Reduction in Blood Glutathione Levels Occurs Similarly in Patients With Primary-Open Angle or Normal Tension Glaucoma

Abstract

Purpose.: To investigate in parallel the systemic glutathione levels of patients suffering from primary open angle glaucoma (POAG) or normal tension glaucoma (NTG) with comparable functional loss.

Methods.: Thirty-four POAG patients, 30 NTG patients, and 53 controls were subjected to blood analysis to detect the level of circulating glutathione in its reduced (GSH) and oxidized (GSSG) forms. Systemic blood pressure (BP) and ocular perfusion pressure (OPP) parameters were also determined.

Results.: Independent of age, POAG and NTG patients demonstrated significantly lower GSH and t-GSH levels than age-matched controls (P < 0.001). Additionally, a lower redox index was found, but in POAG patients only, in comparison to both NTG and control groups (P = 0.020). GSSG levels were, however, similar between all study groups (P > 0.05).

Conclusions.: This study demonstrates, for the first time, that both POAG and NTG patients exhibit lower GSH and t-GSH levels than age-matched controls, indicating a similar general compromise of the antioxidant defense systems may exist in both conditions.

Introduction

In addition to an abnormal intraocular pressure (IOP), other risk factors such as ocular and systemic circulation abnormalities have been linked to the etiology and progression of both primary open-angle glaucoma (POAG) ^1–6 and normal-tension glaucoma (NTG). ^7–10 Moreover, it has been speculated that POAG and NTG patients exhibit similar alterations in both ocular and systemic circulation and that a considerable overlap may in fact exist in the etiology of both POAG and NTG. ¹¹ Nevertheless, questions still remain around the mechanisms which ultimately lead to similar vascular alterations in both of these forms of glaucomatous optic neuropathy (GON). It is possible that explanations are in fact multiple and involve more than one factor, to include the autonomic nervous system ^1,12 and endothelial dysfunctions. ^7,11,13 In addition, oxidative stress represents a variable that seems to play an important role in both types of glaucomatous neurodegeneration ¹⁴ as well as in diseases associated with vascular dysfunctions and abnormal blood flow, including glaucoma. ¹⁵ Indeed, it has been demonstrated that high levels of oxidative stress induce local trabecular damage ^16,17 in POAG patients when compared with age-matched controls. In addition, these patients also demonstrate an impairment of the antioxidant defense systems. ^14,15 The status of these defense systems can be measured using a large variety of techniques, including assessments of various enzymes, micronutrients, vitamins, small molecules, and more. ¹⁸ The antioxidant glutathione (GSH, L-_-glutamyl-L-cysteinylglycine) is among these factors. Glutathione represents a tripeptide consisting of glycine, cysteine, and glutamic acid and prevents the effects of radical oxygen species (ROS) either directly as an antioxidant or indirectly, by maintaining other cellular antioxidants in a functional state. A low level of circulating glutathione results in a higher rate of oxidative reactions, which subsequently may reduce, among others, the bioavailability of nitric oxide (NO) ¹⁹ with important consequence on the normal regulation of systemic hemodynamics ²⁰ and especially on the equilibrium between the endothelial vasoconstrictory and vasodilatory factors. In glaucoma, this could result in a general vasospastic tendency manifested at both peripheral ²¹ and ocular vasculature. ²² Indeed, the presence of endothelial dysfunction has been reported at both systemic macrocirculation ⁴ and retinal microcirculation levels ³ of patients with POAG. However, less is known about the link between oxidative stress and NTG and the few existing studies have pointed toward a completely different situation from that exhibited by POAG patients, namely a compensatory increase in the antioxidant defense mechanisms in NTG individuals comparing to controls. ²³ Based on the above observations, it could be hypothesized that an increase in oxidative stress is not part of the pathogenic process in NTG. However, in light of multiple vascular dysfunctions repeatedly associated with this type of GON, this is a surprising conclusion. Indeed, we have recently demonstrated that newly diagnosed NTG patients showed signs of subclinical vascular abnormalities at both macro- and microvascular levels. ⁷ In addition, we have also shown that there are multiple comparable alterations in both ocular and systemic vascular function between POAG and NTG patients. ¹¹ Such macro- and microvascular changes may contribute to the development of GON regardless of the level of IOP and in addition to other factors oxidative stress could indeed play a pivotal role. The aim of the present study, therefore, was to investigate in parallel the systemic glutathione levels in patients suffering from POAG or NTG with comparable functional loss. 

