Increased Homocysteine Levels in Tear Fluid of Patients with Primary Open-Angle Glaucoma

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1 Originl Pper DOI: / Received: September 20, 2006 Accepted fter revision: July 6, 2007 Published online: April 25, 2008 Incresed Homocysteine Levels in Ter Fluid of Ptients with Primry Open-Angle Glucom J.B. Roedl S. Bleich b U. Schlötzer-Schrehrdt N. von Ahsen c J. Kornhuber b G.O.H. Numnn F.E. Kruse A.G.M. Jünemnn Deprtment of Ophthlmology, University Eye Hospitl, nd b Deprtment of Psychitry nd Psychotherpy, Friedrich-Alexnder University, Erlngen-Nuremberg, nd c Deprtment of Clinicl Chemistry, Georg-August University, Goettingen, Germny Key Words Glucom Homocysteine Ters Vitmins Abstrct Aims: We ssessed homocysteine (Hcy) levels in ter fluid nd plsm of ptients with primry open-ngle glucom (). We determined the ssocition between Hcy levels, dry eye syndrome nd B vitmin sttus. Methods: This prospective cse-control study included 36 ptients with nd 36 controls. Hcy concentrtions were mesured by highperformnce liquid chromtogrphy. Results: Ptients with hd significntly higher men Hcy levels both in ter fluid ( nmol/l; p! 0.001, t test) nd in plsm ( mol/l; p = 0.001, t test) thn control subjects ( nmol/l nd mol/l, respectively). Hcy in ter fluid ws significntly correlted with plsm Hcy in ptients (r = 0.459; p = 0.005, Person s correltion), but not in controls (r = 0.068; p = 0.695). ptients with dry eye disese hd significntly higher Hcy levels both in ter fluid nd plsm thn ptients without dry eye disese. There ws no ssocition between Hcy levels nd B vitmin sttus in subjects with. Conclusions: The study suggests incresed Hcy levels in ter fluid nd plsm of ptients with. Elevted Hcy levels might be risk fctor for nd dry eye syndrome in subjects with glucom. Copyright 2008 S. Krger AG, Bsel Introduction The pthogenesis of primry open-ngle glucom () is still not fully understood. There is ccumulting evidence tht vsculr [1], extrcellulr mtrix [2], nd neurotoxic [3] chnges contribute to the development of besides incresed introculr pressure (IOP). Extrcellulr mtrix nd proinflmmtory chnges re lso involved in oculr surfce disorders seen in including fibrosis of the filtering bleb fter trbeculectomy [4, 5] nd dry eye syndrome [6, 7]. Homocysteine (Hcy) is highly rective mino cid tht cn induce typicl pthologicl chnges found in including poptotic deth of retinl gnglion cells [3], vsculr [8 10], extrcellulr mtrix [8, 11, 12], nd proinflmmtory ltertions [13]. In nd pseudoexfolition glucom (PEXG), elevted plsm Hcy levels were observed in previous studies of our group [14 17]. Moreover, incresed Hcy vlues in queous humor were found both in [17] nd in PEXG [16]. Accordingly, we detected significntly higher prevlence of the most common genetic cuse for hyperhomocysteinemi in [18]. Severl following studies confirmed our results of incresed Hcy levels in PEXG [19, 20]. Hyperhomocysteinemi ws lso shown to be risk fctor for vrious other oculr diseses [21 23]. However, there re in- Fx E-Mil krger@krger.ch S. Krger AG, Bsel /08/ $24.50/0 Accessible online t: Johnnes B. Roedl, MD, Deprtment of Ophthlmology Friedrich-Alexnder University of Erlngen-Nuremberg Schwbchnlge 6, DE Erlngen (Germny) Tel , Fx E-Mil Johnnes.Roedl@ugen.imed.uni-erlngen.de

2 Tble 1. Chrcteristics of ptients nd controls Nongenetic determinnts of Hcy p vlue (differences) Age (men 8 SD), yers Mle sex 17 (48) 15 (42) 0.81 b Cffeinted beverges (>3 per dy) 13 (36) 12 (33) 1.0 b Alcoholic drinks (>1 per dy) 5 (14) 7 (20) 0.54 b Smoking 6 (17) 8 (22) 0.77 b Systemic hypertension 12 (33) 14 (39) 0.81 b Dibetes mellitus 4 (11) 5 (14) 1.0 b Hyperlipidemi 6 (17) 4 (11) 0.74 b Atherosclerotic vsculr disese 4 (11) 3 (8) 1.0 b Figures in prentheses re percentges. t test; b Fisher s exct test. consistent results in the literture regrding plsm Hcy levels of ptients [14, 17, 24 27], possibly due to different ssessments of criticl determinnts of Hcy, such s nutritionl nd B vitmin sttus of enrolled subjects. In order to illuminte the role of Hcy in glucomtous neuropthy nd its possible impliction in oculr surfce disorders ssocited with, we evluted ssocitions between Hcy in plsm, Hcy in ter fluid, nd blood B vitmins. Mterils nd Methods Ptients nd Design In this investigtion, 36 consecutive Cucsin Germn ptients with nd 36 helthy controls without glucom, but concurrent dignosis of ctrct were enrolled. All subjects underwent glucom or ctrct surgery t the Deprtment of Ophthlmology t the University of Erlngen-Nuremberg, Germny. Ptients of the present clinicl tril were not enrolled in ny of our previous studies [14 17]. Informed consent ws obtined from ll prticipnts fter detiled explntion of the purpose nd methods of the tril. The University of Erlngen-Nuremberg Humn Subjects Committee pproved ll protocols of this prospective cse-control study, nd the methods described dhered to the tenets of the Declrtion of Helsinki. All controls nd ptients were thoroughly exmined by slit lmp inspection, pplntion tonometry, fundoscopy, gonioscopy, pupillometry, nd perimetry (Octopus 101, progrm G1). All ptients hd open ngles on gonioscopy, chrcteristic glucomtous optic disk dmge (excvtion, presence of neuroretinl rim thinning, notching, or retinl nerve fiber lyer defects), nd pthologicl cumultive perimetric defect curves, tht is, locl s well s diffuse visul field loss in white-on-white perimetry. In ptients, the men durtion of disese ws yers, the men IOP ws mm Hg, nd the globl indices men defect ws db. Control subjects hd no history of oculr disese (except refrctive error, strbismus, or ctrcts), norml IOP vlues (!21 mm Hg), nd norml eye exmintion, including open ngles, norml ppernce of the optic disks nd norml visul fields. Ech ptient ws evluted for dry eye syndrome bsed on three criteri which hd been used in previous studies [28, 29] : (1) history of, tretment of, or symptoms of dry eye syndrome defined ccording to the Oculr Surfce Disese Index [30] questionnire; (2) ter dynmics which were ssessed by the Schirmer 1 test nd ter brekup time; if one of those two tests ws positive (Schirmer 1 test! 10 mm, or brekup time! 10 s), the ter dynmics were considered bnorml; (3) oculr surfce bnormlities which were identified by positive vitl stining with fluorescein. Ptients with positive results in ll three criteri were considered to hve definite dry eye, wheres ptients with bnormlities in only two of the three criteri were defined to hve probble dry eye disese [28]. Exclusion criteri for controls nd ptients were: medicl history of disorders ssocited with incresed Hcy levels such s thromboembolic, renl, heptic, gstrointestinl or neurologic disese, s well s prior oculr surgery, history of oculr inflmmtion, ge-relted mculr degenertion, dibetic retinopthy, myopi, retinl occlusive disese, rubeosis iridis nd glucom other thn. Ptients tking hormone substitution nd medictions known to ffect Hcy mesurements were excluded. Mtching of controls nd ptients ws performed by demogrphic, common clinicl, nd lifestyle fctors known to cuse incresed Hcy levels ( tble 1 ) [31]. Nutritionl nd lifestyle sttus ws ssessed using the Subjective Globl Nutritionl Assessment test [32]. Since ll ptients nd controls were clssified s well nourished, we used 11.7 mol/l s the primry cutoff vlue to define hyperhomocysteinemi s in previous investigtions [14, 16]. Additionlly, we performed sttisticl nlysis with 15.0 mol/l s the threshold. Twenty-nine of the 36 ptients in the group were on topicl ntiglucomtous therpy: 53% (19/36) of the ptients were on bet-blocker, 47% (17/36) on prostglndin nlog, 36% (13/36) on crbonic nhydrse inhibitor, 19% (7/36) on lph-gonist, 3% (1/36) on cholinergic, nd 3% (1/36) on drenergic topicl mediction. In order to void the possible influence of eyedrops on Hcy mesurements, topicl therpy ws stopped in ll ptients t lest 12 h prior to smpling of reflex ters. 250 Roedl et l.

3 L b o r t o r y An ly s e s All ptients underwent blood nd ter fluid collection t the dy of oculr surgery, ensuring tht the ptients were fsting. Secretion of reflex ters ws incited by the ywn reflex nd by stimultion of the trigeminl nerve with nsl lcohol stimulus. A disposble glss cpillry ws plced ner the cul-de-sc of the eye nd 100 l of ter fluid were collected ccording to Choy et l. [33]. Reflex ter collection with the cpillry tube ws shown to be the method of choice in the study by Choy et l. [33]. They compred bsl ters with reflex ters s sources of ter fluid, nd Schirmer strips with cpillry tubes s collection methods. The Schirmer strips hve the disdvntge tht they re ssocited with direct conjunctivl contct nd therefore risk of vsculr trnsudtion nd cell dmge with lekge of plsm nd intrcellulr constituents [34, 35]. Hcy is found both in plsm nd in cells nd Schirmer strips might therefore led to spuriously high levels of Hcy in ters. The use of cpillry tube for ter collection is much less invsive [34], llows collection of lrger volumes of ter fluid thn with the Schirmer strip [35], nd is therefore the suggested method of choice for ter collection. There is currently no suitble method vilble for collection of bsl ters for biochemicl nlysis [33]. Probes were immeditely plced on ice, promptly centrifuged (2,000 g, 10 min) within 30 min fter collection nd stored t 20 C until biochemicl nlysis ws performed. For nlysis of Hcy in ters, 50 l of undiluted ter fluid were mixed with 14 l of reduction gent (1.43 mol/l sodium borohydride). The smples were then gently vortex-mixed nd incubted for 5 min t room temperture. We dded 50 l of precipittion gent (1.50 mol/l perchloric cid) nd vortex mixed the smples for 30 s. After removl of protein with centrifugtion (12,000 g, 5 min), 40 l of the probes were mixed with 80 l of derivtiztion gent (10.0 mmol/l monobromobimne). The tubes were cpped, mixed, nd incubted t 42 C. The utosmpler (model: Mids, Sprk Systems, Emmen, The Netherlnds) injected 50 l liquots onto 4.6! 150 mm LC8 Supelcosil column (Supelco, Bellefonte, P., USA) with column temperture of 25 C. For determintion of homocysteine-s-bimne, the column ws developed t flow rte of 1.2 ml/min. Homocysteine-bimne dduct ws detected fluorometriclly with n RF-10Axl high-performnce liquid chromtogrphy detector (Shimdzu Corportion, Kyoto, Jpn). The excittion wvelength ws set t 385 nm, nd the emission wvelength t 515 nm. Stndrd curves nd qulity control smples were run with ech btch. For nlysis of Hcy in plsm, 20 l of the diluted plsm probe (1: 10) were mixed with 30 l of wter before the reduction gent ws dded. The following steps of the nlysis were identicl to the preprtion of ter fluid. Vit m i n B 6 concentrtions were nlyzed by high-performnce liquid chromtogrphy (kit by Chromsystems, Munich, Germny), nd serum vitmin B 12 nd folte concentrtions were determined by immunossy (Immulite, DPC Biermnn, Bd Nuheim, Germny). Sttisticl Anlysis All sttisticl tests were two-sided nd p vlue less thn 0.05 ws considered sttisticlly significnt. The Kolmogorov-Smirnov test showed tht Hcy, B vitmins, nd ge were normlly distributed. Differences between ptient groups were evluted by the Fisher exct, ANOVA, nd t tests. Correltion between Hcy in Lcriml fluid Hcy (nmol/l) plsm, Hcy in ter fluid, ge, nd B vitmins ws ssessed using Person s correltion coefficient. In controls nd ptients, univrite nlysis followed by multiple regression nlysis ws executed to find nongenetic determinnts of Hcy. After djustment for confounding fctors, logistic regression nlysis ws done to clculte odds rtios for ssocited with elevted Hcy levels. R e s u l t s Hcy ws detected in ll ter fluid nd plsm smples. Hcy levels in ter fluid nd in plsm were significntly higher in ptients with thn in control ptients ( fig. 1 ; tble 2 ). Elevtion of Hcy both in ter fluid nd in plsm ws ssocited with higher risk for compred to controls in logistic regression model ( tble 2 ). In eyes, significnt correltion with moderte degree of strength could be observed between Hcy in plsm nd Hcy in ter fluid (r = 0.459; p = 0.005; fig. 2 ) s opposed to control subjects in whom no significnt correltion ws found (r = 0.068; p = 0.695; fig. 2 b). The ssocition between Hcy levels in ter fluid nd plsm in ptients ws further demonstrted in generl liner nlysis, in which only the plsm Hcy (p = 0.005) ws significnt predictor of ter fluid Hcy, but not ge (p = 0.316), sex (p = 0.510), or B vitmin levels (B 12 : p = 0.144, B 6 : p = 0.755, folte: p = 0.151). In controls, this s- Plsm Hcy (µmol/l) b Fig. 1. Distribution of Hcy levels in ter fluid ( ) nd plsm ( b ) for controls nd ptients. The box length is the interqurtile rnge with the medin represented by the horizontl line. y = Cse with more thn 1.5 box lengths from the upper edge of the box. Homocysteine in Primry Open-Angle Glucom 251

4 Tble 2. Hcy levels in ter fluid nd plsm were significntly incresed in ptients using t tests nd displyed grded risk fctors for in logistic regression model Vrible p vlue (differences) Odds rtio (logistic regression) Hcy in ter fluid, nmol/l t = 4.52; p < Hcy in plsm, mol/l t = 3.63; p = OR = % CI = p = OR = b 95% CI = p = Hyperhomocysteinemi >11.7 mol/l 22% (8/36) 64% (23/36) p = OR = 6.72 c 95% CI = p = Hyperhomocysteinemi >15.0 mol/l 8% (3/36) 39% (14/36) p = OR = 7.80 c 95% CI = p = Odds rtio for per 1 nmol/l elevtion of Hcy in ter fluid fter djustment for ge, folte, cffeine, nd plsm Hcy. b Odds rtio for per 1 mol/l elevtion of Hcy in plsm fter djustment for ge, folte, nd cffeine. Hyperhomocysteinemi, defined on two different cutoff levels, ws more prevlent in using Fisher s exct tests nd ws significnt risk fctor for compred to controls. c Odds rtio for ssocited with hyperhomocysteinemi. Tble 3. B vitmin nd cretinine levels in controls nd ptients Vrible p vlue (differences) Folte in serum, ng/ml t = 0.70; p = 0.49 Vitmin B 12 in serum, pg/ml t = 0.27; p = 0.79 Vitmin B 6 in plsm, ng/ml t = 0.35; p = 0.73 Cretinine in serum, mg/dl t = 0.70; p = 0.49 No significnt differences were found between controls nd ptients in the bove importnt confounding fctors of hyperhomocysteinemi. socition between ter fluid nd plsm Hcy ws not significnt (p = 0.550). There ws no significnt difference in potentil confounding fctors between ptients nd controls: levels of B vitmins nd cretinine, the most importnt nongenetic determinnts of elevted Hcy, did not differ significntly between ptients nd controls ( tble 3 ). We evluted the reltionship between Hcy levels nd mjor nongenetic determinnts of Hcy in generl liner model for both groups. In ptients, neither Hcy in plsm nor Hcy in lcriml fluid ws significntly relted to ny of the nongenetic determinnts of Hcy. However, in the control group, folte (p = 0.008) demonstrted significnt negtive ssocition, wheres ge (p = 0.009) nd cffeine consumption (p = 0.036) showed signifi- 252 Roedl et l.

5 Lcriml fluid Hcy (nmol/l) Plsm Hcy (µmol/l) Lcriml fluid Hcy (nmol/l) b Plsm Hcy (µmol/l) Fig. 2. Sctterplot grphs with liner regression lines in ptients ( ) nd controls ( b ). Positive correltion between ter fluid nd plsm Hcy (r = 0.459; p = 0.005) in ptients. y = Ptients with topicl ntiglucomtous therpy;! = ptients without topicl ntiglucomtous therpy. b No significnt correltion in controls (r = 0.068; p = 0.695). cnt positive ssocition with plsm Hcy levels in the multiple regression model. Hcy in ter fluid ws negtively ssocited with serum folte (p = 0.047) in the control group. Prevlence of definite dry eye syndrome ws significntly higher in ptients compred to controls [28% (10/36) vs. 8% (3/36), respectively; p = 0.035, Fisher s exct test], wheres prevlence of probble dry eye disese ws not significntly different between both groups [31% (11/36) vs. 14% (3/36), respectively; p = 0.155, Fisher s exct test]. ptients with probble dry eye disese hd significntly higher Hcy levels thn subjects without dry eye syndrome both in ter fluid ( nmol/l vs nmol/l; t = 2.33, p = 0.026) nd in plsm ( mol/l vs mol/l; t = 3.09, p = 0.004). In the control group, ptients with dignosis of dry eye did not hve significntly incresed Hcy levels compred to subjects without dry eye disese in ter fluid ( nmol/l vs nmol/l; t = 1.18, p = 0.245) or plsm ( mol/l vs mol/l; t = 1.77, p = 0.368). After exclusion of ll nd control ptients with dry eye disese, ptients with still hd significntly higher Hcy levels compred with control ptients both in plsm ( mol/l vs mol/l; t = 2.86, p = 0.006) nd ter fluid ( nmol/l vs nmol/l; t = 3.16, p = 0.003). Furthermore, ptients with dry eye syndrome hd higher Hcy levels in ter fluid thn control ptients with dry eye syndrome ( nmol/l vs nmol/l; t = 3.0, p = 0.010). Both in ptients with (n = 16) nd without dry eye disese (n = 56), Hcy in ter fluid ws significntly correlted with Hcy in plsm (r = 0.509, p = 0.044, nd r = 0.272, p = 0.043, respectively). Topicl therpy ws bndoned 12 h prior to collection of reflex ters, in order to void contmintion of ter fluid with eyedrops nd thus interference of Hcy mesurements. Despite these precutions, possible influence of chronic ntiglucomtous mediction on Hcy levels could not be ruled out. Therefore, we dditionlly evluted possible ssocitions between topicl therpy prior to the 12-hour puse nd Hcy levels in ter fluid nd dry eye disese. t Tests for the vrious types of ntiglucomtous eyedrops were performed nd no significnt ssocition between Hcy levels in ter fluid nd ny topicl therpy could be observed (t tests; p in ll types of eye mediction; tble 4 ). Furthermore, the 29 ptients with nd the 7 ptients without topicl ntiglucomtous drugs showed no significnt difference in Hcy levels of ter fluid ( nmol/l vs nmol/l; t = 0.559, p = 0.580). Besides tht, Hcy levels in ter fluid nd the number of different topicl drugs used for glucom therpy were not significntly ssocited (x = number of different ntiglucomtous eyedrops; n = number of ptients): x = 0 (n = 7): nmol/l; x = 1 (n = 12): nmol/l; x = 2 (n = 9): nmol/l; x = 3 (n = Homocysteine in Primry Open-Angle Glucom 253

6 Tble 4. Assocition between Hcy levels in ter fluid nd therpy with different ntiglucomtous eyedrops in ptients (n = number of ptients) 4): nmol/l; x = 4 (n = 4): nmol/l; p = 0.830, F = 0.367, one-wy ANOVA. No ssocition ws found between ny of the ntiglucomtous eye medictions nd dry eye syndrome (Fisher s exct test: betblockers, p = 1.0; prostglndin nlogs, p = 0.281; crbonic nhydrse inhibitors, p = 0.475; lph-gonists, p = 1.0; cholinergics, p = 0.306; drenergics, p = 1.0). Furthermore, the number of different ntiglucomtous drugs ws not ssocited with dry eye syndrome (p = 0.302; 2 test). Discussion Yes No p vlue Bet-blockers (n = 19) (n = 17) Hcy in ter fluid, nmol/l t = 0.431; p = Prostglndin nlogs (n = 17) (n = 19) Hcy in ter fluid, nmol/l t = 0.353; p = Crbonic nhydrse inhibitors (n = 13) (n = 23) Hcy in ter fluid, nmol/l t = 0.401; p = Alph-gonists (n = 7) (n = 29) Hcy in ter fluid, nmol/l t = 0.528; p = Cholinergics (n = 1) (n = 35) Hcy in ter fluid, nmol/l t = 0.903; p = Adrenergics (n = 1) (n = 35) Hcy in ter fluid, nmol/l t = 0.300; p = Topicl therpy ws stopped 12 h prior to smpling of reflex ters to void contmintion of ter fluid. Chronic ntiglucomtous tretment prior to this 12-hour puse ws not ssocited with Hcy levels in ters (t tests; p > 0.3 in ll types of topicl mediction). In the current study, incresed Hcy concentrtions in both ter fluid nd plsm of ptients were detected. The mesured men plsm Hcy levels in ptients nd controls of the present study were similr to vlues found in other diseses with Hcy s risk fctor including vsculr [9, 10] nd oculr [19 23] disorders. However, in the study by Wng et l. [25], who found no significnt differences between Hcy levels in nd controls, the men Hcy vlue of the control subjects ws bove most thresholds for hyperhomocysteinemi. It ws t lest 1 mol/l higher compred to controls of other investigtions [9, 19 23] including 27 studies evluted by Boushey et l. [10] in met-nlysis. Furthermore, different ethnicities of the enrolled ptients nd different evlution of criticl nongenetic determinnts of Hcy [31], such s ssessment of nutritionl sttus, B vitmin nd cretinine levels, might be responsible for the inconsistent results in the literture. Hcy in plsm showed significnt nd modertely strong correltion with Hcy in ter fluid in ptients, but not in controls. Furthermore, ptients with dry eye syndrome hd significntly higher Hcy levels in ter fluid nd plsm thn ptients without dry eye disese. Subjects with suffered significntly more often from definite dry eye syndrome thn control ptients. To our knowledge, this is the first study evluting the possible clinicl role of Hcy in ter fluid. Hcy cn induce deposition of extrcellulr mtrix components, fibrosis [11], oxidtive stress, nd production of those subtypes of mtrix metlloproteinses [8], trnsforming growth fctors, collgens [12], nd proinflmmtory fctors [13], which were found to be incresed in dry eye syndrome [7] nd insufficient filtering blebs [4, 5]. Therefore, further studies re wrrnted to investigte whether incresed Hcy levels in ter fluid might be implicted in dry eye syndrome or filure of filtering blebs in eyes. The result of previous investigtion [18], which showed tht hyperhomocysteinemi in might be cused by n endogenous genetic vrint, is indirectly supported by our finding tht elevted Hcy levels in plsm nd ter fluid in were not ssocited with low B vitmin levels or other importnt nongenetic determinnts of Hcy, such s incresing ge or cffeine consumption, s opposed to controls. Similr ssocitions were found for Hcy in queous humor nd nongenetic determinnts in previous study [17]. The study hs limittions. Due to the prospective study design nd the complex nture of collecting ter fluid, the study popultion in this investigtion ws reltively smll (n = 72), especilly the number of ptients with dry eye syndrome (n = 16). Further studies re needed with lrger ptient numbers to confirm the possible 254 Roedl et l.

7 ssocition between incresed Hcy levels in ter fluid nd dry eye syndrome in ptients. In conclusion, our results suggest tht elevted Hcy levels in ter fluid nd plsm might be potentil risk fctors for nd dry eye disese in ptients with glucom. Therpy of high Hcy levels by dministrtion of systemic or topicl B vitmin preprtions would be sfe, effective nd inexpensive [10, 12], nd might therefore ttenute the possible negtive effects of Hcy. However, there re conflicting results in the literture regrding plsm Hcy levels in. Thus, further investigtions re necessry to elucidte the potentil clinicl role of Hcy in glucom ptients, such s the possible ssocition between elevted Hcy vlues nd the higher prevlence of systemic vsculr dysregultions or oculr surfce disorders in. References 1 Emre M, Orgul S, Guglet K, Flmmer J: Oculr blood flow ltertion in glucom is relted to systemic vsculr dysregultion. 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