and L-arginine (C) in advanced glaucoma patients (n = 211) and normal controls (n = 295). The mean value is given inside each bar. Error bars indicate 95% CI of the mean. association was observed with the DDAH2 SNP rs Similarly, neither SNP was associated with glaucoma status (Table 3). DISCUSSION In this study, serum ADMA and SDMA levels were found to be elevated in advanced glaucoma patients compared with ageand sex-matched normal controls. This study is the first to report such an association and provides further evidence for a role of the NO pathway in glaucoma pathogenesis. Furthermore, we found DDAH1 genetic variants to be associated with serum ADMA level in normal controls, but not glaucoma patients. Our previous work 18 revealed that the genetic variation in the DDAH1 and DDAH2 genes was significantly associated with ADMA serum level in patients with type 2 diabetes, particularly those without diabetic retinopathy. Together, these results suggest that the DDAH genes are involved in the regulation of ADMA levels under normal circumstances and that this regulation is partly controlled by genetic variants in these genes. It also suggests that as disease processes progress, ADMA becomes dysregulated, potentially independently of the DDAH genetic variation. This cohort of patients does not have high levels of chronic metabolic disease (e.g., diabetes or cardiovascular disease); however, there are high systemic levels of ADMA and SDMA observed here, similar to that seen in other metabolic conditions. We propose that these patients have a propensity to elevated ADMA/SDMA, conferred either through genetic or environmental risk factors. The observed elevated dimethylarginines may then lead to a pathogenic state characterized by reduced NOS activity, leading to disease. The determination of TABLE 2. P Values for Association of DDAH1 and DDAH2 Variants with Log(ADMA) in Advanced Glaucoma Cases and Controls* rs (DDAH1) rs (DDAH2) Unadjusted Adjusted Unadjusted Adjusted Glaucoma Controls Glaucoma + Controls * P values for unadjusted and age/sex adjusted results are presented. which disease, or combination of diseases, manifests in patients with elevated ADMA/SDMA may be dependent on other underlying genetic and environmental risk factors and tissue-specific effects. NOS is constitutively expressed throughout the eye. 22 Both constitutive isoforms of NOS (enos and nnos) as well as NO production were detected in rat retinal ganglion cells both in vivo and in vitro. 23 Inhibition of NOS was shown to reduce ischemic damage in rat retinas induced by transiently elevated IOP. 5 NO has been found to be involved in the regulation of IOP, ocular blood flow, and also retinal ganglion cell death. 24 It has been suggested that the altered function of the regulatory system of the endothelium could be a vascular risk factor for the pathogenesis of glaucoma. 25,26 The failure of adequate perfusion pressure and stable blood flow at the optic nerve head could be one of the underlying pathogenic mechanisms for glaucomatous optic neuropathy. 27 Polak and colleagues 28 investigate the response of ocular blood flow in relation to NOS levels in patients with glaucoma compared with controls. They observe an abnormal blood flow in patients with glaucoma and thus provide the first in vivo evidence for disruption of the ocular NO pathway in glaucoma patients. Oxidative and nitrative stresses are likely to have an important role in glaucoma. 29 Oxidative stress is caused by accumulation of reactive oxygen species, which can react with NO to form peroxynitrite, thereby inducing apoptotic pathways. Increased NO and oxidative stress have been reported in neurodegenerative conditions such as Alzheimer disease, along with a decrease in ADMA level. 4 The current study shows elevated ADMA in glaucoma patients, implying a reduction in NO availability and a potential increase in apoptotic events as a consequence. Although it is yet to be investigated, the lack of NO availability may lead to increased levels of NOS to compensate. Excessive production of inos can be toxic in the central nervous system 30,31 and retinal ganglion cells. 32 Our current results could be indicative of a potential compensatory mechanism to decrease pathological NO elevation and thus prevent damage of retinal ganglion cell axons. However, NO is a modulator with both pro- and anti-apoptotic mechanisms dependent on the context. 33 Thus, the precise pathogenic role in glaucoma requires further elucidation. Nonetheless, it is clear that an optimal level of NO is essential for functionality of retinal ganglion and endothelial cells and that ADMA and SDMA are involved in the regulation of this important metabolite.

4 1926 Javadiyan et al. IOVS, April 2012, Vol. 53, No. 4 TABLE 3. Association of DDAH SNPs with Glaucoma Status* Gene SNP Allele 1 Allele 2 Frequency Cases Frequency Controls P Value DDAH1 rs C T DDAH2 rs A C * Frequencies of Allele 1 in cases and controls are shown. In summary, we have observed for the first time, elevated levels of serum ADMA and SDMA in patients with glaucoma. These data provide a link at the clinical level between the NO pathway and poor disease outcomes. This study is limited to advanced glaucoma, so it is not possible to determine if this is a result of disease or a contributing cause; however, it forms the basis of hypotheses moving forward to further explore the role of this pathway in the clinical setting. Further work is required to determine whether this finding will hold true in glaucoma patients with less severe earlier stage disease. ADMA/SDMA may have potential as a novel biomarker for progression risk in glaucoma, although it is not yet known if the magnitude of the difference observed here is clinically significant or if such a test could have sufficient sensitivity and specificity. An understanding of the role of ADMA/SDMA and NO in glaucoma pathogenesis is likely to be useful in the quest for novel therapeutics designed to target particular pathways involved in disease processes. Any such therapies designed to increase NO production may have to work against or counteract a background of endogenous NOS inhibition in glaucoma patients. Acknowledgments The authors thank SA Pathology for their donation of materials for the measurement of serum metabolites. References 1. Quigley HA, Broman AT. The number of people with glaucoma worldwide in 2010 and Br J Ophthalmol. 2006;903: Gupta N, Yucel YH. Glaucoma as a neurodegenerative disease. Curr Opin Ophthalmol. 2007;182: Quigley HA. Glaucoma. 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5 IOVS, April 2012, Vol. 53, No. 4 Elevated Dimethylarginines in Advanced Glaucoma Aslan M, Cort A, Yucel I. Oxidative and nitrative stress markers in glaucoma. Free Radic Biol Med. 2008;454: Sawada A, Becker B. Inhibition of nitric-oxide synthase 2 by aminoguanidine provides neuroprotection of retinal ganglion cells in a rat model of chronic glaucoma. Proc Natl Acad Sci U SA. 1999;9617: Dawson VL, Dawson TM. Nitric oxide in neuronal degeneration. Proc Soc Exp Biol Med. 1996;2111: Toda N, Nakanishi-Toda M. Nitric oxide: ocular blood flow, glaucoma, and diabetic retinopathy. Prog Retin Eye Res. 2007; 263: Choi BM, Pae HO, Jang SI, Kim YM, Chung HT. Nitric oxide as a pro-apoptotic as well as anti-apoptotic modulator. J Biochem Mol Biol. 2002;351: