Introduction: To measure copper (Cu), lysyl oxidase (LOX) activity, and collagen levels in aqueous humour (AH) of primary glaucoma patients and correlate with clinical parameters. Methods: 120 patients with 40 each of primary open angle glaucoma (POAG), primary angle closure glaucoma (PACG), and cataract controls were recruited in this case-control study. AH samples were collected during the trabeculectomy and cataract surgeries. Cu levels were measured using an atomic absorption spectrophotometer. LOX unit activity was determined by Amplex Red assay and collagen concentration by Sirius red assay. Results: Significantly higher levels of Cu expressed as median (IQR) µmol/L were observed in POAG (p = 0.008) and PACG (p = 0.005) compared to controls. The LOX activity was increased in POAG and PACG (p = 0.04) compared to controls represented as median (IQR) µmol/min. The collagen levels given as median (IQR) mg/ml showed an insignificant increase in POAG and PACG compared to controls (p = 0.78). The LOX unit activity was correlated with visual field index (VFI), which showed a significant increase with the progression of the diseases (p < 0.05), whereas Cu levels were negatively correlated with LOX activity in AH. Cu and LOX activity showed weak correlation with YAG peripheral iridotomy (YAGPI), duration of anti-glaucoma medications, and highest preoperative intraocular pressure. Conclusion: Elevated Cu and LOX activity was observed in both POAG and PACG groups compared to controls. LOX activity showed notable increase with VFI as the severity of the disease. Although Cu levels are increased in glaucoma, it’s insufficient to significantly increase the activity of LOX.

Glaucoma is an optic neuropathy, associated with elevated intraocular pressure (IOP) due to increased aqueous outflow resistance in the trabecular meshwork (TM). Increased deposition of the extracellular matrix (ECM) in the TM appears to be responsible for the glaucomatous IOP [1]. The ECM represents a heterogeneous group of macromolecules including collagen, non-collagenous glycoproteins, elastic fibres, and proteoglycans. The ECM is under continuous remodelling by synchronous degradation and synthesis of matrix components with different turnover rates. The structural integrity of the ECM depends on the collagen and elastin cross-links [2].

Copper (Cu) is an essential micronutrient involved in various physiological functions that includes free radical detoxification, mitochondrial respiration, iron metabolism, maturation of neuropeptides, connective tissue formation, and pigmentation [3]. The redox potential of Cu ions makes it a useful biological co-factor for various enzymes such as ceruloplasmin, superoxide dismutase, lysyl oxidase (LOX), and cytochrome oxidase. Cu promotes wound healing and fights infection. Disorders of Cu metabolism have been associated with liver necrosis, carcinogenesis, Menkes and Wilson’s disease [4, 5].

LOX (EC 1.4.3.13; protein-lysine 6-oxidases) is a Cu-dependent amine oxidase that initiates the covalent cross-linking of collagen and elastin in the ECM via generation of aldehydes on lysine residues [6, 7]. The formation of collagen or elastin cross-links leads to an increase in tensile strength and structural integrity, which is essential for normal connective tissue function, embryonic development, and adult tissue remodelling [8]. LOX is secreted as a 50 kDa proenzyme and proteolytically cleaved by bone morphogentic protein 1 (BMP-1) into 32 kDa catalytically active enzyme [9]. LOX expression is regulated by hypoxia-inducible factor-1, IL-1β, IL-6, interferon-γ, transforming growth factor β (TGF-β), tumour necrosis factor α, platelet-derived growth factor, and fibroblast growth factor [10]. LOX expression is inhibited by β-aminopropionitrile, heparin, homocysteine thiolactone, and diethyl pyrocarbonate [11].

Increased Cu in aqueous humour (AH) and serum is reported in unclassified glaucoma [12]. We have reported increased protein and transcript levels of lysyl oxidase like-2 (LOXL2) and elastin in AH and Tenon’s tissue of primary open angle glaucoma (POAG), primary angle closure glaucoma (PACG) compared to cataract controls in a previous study [13]. As LOX is a key player in the formation of ECM, aberrant LOX expression or enzymatic activity cause altered ECM remodelling in glaucoma. There is no report that simultaneously measured Cu, LOX activity, and collagen in AH of POAG and PACG. Therefore, we aimed to determine the Cu concentration, total LOX activity, and collagen concentration in AH of patients with primary glaucoma and cataract as controls.

Patient Selection and Collection of Samples

This research was conducted ethically in accordance with the World Medical Association Declaration of Helsinki. A case-control study was conducted between March 2014 and August 2017, after approval from the Ethics Sub-Committee (Institutional Review Board), Vision Research Foundation (reference number: 289-2011-P). The subjects have given their written consent to participate in the study. Patients undergoing trabeculectomy with or without cataract surgery and cataract surgery alone (controls) were enrolled in this study. The recruitment process of cases to the study was consecutively by presentation to the glaucoma service. Participants were >18 years old; glaucoma patients were either POAG or PACG, diagnosed based on the presence of optic disc and visual field damage in the presence of a gonioscopically open or closed angle. PACG patients (n = 37) had undergone a YAG peripheral iridotomy (YAGPI) with duration between <1 year and 15 years before trabeculectomy. Age-matched cataract patients without history of previous surgery or laser, anti-glaucoma medications (AGMs) use, elevated IOP, or optic disc changes suggestive of glaucoma with open or closed angles were included as controls. Patients with other ophthalmic conditions, use of systemic steroids, and pregnancy or lactation were excluded from the study (Table 1). AH (≤100 µL) was collected through an anterior chamber limbal paracentesis at the beginning of the surgical procedures, using a 30 gauge needle. The AH samples were transported on ice, centrifuged at 3,000 rpm for 10 min at 4°C, aliquoted, and further, immediately within 30 min, frozen in −80°C deep freezer until processing.

Table 1.

Clinical demographics and baseline information of the patients

ParametersCataract (n = 40)POAG (n = 40)PACG (n = 40)p value
Age, years, mean±SD 61.4±10.3 60.7±10.4 62.3±8.8 0.85* 
Gender, n (%)MaleFemale 22 (55)18 (45) 35 (88)05 (12) 23 (58)17 (42) 0.0028a 
Preoperative topical AGMs, n (%) Nil   0.24a 
 Beta-blockers  36 (90) 35 (87)  
 Prostaglandins analogues  32 (80) 33 (82)  
 Alpha adrenergics  24 (60) 24 (60)  
 Carbonic anhydrase inhibitors  19 (47) 15 (37)  
 Cholinergics  00 (00) 05 (12)  
Preoperative topical AGMs, n (%) Nil   0.69a 
 Two  12 (30) 13 (32)  
 Three  25 (63) 22 (55)  
 Four  03 (07) 05 (13)  
Duration of AGMs, years, range (mean±SEM) 0.1–26 (5.7±0.8) 0.2–21.3 (6.6±0.8) 0.33b 
Duration of preoperative AGMs, n (%) Nil   0.54a 
 ≤1 years  06 (15) 06 (15)  
 >1–5 years  18 (45) 12 (30)  
 >5–10 years  09 (22) 12 (30)  
 >10 years  07 (17) 10 (25)  
Systemic illness, n (%)    0.52a 
 Hypertension 21 (52) 17 (42) 17 (42)  
 Diabetes mellitus 19 (47) 18 (45) 18 (45)  
 CVD/IHD 05 (12) 07 (17) 03 (07)  
 Thyroid 04 (10) 01 (02) 04 (10)  
 Hypercholesterolaemia 02 (05) 06 (15) 01 (02)  
 No systemic illness 09 (22) 14 (35) 10 (25)  
CDR (mean±SD) (0.49±0.12) (0.83±0.07) (0.82±0.09) <0.0001* 
Number of eyes with prior laser iridotomy (YAGPI), n (%) Nil Nil 37 (93) 
Duration between YAGPI and trabeculectomy, n (%) Nil Nil  
 ≤1 years   12 (32)  
 >1–5 years   07 (19)  
 >5–10 years   10 (27)  
 >10 years   08 (22)  
VFI (%), range (mean±SEM) 4–95 (51.6±4.8) 4–98 (46.83±4.9) 0.61b 
VFI, n (%)   0.34a 
 1–25%  10 (25) 16 (40)  
 26–50%  09 (22) 04 (10)  
 51–75%  10 (25) 10 (25)  
 76–100%  11 (28) 10 (25)  
Preoperative IOP, mm Hg, range (mean±SD) 10–34 (18.6±6.6) 11–28 (17.6±4.4) 0.016b (POAG Pre vs. Post-IOP) 
Post-operative IOP, mm Hg, range (mean±SD) 4–30 (14.9±5.3) 4–24 (14.9±3.9) 0.017b (PACG Pre vs. Post-IOP) 
Preoperative highest IOP, mm Hg, range (mean±SD) 12–48 (24.7±8.5) 12–50 (25.2±8.3) 0.64b 
Preoperative highest IOP, n (%)   0.36a 
 12–20 mm Hg  18 (45) 14 (35)  
 21–50 mm Hg  22 (55) 26 (65)  
ParametersCataract (n = 40)POAG (n = 40)PACG (n = 40)p value
Age, years, mean±SD 61.4±10.3 60.7±10.4 62.3±8.8 0.85* 
Gender, n (%)MaleFemale 22 (55)18 (45) 35 (88)05 (12) 23 (58)17 (42) 0.0028a 
Preoperative topical AGMs, n (%) Nil   0.24a 
 Beta-blockers  36 (90) 35 (87)  
 Prostaglandins analogues  32 (80) 33 (82)  
 Alpha adrenergics  24 (60) 24 (60)  
 Carbonic anhydrase inhibitors  19 (47) 15 (37)  
 Cholinergics  00 (00) 05 (12)  
Preoperative topical AGMs, n (%) Nil   0.69a 
 Two  12 (30) 13 (32)  
 Three  25 (63) 22 (55)  
 Four  03 (07) 05 (13)  
Duration of AGMs, years, range (mean±SEM) 0.1–26 (5.7±0.8) 0.2–21.3 (6.6±0.8) 0.33b 
Duration of preoperative AGMs, n (%) Nil   0.54a 
 ≤1 years  06 (15) 06 (15)  
 >1–5 years  18 (45) 12 (30)  
 >5–10 years  09 (22) 12 (30)  
 >10 years  07 (17) 10 (25)  
Systemic illness, n (%)    0.52a 
 Hypertension 21 (52) 17 (42) 17 (42)  
 Diabetes mellitus 19 (47) 18 (45) 18 (45)  
 CVD/IHD 05 (12) 07 (17) 03 (07)  
 Thyroid 04 (10) 01 (02) 04 (10)  
 Hypercholesterolaemia 02 (05) 06 (15) 01 (02)  
 No systemic illness 09 (22) 14 (35) 10 (25)  
CDR (mean±SD) (0.49±0.12) (0.83±0.07) (0.82±0.09) <0.0001* 
Number of eyes with prior laser iridotomy (YAGPI), n (%) Nil Nil 37 (93) 
Duration between YAGPI and trabeculectomy, n (%) Nil Nil  
 ≤1 years   12 (32)  
 >1–5 years   07 (19)  
 >5–10 years   10 (27)  
 >10 years   08 (22)  
VFI (%), range (mean±SEM) 4–95 (51.6±4.8) 4–98 (46.83±4.9) 0.61b 
VFI, n (%)   0.34a 
 1–25%  10 (25) 16 (40)  
 26–50%  09 (22) 04 (10)  
 51–75%  10 (25) 10 (25)  
 76–100%  11 (28) 10 (25)  
Preoperative IOP, mm Hg, range (mean±SD) 10–34 (18.6±6.6) 11–28 (17.6±4.4) 0.016b (POAG Pre vs. Post-IOP) 
Post-operative IOP, mm Hg, range (mean±SD) 4–30 (14.9±5.3) 4–24 (14.9±3.9) 0.017b (PACG Pre vs. Post-IOP) 
Preoperative highest IOP, mm Hg, range (mean±SD) 12–48 (24.7±8.5) 12–50 (25.2±8.3) 0.64b 
Preoperative highest IOP, n (%)   0.36a 
 12–20 mm Hg  18 (45) 14 (35)  
 21–50 mm Hg  22 (55) 26 (65)  

AGM, anti-glaucoma medication; YAGPI, YAG peripheral iridotomy; VFI, visual field index; IOP, intraocular pressure; CDR, cup-to-disc ratio; SD, standard deviation; NA, not applicable.

p < 0.05 were considered statistically significant.

* Kruskal-Wallis.

aχ2 test.

bMann-Whitney test.

Atomic Absorption Spectroscopy

Cu levels in AH [14‒17] were measured by atomic absorption spectroscopy using AAnalyst 700 (Perkin Elmer, USA). About 25 µL of AH was added to 50 µL of 30% hydrogen peroxide (H2O2) and kept for digestion in hot air oven for 3 h at 75°C. The digested samples were ashed with 50 μL of nitric acid (Fluka, TraceSELECT). The ashed product was extracted using 500 μL of 0.2% HNO3 centrifuged at 2,500 rpm for 10 min; the supernatant was taken for Cu estimation by atomic absorption spectroscopy at 324.8 nm using a hollow cathode lamp. The slit was maintained at 0.7 nm; Cu was atomized at 2,300°C using a graphite furnace system and detected by a spectrophotometer. Standard Cu (Perkin Elmer) was used for calibration. All measurements were taken in triplicates.

LOX Activity Assay

LOX enzyme activity was measured by Palamakumbura’s modified protocol [18, 19]. Active LOX in the sample reacts with the pseudosubstrate cadaverine and the H2O2 generated in the reaction is able to oxidise the Amplex Red to resorfurin, a fluorescence product. The assay reaction mixture consisted of 50 mm sodium borate (pH 8.2), 50 µm Amplex Red (N-acetyl-3,7 dihydroxyphenoxazine, Molecular Probes, Invitrogen), 0.1 U/mL horseradish peroxidase, and a naturally occurring diamine substrate, 10 mm 1,5-diaminopentane, cadaverine (Sigma, St. Louis, USA). Briefly, 5 µL of AH was added to the final reaction mixture. Resorfurin is the final product of Amplex Red assay, whose fluorescence is measured in kinetic mode at 37°C at 563 nm excitation, and the emission was read at 587 nm using a SpectraMax plate reader (Molecular Devices, USA). A standard graph was obtained using H2O2 (0.2–1.0 µm). The relative fluorescence unit obtained in test samples was calculated based on the standard and was expressed as unit activity (micromoles H2O2/minute). We measured only total LOX activity and did not perform specific assays for the LOX isoforms.

Sirius Red – Collagen Assay

The Sirius red 96-well plate assay protocol was modified from Kliment et al. [20]. In brief, Collagen I (Sigma C3511-100 mg) was used as standard, ranging from 0.3 to 5 mg/mL. About 20 µL of AH was mixed with equal volume of 10% tricholoroacetic acid and centrifuged at 10,000 rpm for 5 min for precipitation. The supernatant was discarded, and the pellet was suspended in PBS and added to well. The plate was incubated at 37°C humidified overnight and 24 h in dried condition at 37°C. It was subsequently washed three times with distilled water. To this, 0.1% Sirius red stain (Direct Red 80, Sigma) in saturated picric acid was added and incubated for 1 h at RT on a rocker. The plate was washed thrice with 5% acetic acid and incubated with 0.1 m NaOH for 30 min at RT on a rocker. The absorbance was read on a SpectraMax M2e (Molecular Devices, USA) at 550 nm.

Statistical Analysis

The data were processed and analysed using statistical analysis software GraphPad® Prism 5 (GraphPad Software, Inc., San Diego, CA). For non-parametric multiple comparisons, Kruskal-Wallis test followed by Mann-Whitney test was done. For non-parametric correlation, Spearman analysis was performed. Data were expressed as median (IQR) or mean ± SEM. p < 0.05 was considered to be statistically significant.

Increased Cu Levels, LOX Activity, and Collagen Levels in AH of POAG and PACG

The Cu levels represented as median (IQR) µmol/L were 0.53 (0.49) in cataract, 0.85 (0.56) in POAG, and 0.82 (0.87) in PACG, respectively. Cu concentration was significantly higher in AH of POAG (p = 0.008) and PACG patients (p = 0.005) compared to cataract controls (shown in Fig. 1a). The LOX activity was 0.40 (0.45) in cataract, 0.47 (0.61) in POAG, and 0.52 (0.52) in PACG median (IQR) µmoL/min. The activity of LOX was significantly increased in AH of PACG (p = 0.04), while in POAG it showed increased activity without any statistical difference compared with cataract controls (shown in Fig. 1b). The collagen levels were 0.20 (0.33) in cataract, 0.32 (0.20) in POAG, and 0.27 (0.21) in PACG median (IQR) mg/mL. The collagen levels showed a non-significant increase in AH of POAG and PACG compared with cataract control (p = 0.78) (shown in Fig. 1c). Although there was a significant increase in Cu and LOX unit activity of POAG and PACG group, there was insignificant negative correlation observed between Cu and LOX unit activity overall in AH of all the groups (r = −0.098; p = 0.35; n = 90) (shown in Fig. 1d). However, this warrants a study in a larger sample size with detection of LOX isoforms.

Fig. 1.

Box plot showing the Cu concentration (a), LOX activity (b), collagen levels (c) in AH of cataract control, POAG, and PACG. *indicates p value comparison between control and test groups. Kruskal-Wallis test was performed to test the significance within the group, followed by Mann-Whitney U-test between groups. Box boundaries mark the 25th and 75th percentile of each distribution. Black line inside each box marks the 50th percentile or median. The whiskers above and below the hinges mark the largest and smallest observed values. d Correlation between Cu and LOX unit activity in all groups. Spearman non-parametric correlation was performed. p < 0.05 is statistically significant. AH, aqueous humour.

Fig. 1.

Box plot showing the Cu concentration (a), LOX activity (b), collagen levels (c) in AH of cataract control, POAG, and PACG. *indicates p value comparison between control and test groups. Kruskal-Wallis test was performed to test the significance within the group, followed by Mann-Whitney U-test between groups. Box boundaries mark the 25th and 75th percentile of each distribution. Black line inside each box marks the 50th percentile or median. The whiskers above and below the hinges mark the largest and smallest observed values. d Correlation between Cu and LOX unit activity in all groups. Spearman non-parametric correlation was performed. p < 0.05 is statistically significant. AH, aqueous humour.

Close modal

Analysis of Cu and LOX Activity with the Clinical Parameters

Association of Visual Field Index with Cu and LOX Activity

Visual field index (VFI) can range between 100% (normal visual field) and 0% (perimetrically blind eye) [21, 22]. Of the n = 80 eyes with glaucoma, n = 61 had phacoemulsification combined with trabeculectomy. Since there were eyes with significant cataract, VFI is less sensitive to cataract-induced change than VF mean deviation. Hence, we had used VF index rather than VF mean deviation.

We categorized the VFI based on the severity of the disease (i.e., 1–25%, 26–50%, 51–75%, and 76–100%). Cu levels showed increased significance with VFI 1–25% (p = 0.006) and 76–100% (p = 0.0005) compared to the controls (shown in Fig. 2a), but Cu levels revealed a weak positive correlation with r = 0.28, p = 0.14, n = 60 (shown in Fig. 2c), suggesting there was no net increase in Cu as the disease progressed. Similarly, we observed that LOX activity increased with VFI 26–50% significantly (p = 0.008), and at VFI 1–25%, a non-significant increase was noticed compared to the controls (shown in Fig. 2b). The correlation between LOX activity and VFI showed that the activity increases significantly (p < 0.05) with the progression of the disease (shown in Fig. 2d).

Fig. 2.

a, b Bar graph showing Cu and LOX unit activity plotted against VFI. *indicates p value comparison between control and test groups. Kruskal-Wallis test was performed to test the significance within the group, followed by Mann-Whitney U-test between groups. c, d Correlation between Cu and LOX unit activity and VFI in glaucoma. Spearman non-parametric correlation was performed. p < 0.05 is statistically significant. VFI, visual field index.

Fig. 2.

a, b Bar graph showing Cu and LOX unit activity plotted against VFI. *indicates p value comparison between control and test groups. Kruskal-Wallis test was performed to test the significance within the group, followed by Mann-Whitney U-test between groups. c, d Correlation between Cu and LOX unit activity and VFI in glaucoma. Spearman non-parametric correlation was performed. p < 0.05 is statistically significant. VFI, visual field index.

Close modal

Association of YAGPI with Cu and LOX Activity

As YAGPI is the first line of treatment in PACG, the duration after YAGPI and Cu levels in PACG were correlated; we stratified the duration of years between YAGPI and trabeculectomy (i.e., 0–1 year, 1–5 years, 5–10 years, >10 years) and observed that Cu levels were significantly increased between 0 and 1 year and >10 years with p = 0.02. However, we observed a weak negative correlation with r = −0.04, p = 0.82, and n = 27, indicating that the YAGPI did not contribute to increased Cu concentration in AH. Likewise, the LOX unit activity was increased without any significance in duration after YAGPI when compared to the controls (p = 0.91). The correlation between LOX activity and YAGPI showed a weak positive correlation with r = 0.01, p = 0.93, and n = 37, disclosing that YAGPI does not influence increased LOX activity (shown in online suppl. Fig. 1; for all online suppl. material, see https://doi.org/10.1159/000531247).

Association of the Duration of the AGMs with Cu and LOX Activity

The duration of the AGMs was segregated as 0–1 year, 1–5 years, 5–10 years, and >10 years. We observed that Cu levels were significantly increased (p < 0.05) in 0–1 year, 5–10 years, and >10 years durations of the AGMs compared to the controls. However, there was a weak positive correlation in Cu with increasing duration of the AGMs (r = 0.05, p = 0.69, n = 60), whereas LOX unit activity showed non-significant increase compared to the controls with increasing duration of the AGMs. It showed a weak negative correlation in LOX activity (r = −0.17, p = 0.11, n = 80). This clarifies that duration of AGMs does not increase the Cu and LOX activity levels in the AH of primary glaucoma (shown in online suppl. Fig. 2).

Association of the Highest Preoperative IOP with Cu and LOX Activity

We grouped highest preoperative IOP into two groups with 12–20 and 21–50 mm Hg. We observed that Cu was significantly increased (p < 0.01) in both the IOP ranges compared with the controls. But there was a weak positive correlation between Cu and IOP, with r = 0.01, p = 0.93, n = 60. The LOX activity showed a non-significant increase compared to the controls. But it showed a weak positive correlation between LOX activity and IOP, with r = 0.006, p = 0.95, n = 80 (shown in online suppl. Fig. 3). As the collagen levels were measured in a smaller number of samples (n = 10) in each group, the data were insufficient to run any statistical analysis for clinical correlation.

The Cu concentration is found to be within 0.018–0.080 pg/mL (0.28–1.3 μmol/L) in the AH of normal human subjects. In the present study, we have selected patients with primary glaucoma who showed increased Cu concentration compared to controls. Elevated Cu levels may be due to the abnormalities in AH secretion, difficulties in drainage through Schlemm’s canal, or breakdown of the blood-aqueous barrier (BAB) as a result of trauma, infection, and inflammation [14]. Aydin et al. [15] discussed augmented vascular permeability results in break of the BAB, with the transfer of Cu into the AH. Increased concentration of Cu was reported in the lens, serum, and AH confirmed that Cu may play a role in matrix formation in pseudoexfoliation syndrome patients [16]. The trace element Cu is able to generate oxidative stress via Fenton’s reaction or reduction of glutathione. Glutathione plays a critical role in protection of cells against oxidative damage [23, 24]. Increased Cu results in generation of hydroxyl radicals, causing DNA strand breaks [15, 25]. Cu accumulation is associated with fibrosis in the human liver and oral submucous tissue in humans, renal fibrosis, and lung fibrosis in rats [26]. It is reported that abnormal concentrations of Cu and iron in the eye may be due to depolymerization of hyaluronic acid which blocks the outflow of AH through the TM, leading to increase in IOP [27]. Although we reported the levels of Cu to be elevated in primary glaucoma cases, we found that they negatively correlated with LOX, revealing that the levels of Cu were not sufficient to make LOX more active.

Inflammation in the anterior chamber was found in eyes with PACG before YAGPI with laser flare meter assessments of aqueous flare, and cells indicated inflammation due to the impairment of the BAB [28]. YAG laser iridotomy is performed to break the pupillary block [29] in the eyes with PACG, which would result in the disruption of the BAB. We observed a significantly increased level in AH Cu, between 0 and 1 year duration, since the YAGPI was performed, indicating a compromised BAB. In a previous study, we had correlated the total protein in AH and the duration of year between YAGPI and trabeculectomy in PACG (n = 61) to check if the increase in AH protein levels is attributed to the effect of YAGPI. However, no significant clustering (p = 0.2515, r = −0.1491, R2 = 0.0222) was observed. This suggests that increased protein is more reflective of the pathological condition rather than the treatment interventions [30]. However, the blood level of Cu was not measured in this study.

In healthy ocular tissues, LOX is detected in the AH [31], vitreous, iris, ciliary body, lens, choroid, retinal pigment epithelium, and retina [19]. LOX is an important factor in cross-linking and stabilizing collagen, and elastin fibres are associated with ECM remodelling in various ocular diseases [32‒34]. The specific activity of LOX in AH is reported to be significantly lower in eyes with PXF compared with cataract controls [31]. LOXL2 is a candidate susceptibility gene for the population-specific genetic risk of POAG [35]. LOXL2 levels in AH and Tenon’s tissue were significantly increased in POAG patients with bleb failure [36]. We have also reported that LOXL2 levels were increased significantly in POAG and PACG eyes in AH and Tenon’s tissue, showing a significant increase in PACG [13]. Reports suggest upregulation of LOXL2 with activation of TGF-β in POAG Tenon’s tissue [36]. In our study, LOX activity levels were significantly increased in PACG compared to controls. Repeated appositional closure in PACG could result in influencing changes in the TM [37]. Sihota et al. [38] had highlighted the changes in acute and chronic PACG with extensive changes in the trabecular region. About n = 25 had peripheral anterior synechiae in PACG on gonioscopic evaluation. Peripheral anterior synechiae may reduce outflow of AH, which leads to elevated IOP. Since TGF-β isoforms are increased in the AH of glaucoma patients, increased LOX activity might be responsible for an increased outflow resistance of AH by inducing deposition of ECM material within the TM, increasing its stiffness, and thereby elevating intraocular pressure [7, 39]. Bergen et al. [7] had reported administration of LOXL2 mAb increased bleb area and survival. These studies implicate the role of LOX in ECM remodelling and its therapeutic implication.

Remodelling of collagen in the outflow pathway may change mechanical tissue characteristics, causing increased AH outflow resistance and leading to elevation of IOP. Several modifications in collagen expression and transcription have been identified in the TM of POAG. Type 1 collagen is the important constituent within the TM and uveoscleral outflow pathways [40]. Aihara et al. [41] induced type 1 collagen mutations in mice which led to an elevation of IOP, suggesting an association between IOP regulation and collagen turnover. We observed an increased total collagen level in AH which was not statistically significant. This requires larger sample size to draw any conclusion. A mass spectrometry study on low-abundant protein may help in identifying the isoforms of collagen present in AH. It may be helpful to estimate various collagens as we have only measured the total collagen in this study.

VFI is determined to calculate the rate of progression of disease, and it stages the functional damage of glaucoma. It closely reflects the loss of retinal ganglion cells [21, 22]. In this study, we report that the LOX activity is significantly increased with decreasing VFI, i.e., progression of the disease. We also observed an increase in the Cu and LOX activity in YAGPI, duration of disease, and highest preoperative IOP. But Cu showed a weak positive correlation with VFI, duration of the disease, and highest preoperative IOP and a weak negative correlation was observed in YAGPI. Likewise, LOX activity showed a weak positive correlation with YAGPI and highest preoperative IOP, and a weak negative correlation was observed in duration of the disease. The limitations of the study include small sample size, lack of serum sampling, and LOX isoform assessment.

This is the first study to determine the activity of LOX in AH of primary glaucoma with a significant increase in PACG. Cu levels were significantly increased in POAG and PACG compared to the controls. LOX activity significantly increased with the severity of the disease based on VFI. Other clinical parameters such as YAGPI, duration of the disease, and highest preoperative IOP did not show any significant correlation. Although Cu levels are increased in glaucoma, they are insufficient to significantly increase the activity of LOX. We had measured LOX activity and collagen; probably analysing LOX and collagen isoforms would throw more light on the clinical association, which could be warranted with a larger sample size.

This research was conducted ethically in accordance with the World Medical Association Declaration of Helsinki. This study protocol was reviewed and approved by the Vision Research Foundation, Ethics Sub-Committee (Institutional Review Board), reference number: 289-2011-P. Subjects had given their written informed consent to participate in the study.

The authors have no conflicts of interest to declare.

The financial assistance for the study was provided by Indian Council of Medical Research under the project – 5/4/6/4/oph/12-NCD-II.

Sampath Nikhalashree performed all the in vitro experiments, acquired and interpreted data, and prepared the manuscript. Ronnie George, Balekudaru Shantha, and Lingam Vijaya provided surgically excised Tenon’s tissue for the study and reviewed the manuscript. Konerirajapuram Natarajan Sulochana designed the experiments, interpreted the data, gave intellectual inputs for the work, and reviewed the manuscript. Karunakaran Coral conceived the idea, designed the experiments, interpreted the data, and prepared and reviewed the manuscript. All authors read and approved the final manuscript.

All data generated or analysed during this study are included in this article and/or its supplementary material files. Further enquiries can be directed to the corresponding author.

1.
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Kelley
MJ
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