Materials and Methods

Successive, newly diagnosed, and previously untreated POAG and NTG patients were recruited for the below investigations from two local UK National Health System (NHS) Trusts. Only those patients identified as having glaucomatous cupping of the optic disc on fundoscopic assessment, normal open anterior chamber angles by gonioscopy, and visual field (VF) defects consistent with the diagnosis of glaucoma using a commercial visual field analyser (SITA 24-2, Humphrey Field Analyzer; Carl Zeiss Meditec, San Leandro, CA) were included. Only reliable VF plots, with <20% fixation losses and <33% false positive and false negative responses, were considered. Classification as POAG was based on an IOP measurements consistently above 21 mm Hg on diurnal testing with applanation tonometry (measurements taken every 2 hours across an 8-hour period). Classification as NTG was based on an IOP measurement consistently less than or equal to 21 mm Hg on diurnal testing with applanation tonometry. Patients with closed iridocorneal angles, evidence of secondary glaucoma, pseudoexfoliation, history of intraocular surgery, cataract, or any form of retinal or neuro-ophthalmological disease that could result in visual field defects were excluded from the study. Furthermore, newly diagnosed POAG and NTG patients with more advanced visual field loss, defined as a mean deviation (MD) score of < −8.00 dB, were also excluded from the study. 

Age-matched healthy controls were recruited by inviting the participation of patients' spouses and friends, as well as through promotion of the study at the Aston University Health Clinics, Birmingham, UK. All healthy controls were screened for glaucoma and other ocular disease and were excluded if any signs consistent with glaucomatous optic neuropathy, ocular hypertension, or retinal disease were found. 

Other exclusion criteria for all the groups were smoking or a history of any chronic systemic disease with presumed abnormal circulating glutathione levels, including autoimmune diseases, ²⁴ alcoholic liver disease, ²⁵ cancer, ²⁶ and diabetes mellitus. ²⁷ In addition, subjects were also excluded if they suffered from cardio- or cerebrovascular disease; coronary artery disease (heart failure, arrhythmia, stroke, and transient ischemic attacks); inflammatory conditions (rheumatoid arthritis and systemic lupus erythematosus); or were receiving hormone replacement therapy or antioxidant, vitamins, or iron supplements. Mild, well-controlled hypertension was not a criterion for being included or excluded from the study. 

Sixty previously untreated POAG, 55 NTG patients and 68 controls were screened during the study period. However, after this strict selection, the final experimental group was narrowed to 34 POAG patients, 30 NTG patients, and 53 controls. Ethical approval for the study was received from Heart of England and Sandwell and West Birmingham NHS Research Ethics Committees, as well as the Aston University Ethics Committee. Informed consent was obtained from all participants before entry to the study. All procedures were designed and conducted in accordance with the tenets of the Declaration of Helsinki. 

Subjects were instructed to fast after 9 PM on the evenings before being tested. On the morning of the test, subjects were requested to have only a light breakfast such as simple toast. They were also asked to avoid any cooked breakfast, meat, cereal, fresh fruits, or fruit juice. In addition, subjects were asked to abstain from caffeinated beverages and chocolate and from alcohol for at least two hours before the visit. 

Investigations

Blood Pressure (BP) Measurement.

Blood pressure was measured in each subject in the morning between 8 and 9 AM with a BP monitor (UA-779; A&D Instruments Ltd., Oxford, UK). In preparation for this measurement, each subject rested in a sitting position for approximately 10 minutes in a quiet room to achieve sufficient mental and physical calm. The systolic BP (SBP) and diastolic BP (DBP) were measured three times (1 minute apart). The average readings for SBP and DBP were then used to calculate the mean BP (MBP) using the formula: MBP = 2/3(DBP) + 1/3(SBP). 

Blood Sampling and Analyses.

In order to avoid variations and GSH loss, we have paid particular attention to blood collection, initial processing, and storage. Blood samples were collected at the same time (between 9 AM and 10 AM) and processed in the same way and time interval from collection in all individuals. 

A total of 7 mL of blood was collected in EDTA tubes (to prevent oxidation) ^11,28 by venipuncture to the antecubital vein using a butterfly needle and syringe to avoid hemolysis ²⁹ ; 30 μL of blood was then transferred into centrifuge tubes for the initial processing. The GSH was released from the blood cells by protein precipitation and cellular disruption achieved by addition of 33.3 μL of 5-sulphosalicylic acid (SSA), 1 g/mL within 10 minutes from the blood collection. ³⁰ The addition of acid minimizes artifactual sample oxidation and removes interfering protein thiols. 

Each sample was then diluted with 936.7 μL sodium phosphate buffer (pH 7.5) and the content of each tube was rapidly centrifuged at 13,000 rpm for 5 minutes; 150 μL of supernatant were then collected into clean centrifuge tubes and immediately cooled at −70°C. Samples stored at this temperature are stable for at least 2 months and can be transported on dry ice without deterioration. ³¹ In our hands, GSH loss is less than 5% over this time period. ³²  

GSH Assay.

The total GSH levels (t-GSH) were assessed by the glutathione reductase-DTNB (5.5 dithiobis-2-nitrobenzoic acid) recycling procedure, as described in previous studies. ^15,28 Standards were prepared from 0 to 80 μM in 20-μM increments using 10 mM GSH solution. The standards contained the same final concentrations of SSA as for the samples. To each well of a 96-well plate, NADPH (0.3 mg/mL)—dissolved in 150 μL of 125 mM sodium phosphate with 6.3 mM EDTA pH 7.5, also known as daily buffer—50 μL of DTNB solution, and 25 μL of standards or samples were added in quadruplicate and the plate was incubated at 37°C for 3 minutes. Finally, 25 μL of GSH reductase (GSR) were added to the previous mixture and the plate was read at 410 nm using a 96-well plate reader. A standard curve of GSH was then generated using a linear regression program (Microsoft Excel; Microsoft Corporation, Redmond, WA) as previously reported. ¹⁵  

GSSG Assay.

The GSSG levels were assessed using a glutathione reductase-DTNB recycling assay. ³³ The reagents used in this assay were those already described above for GSH assay and, in addition, triethanolamine (TEA) and 2-vinyl pyridine (2-VP). TEA prevents a local high local pH and oxidation while 2-VP is used for derivitization of GSH. GSSG standards were prepared from 0 to 10 μM in 1-μM increments; 100 μL of standards and samples were transferred into separate centrifuge tubes and 2 μL 2-VP was added to each tube. TEA was then used to adjust the pH of the standards/samples to a pH of 7.5. The assay was carried out as for GSH assay described above. Finally, a standard curve of GSSG was then generated using a linear regression program (Microsoft Corporation) as previously reported. ¹⁵  

The GSH levels (t-GSH − [2 × GSSG]) and the redox index (defined as the GSH/GSSG ratio) were then calculated. 

Statistical Analysis

The statistical analysis was performed using commercial statistical software (Statistica, version 9.0; StatSoft, Tulsa, OK). Data are expressed as mean ± SD. The Kolmogorov-Smirnov test was used to determine the distribution of the data. Multivariate analysis was performed to determine the influence of age, sex, and systemic BP on blood glutathione levels. Differences between groups were subsequently assessed using one-way ANOVA or ANCOVA, as appropriate, followed by Tukey's post hoc analysis. P values of less than 0.05 were considered statistically significant. 

Results

Thirty-four POAG patients (17 men and 17 women); 30 NTG patients (9 men and 21 women); and 53 controls (30 men and 23 women) were included in the present study. The characteristics of the study groups are given in Table 1. There was no significant difference in age and systemic BP between the two groups of glaucoma patients and control subjects (all P > 0.05); however, there were significant differences in IOP (P < 0.001) with POAG patients exhibiting higher values than both NTG patients and controls (all P < 0.001, post hoc Tukey's HSD test). In addition, there was no overall significant difference between the number of men and women included in the study groups (P = 0.063). 

Table 1. 

 

Summary of the Systemic Characteristics of the Three Study Groups

	POAG	NTG	Controls	P Value	Significance
N	34	30	53	–	–
Sex	17 M, 17 F	9 M, 21 F	30 M, 23 F	0.064
Age, y	64.85 ± 8.95	62.07 ± 11.81	61.02 ± 6.60	0.145	–
SBP, mm Hg	134.88 ± 13.14	127.96 ± 11.18	126.67 ± 16.14	0.056	–
DBP, mm Hg	77.90 ± 11.43	73.06 ± 9.82	75.16 ± 10.59	0.249	–
MBP, mm Hg	96.89 ± 11.12	91.36 ± 9.07	92.33 ± 11.99	0.138	–
IOP, mm Hg	25.44 ± 3.63	17.76 ± 2.56	16.60 ± 3.34	<0.001*	1 > 2, 3
OPP	47.27 ± 7.48	48.80 ± 6.31	50.27 ± 8.21	0.273	–
MD	−2.03 ± 5.04	−2.95 ± 3.56	–	0.469	–

P  < 0.05 is considered a significant difference (*).

Effect of Age, Sex, and BP on Blood Glutathione Levels

A forward stepwise multiple regression analysis has revealed that age had a negative effect on GSH in both the control and NTG groups (β = −0.33, P = 0.013, and β = −0.44, P < 0.001, respectively, Fig. 1). In addition, age also had a negative effect on the t-GSH in the NTG patients (β = −0.45, P < 0.001, Fig. 2) and redox index in the control group (β = −0.37, P = 0.001, Fig. 3). In the POAG patients, however, all measured blood glutathione levels were not significantly influenced by age (all P > 0.05). 

Figure 1

 

(A) Age influence on GSH levels in NTG patients. (B) Age influence on GSH levels in control subjects.

Figure 2. 

 

Age influence on t-GSH levels in NTG patients.

Figure 3. 

 

Age influence on redox index in controls.

There was no significant association between blood glutathione levels and sex or systemic BP values in either of the study groups (all P > 0.05). Sex differences in blood glutathione levels are outlined in Table 2. 

Table 2. 

 

Sex Differences in Blood Glutathione Levels Between the Three Study Groups

	POAG			NTG			Controls
	Men 17	Women 17	P Value	Men 9	Women 21	P Value	Men 30	Women 23	P Value
GSH, nmol	236.67 ± 129.68	221.34 ± 134.41	0.741	306.79 ± 276.02	338.33 ± 276.02	0.753	455.83 ± 229.87	411.56 ± 220.87	0.507
GSSG, nmol	36.22 ± 26.24	46.30 ± 20.94	0.233	40.27 ± 22.26	39.86 ± 21.65	0.963	40.15 ± 24.59	43.26 ± 24.81	0.668
t-GSH, nmol	272.89 ± 120.93	267.65 ± 137.10	0.908	347.05 ± 147.64	368.67 ± 272.28	0.748	495.97 ± 234.70	454.83 ± 225.51	0.546
GSH/GSSG, redox index	8.84 ± 6.54	6.64 ± 4.45	0.244	10.05 ± 6.56	10.05 ± 9.72	0.778	14.77 ± 10.34	11.74 ± 7.45	0.270

Intergroups Differences in Blood Glutathione Levels

Table 3 shows the measured blood glutathione parameters in all 3 study groups. After correcting for age influences in an ANCOVA model, GSH and t-GSH levels were significantly lower in both POAG and NTG groups comparing with controls (P < 0.001, F = 8.74; and P < 0.001, F = 8.69, respectively). In addition, redox index was also reduced but only in POAG as compared with both NTG and control individuals, which were comparable (P = 0.020, F = 4.04). There were no significant differences between study groups in GSSG levels (P > 0.05). 

Table 3. 

 

Intergroups Differences in Blood Glutathione Levels (ANCOVA and Post Hoc Test)

	POAG	NTG	Controls	P Value	Significance
N	34	30	53	–	–
GSH, nmol	228.77 ± 130.29	328.19 ± 240.52	437.38 ± 224.77	<0.001	1, 2 < 3
GSSG, nmol	41.42 ± 24.00	39.98 ± 21.44	41.44 ± 24.47	0.938	–
t-GSH, nmol	270.19 ± 127.51	368.52 ± 11.18	478.70 ± 229.40	<0.001	1, 2 < 3
GSH/GSSG, redox index	7.15 ± 5.60	10.71 ± 8.71	13.50 ± 9.28	0.020	1 < 2, 3

P  < 0.05 is considered a significant difference. 1 = POAG group; 2 = NTG group; 3 = normal controls.

Discussion

The present study assessed the blood circulating GSH and GSSG levels in newly diagnosed and previously untreated patients diagnosed with either POAG or NTG. Our results disclosed that independent of age, POAG and NTG patients demonstrated significantly lower GSH and t-GSH levels than age-matched controls. In addition, only POAG patients have demonstrated a lower redox index than both NTG and control groups. GSSG levels were, however, similar between all study groups. 

GSH is among the most efficient substance that cells and tissues can use in their defense against oxidative stress ³⁴ and is representative of the redox environment of the cell. Age and disease, however, act to decrease the amount of GSH available in the organism. It has been demonstrated that at least half of the apparently healthy elderly individuals show low blood GSH levels. ^35–37 Indeed, our results show a negative correlation between age and both GSH levels and redox index in controls. This effect was, however, lost in the POAG patients group. In the general population, age is strongly associated with increased oxidative stress and reduced antioxidant status. ³⁸ In addition, it also results in reduced glutathione synthesis. ³⁹ The loss of the correlation between age and GSH in POAG patients, demonstrated in the present study, could indicate that other factors are influencing GSH levels in this cohort and this result is consistent with our previous observations. ¹⁵ Nevertheless, a surprising result of the present study was that similarly to the healthy controls, age also had also a negative effect on both GSH and t-GSH levels in the NTG patients group. It is possible that in this type of patient, age still has a strong and independent influence on the blood glutathione levels despite the fact that our NTG patients exhibited much lower GSH and t-GSH levels than the controls and comparable with those recorded in the POAG individuals. Therefore, it cannot be excluded that in the NTG group, the age effect on GSH synthesis is accelerated by other noxious variables that result in further GSH depletion and that are specific to this type of glaucoma. The implications of this observation will be explored later in this article. 

Although some studies report sex differences in plasma GSH levels, ⁴⁰ others did not confirm it in either plasma or blood GSH. ⁴¹ In a previous report, we have also shown that in healthy controls, men have higher levels of GSH and t-GSH than women. ¹⁵ However, in a slightly younger cohort, we were not able to record again such difference. ³² As the methodology used by these later two papers was identical, this discrepancy could only occur due to the age difference of the subjects included in each of these studies. In the present report, in a similar age group, and in agreement with Lu et al. and Michelet et al., we also could not find this time any difference between men and women with respect to blood glutathione levels in either of our study groups. It seems, therefore, that sex has a less consistent effect on the blood glutathione levels than other confounding variables such age and more research will be necessary to clarify this issue. 

The main finding of the present report represents that both POAG and NTG groups show similarly lower GSH and t-GSH levels than age-matched controls. Although we have previously demonstrated an abnormal GSH level in the blood of patients suffering from POAG, ¹⁵ this represents the first study to assess in parallel blood glutathione levels in patients suffering from either POAG or NTG, with similar functional loss. 

It has previously been shown that oxidative stress exists at high levels at both ocular ^{16,17,42–47} and systemic levels, ¹⁵ and plays an important role in glaucomatous neurodegeneration. ¹⁴ In addition, POAG patients also demonstrate an impairment of the antioxidant defense systems. ^14,15 Nevertheless, in NTG patients, these things are less clear. Existing reports show that these patients exhibit either a higher antioxidant response ²³ or similar total glutathione circulating levels to normal controls. ⁴⁸ These results are somehow surprising. It is widely recognized that NTG is associated with both ocular and systemic vascular dysfunctions ^7,11,49–51 and among other factors, a short supply of NO or a rapid deactivation of the existing reserves have been hypothesized as playing a causative role in the process. ⁷ As for the cause of this NO depletion, a dominant mechanism involved in reducing vascular NO is rapid oxidative inactivation in the presence of excessive ROS with an increased formation of peroxynitrite anion (ONOO⁻), ⁵² a highly toxic reactive nitrogen species (RNS). In addition, excessive amounts of ROS uncouple endothelial nitric oxide synthase (e-NOS) and result in endothelial dysfunction. ⁵³ Therefore, the neurodegeneration and vascular dysfunction associated with either POAG or NTG could be at least in part due to intracelullar oxidation and low NO bioavailability as a direct result of high levels of ROS or RNS found in these two forms of the disease. ^23,54 This would imply, however, that the antioxidant defense systems of these patients are defective and unable to cope with free radicals' overload. Although this might seem the case in POAG, ¹⁵ Yuki et al. ²³ has shown that in NTG patients, an increase in the systemic oxidative stress triggers a compensatory activation of antioxidant substances. However, our present results show the opposite, with NTG patients showing similar reduction in GSH and t-GSH to those with POAG. It is possible that the various methods used to assess antioxidant status are partially responsible for this lack of consistency between various reports. However, in the present study, we used a validated method for measuring blood levels of GSH and GSSG. ^15,28,55 In addition, glutathione is a major antioxidant substance and has a substantial contribution to the body's overall ROS scavenging activities. ⁵⁶ Therefore, we are confident that the assessment of this parameter offers a good estimation of the antioxidant defense level samples of patients such as those included in the present paper. It is also possible that the discrepancies observed between the results of our study and those published by Yuki et al. and Park et al. are the result of differences in diet or ethnicity. Our sample consisted only of White European subjects born in the UK and no attempt was made to change their usual diet. More research is, therefore, necessary to establish possible differences between various populations with respect to their antioxidant defense systems and glaucoma pathogenesis. 

One interesting observation of the present study is that despite differences in blood GSH between the glaucoma patients and controls, GSSG levels were similar. This is, however, in accordance to our previously published results. ¹⁵ It seems that the most plausible explanation for this apparent contradiction could be that in our glaucoma patients, the mechanisms responsible for the normal redox cycle were defective and have not allowed a reduction of part or all GSSG back to GSH in order to rebuild the GSH reserves. In addition, a possible defective GSH synthesis could also be implicated; this later hypothesis is, however, still controversial in glaucoma. ¹⁵ Future studies are necessary to allow a better understanding of the mechanisms responsible for the above results. 

Conclusion

In conclusion, our study demonstrates for the first time and in parallel, that both POAG and NTG patients demonstrate lower GSH and t-GSH levels than age-matched controls suggesting a similar general compromise of the antioxidant defense systems. Whether this systemic imbalance is among the risk factors that leads to the similar vascular alterations in these two forms of GON is still to be determined. 

Acknowledgments

Disclosure: D. Gherghel, None; S. Mroczkowska, None; L. Qin, None